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Isolation and identification of 0-linked--N-acetylglucosamine modified proteins (O-GlcNAc) in the developing Xenopus laevis oocytes
h [electronic resource] /
by Sreelatha Paspuleti.
[Tampa, Fla.] :
University of South Florida,
Thesis (M.S.)--University of South Florida, 2005.
Includes bibliographical references.
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ABSTRACT: Oocyte development in Xenopus laevis spans six morphologically distinct stages (stage I-VI), and is associated with a decrease in protein O-GlcNAc levels. As a first step in elucidating the role of O-GlcNAc in developing oocytes, initial efforts were focused on isolation and identification of fifteen modified proteins that decrease during oocyte development. Stage I oocytes due to their high amounts of these proteins, were used as starting material for purification. Multiple affinity and specific antibody based purification technique were initially used in an attempt to enrich the O-GlcNAc proteins. Due to the unique properties of the proteins ultimately identified, these techniques were unable to provide sufficient material for sequencing. However, differential centrifugation coupled with 2D-gel electrophoresis was highly successful. The majority of isolated proteins were strongly basic in nature with pIs 8-10.Coomassie stained bands from 2D-analysis were trypsin digested, and peptides were sequenced by mass spectroscopy (Finnigan LCQ). Mass data were interpreted by Bioworks software, and protein sequences were compared to multiple protein databases. Initially, six proteins were identified as Thesaurin a (42Sp50), cytoplasmic mRNA binding protein p54, y-box homolog, Xp 54 (ATP dependent RNA helicase p54), Vg1 RNA binding protein variant A, Zygote arrest 1(Zar1) and Poly (A) binding protein (PABP). Thesaurin a, the main component of 42S particle of previtellogenic oocytes (stages I-III) is involved in tRNA storage and possess low tRNA transfer activity; y-box factor homolog and Xp54 are present in oocyte mRNA storage ribonucleoprotein particles; Vg1 RBP variant A associates mVg1 RNA to microtubules in order to translocate to the vegetal cortex; Zar1 is involved in oocyte-to-embryo transition; and PABP initiates mRNA translation.This study is the first to characterize these oocyte specific proteins as O-GlcNAc modified proteins. Overall, the presence of several O-GlcNAc proteins in oocytes, the reduction in their levels/ O-GlcNAc levels, and the variation in maturation time in the presence of HBP-flux modulators in developing oocyte indicates O-GlcNAc may play important roles in metabolism, cell growth and cell division of X. laevis oocytes. Therefore, identifying the remainder of these proteins and elucidating the O-GlcNAc role in their function is a worthwhile pursuit.
Adviser: Potter, Robert.
Cytoplasmic mRNA binding protein p54.
Vg1 RNA binding protein variant A.
Zygote arrest 1.
Two-dimensional gel electrophoresis.
t USF Electronic Theses and Dissertations.
Isolation and Identification of Olinked-N-acetylglucosamine Modified Proteins ( OGlcNAc) in the Developing Xenopus laevis Oocyte by Sreelatha Paspuleti A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Dr. Robert Potter, Ph.D. Committee Members: Dr. David Merkler, PhD Dr. Larry Solomonson, PhD Date of Approval: November 8, 2004 Keywords:,Thesaurin a, Cytoplasmic mRNA binding protein p54, Vg1 RNA binding protein variant A, Zygote arrest 1,Two-dimensional gel electrophoresis. Copyright 2004 Sreelatha Paspuleti
Dedication To those who have given me the confiden ce, ability, love and encouragement to complete this thesis; I dedicate this new scientific information to my late sister Sunitha, my parents (P. S. Pandu Ranga Rao and P.V. Roopavathi), my aunt (P. V. Vijayakumari), my brothers (Sreedhar and Shashidhar), my si sters (Swapna and Supriya) and my beloved husband (Ravi Jonna).
ACKNOWLEDGEMENTS I wish to acknowledge and thank those who helped me throughout my study at the University of South Florida. I thank my ma jor Professor Robert Potter for his guidance and support throughout the project. I thank my senior, Dr. Stephen A Whelan who is currently post-doctoral fellow in Dr. G. W. Hart laborator y at John Hopkins University for the protein sequencing and identification. I am also indebted to all my friends in the laboratories and the department, who were plentiful source of encouragement. I thank my brother, Sreedhar Paspuleti for encouraging me to continue my studies in US. The selfless attitude of my husband, Ravi Jonna, has allowed me to pursue my goal of finishing this thesis and I appreciate his patience, love and affection. I thank all of the USF Professors who o ffered their time for discussions and their laboratories to me. These include Dr. Larry P Solomonson, Dr. Sidney Pierce, Dr. David Merkler, and Dr, Ted Gauthier. Additionally, I thank the chemistry department at USF for the financial support throughout my study.
i Table of contents List of TablesÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â….. iii List of FiguresÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…. iv AbstractÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…vi Chapter 1 Introduction: The use of Xenopus laevis as a model system for cell developmental studies involving OGlcNAc modified proteinsÂ…Â…Â…Â…Â…Â…. 1 1.1 Statement of problemÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â… 1 1.2 Introduction to Xenopus laevis oocyteÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…. 3 1.2.1 OogenesisÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…... 3 1.2.2 Oocyte developmentÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â….. 6 1.2.3 Protein synthesis during oogenesisÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â… 8 1.2.4 Gluconeogenic metabolismÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â….. 10 1.3 Introduction to Olinked-N-linked acetylglucosamine modificationÂ…Â…...11 1.4 Earlier findings of the studies on OGlcNAc modification in oocytes of Xenopus laevis Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…. 20 Chapter 2: Materials and MethodsÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â… 22 2.1 MaterialsÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…. 22 2.1.1 ReagentsÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…... 22 2.1.2 EquipmentÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…... 23 2.1.3 AnimalsÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â… 23 2.1.4 Buffers and solutionsÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…... 23 2.2 MethodsÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…...27 2.2.1 Oocyte harvesting and isolationÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…... 27 2.2.2 Homogenization of the oocytesÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…... 28 2.2.3 Protein estimationÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â… 29 2.2.4 Immunoprecipitation with RL-2 antibodyÂ…Â…Â…Â…Â…Â…Â…Â…Â…... 29 2.2.5 Immunoaffinity purific ation using CTD110.6 antibodyÂ…Â…Â…Â…. 30 2.2.6 Affinity chromatographyÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…. 31 2.2.7 Differential sedimentation for enrichment of OGlcNAc modified proteinsÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…. 32 2.2.8 One-dimensional gel electrophoresisÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…... 32 2.2.9 Two-dimensional gel electrophoresisÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…...33 2.2.10 Gel staining methodsÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…..35 18.104.22.168 Coomassie stainingÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…..35
ii 22.214.171.124 Silver stainingÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…..35 2.2.11 Gel dryingÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…...36 2.2.12 ImmunoblottingÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…..36 126.96.36.199 CTD110.6 immunoblottingÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…. 37 188.8.131.52 RL-2 immunoblottingÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…. 37 2.2.13 Wheat germ agglutinin affinity blottingÂ…Â…Â…Â…Â…Â…Â…Â…Â…... 38 2.2.14 Membrane staining methodsÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…. 39 184.108.40.206 India ink stainingÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â… 39 220.127.116.11 Ponceau S stainingÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â….. 39 18.104.22.168 Coomassie stainingÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…..40 2.2.15 Membrane stripping methodsÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â… 40 2.2.16 Identification of the protein bands of interestÂ…Â…Â…Â…Â…Â…Â…Â… 40 2.2.17 Mass spectrometric peptide sequencing and identification of proteinsÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…... 41 Chapter 3 Results and DiscussionsÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…... 43 3.1 Confirmation of the presence of high OGlcNAc modified proteins in stage I oocytes compared to st age VI oocytes per unit massÂ…Â…Â…Â…Â…Â….. 43 3.2 Two-dimensional analysis of stage I and VI oocytesÂ…Â…Â…Â…Â…Â…Â…Â…Â…. 47 3.3 Immunoprecipitation with RL-2 antibodyÂ…Â…Â…Â…Â…Â….............................. 54 3.4..Immunoaffnity purificati on using CTD110.6 antibodyÂ…Â…Â…Â…Â…Â…Â…Â….. 57 3.5 Affinity chromatographyÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â… 61 3.6 Differential sedimentationÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â….. 65 3.7 Identification and selection of the modified bands for sequencing.......Â…Â…. 70 3.8 Mass spectrometry and database searchÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…. 73 Chapter 4 Conclusion Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…86 ReferencesÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â….. 88 AppendicesÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…... 100 Appendix A AbbreviationsÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…. 101 Appendix B Structures of commonly used compoundsÂ…Â…Â…Â…Â…Â…Â…Â…Â….. 105 Appendix C Mass dataÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â… 108
iii List of Tables Table 22.214.171.124 Composition of a Xenopus oocyte, fully grown and without follicle cells... 9 Table 1.3.1 List of the OGlcNAc modified proteins classified based on their FunctionsÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…...14-16 Table 3.8.1 Mass data of a pep tide of Vg1 RBP variant AÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…... 75 Table 3.8.2 Peptide sequences of Vg1 RBP variant AÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…..75
iv List of Figures Figure 1.2.1 Female Xenopus laevis (South African clawed frog)Â…Â…Â…Â…Â…Â…Â…Â…Â…. 4 Figure 126.96.36.199 The different stages of Xenopus laevis oocytes during oogenesisÂ…Â…Â…...5 Figure 1.3.1 Olinked-N-acetylglucosamine ( OGlcNAc modified) modified protein. 13 Figure 1.3.2 Schematic diagram of the he xosamine biosynthetic pathway and the dynamic processing of OGlcNAc modification by O -GlcNAc transferase and OGlcNAcaseÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…. 18 Figure 3.1.1 CTD110.6 immunoblot of one-dimensi onal gel separation of proteins from ooc ytes at stages I, II, III and VIÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â… 44 Figure 3.1.2 Competition with 15mM N-Acetylgl ucosamine to show specificity of OGlcNAc modified to CTD110.6 antibodyÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â… 45 Figure 3.1.3 RL-2 immunoblot of one-dimensi onal gel electrophoretic separation of proteins from oocytes at stage IÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â… 46 Figure 3.2.1 Two-dimensional ge l electrophoresis of oocyte pr oteins (stage I) using isoelectric fo cusing with pH range 3-10 in the horizontal dimension and SDS-PAGE (10%) in the vertical dimensionÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â… 49 Figure 3.2.2 Two-dimensional gel electrophore sis of oocyte proteins (stage VI) by isoelectric focusi ng with pH range 3-10 in the horizontal dimension and SDS-PAGE (10%) in the vertical dimensionÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…... 50 Figure 3.2.3 Two-dimensional gel electrophore sis of oocyte proteins (stage I) by isoelectric focu sing with pH range 6-11 in the horizontal dimension and SDS-PAGE (10%) in the vertical dimensionÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…....52 Figure 3.2.4 Two-dimensional gel electrophore sis of oocyte proteins (stage VI) by isoelectric focu sing with pH range 6-11 in the horizontal dimension and SDS-PAGE (10%) in the vertical dimensionÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…... 53 Figure 3.3.1 One-dimensional gel electropho resis of RL-2 immunoprecipitate of stage I oocytesÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â….. 56
v Figure 3.4.1 One-dimensional gel electropho resis of the CTD110.6 immunoaffinity purified proteins of stage I oocytesÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â….. 58 Figure 3.4.2 One-dimensional and two-dimens ional gel electrophores is of CTD110.6 immunoprecipitate (Batch-wise incuba tion method) of stage I oocytesÂ….. 60 Figure 3.5.1 WGA Affinity blot of the oocytes at stages I and VIÂ…Â…Â…Â…Â…Â…Â…Â…... 62 Figure 3.5.2 One-dimensional gel electrophoresi s of affinity purified proteins from oocytes at stageIÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â… 64 Figure 3.6.1 One-dimensional gel electrophoresis of proteins from oocytes at stage I fractionated by Differential SedimentationÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â….. 66 Figure 3.6.2 Two-dimensional gel electrophore sis of pellet at 100,000 x g from stage I oocytes by isoel ectric focusing with pH 6-11 in the horizontal dimension and SDS-PAGE (10%) in the vertical dimensionÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â… 68 Figure 3.6.3 Two-dimensional gel electrophore sis of pellet at 100,000 x g from stage I oocytes by isoe lectric focusing with pH 3-10(NL) in the horizontal dimension and SDS-PAGE (10%) in the vertical dimensionÂ…Â…Â…Â…Â…...69 Figure 3.7.1 Scheme for Isolati on and Identification of the OGlcNAc modified proteinsÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…. 71 Figure 3.7.2 Two-dimensional gel electrophore sis of pellet at 100,000 x g from stage I oocytes by isoel ectric focusing with pH 6-11 in the horizontal dimension and SDS-PAGE (10%) in the vertical dimensionÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â… 72 Figure 3.8.1 General fragmenta tion pattern of peptide and sequence nomenclature for mass ladderÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…...74 Figure 3.8.2 MS/MS spectrum of a protein ba nd # 9a identified as Vg1 RNA binding protein variant AÂ…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…... 74 Figure 3.8.3 Sequence of Vg1 RNA binding protein variant AÂ…Â…Â…Â…Â…Â…Â…Â…Â…... 76
vi Isolation and Identification of the Olinked-N-aetylglucosamine ( OGlcNAc) Modified Proteins in the Developing Oocytes of Xenopus laevis Sreelatha Paspuleti ABSTRACT Oocyte development in Xenopus laevis spans six morphologically distinct stages (stage I-VI), and is associated with a decrease in protein OGlcNAc levels. As a first step in elucidating the role of OGlcNAc in developing oocytes, initial efforts were focused on isolation and identification of fifteen modi fied proteins that decrease during oocyte development. Stage I oocytes due to their hi gh amounts of these proteins, were used as starting material for purification. Multiple a ffinity and specific antibody based purification technique were initially used in an attempt to enrich the OGlcNAc proteins. Due to the unique properties of the proteins ultimat ely identified, these techniques were unable to provide sufficient material for sequen cing. However, differential centrifugation coupled with 2D-gel electrophoresis was hi ghly successful. The majority of isolated proteins were strongly basic in nature with pIs 8-10. Coomassie stained bands from 2Danalysis were trypsin digested, and pe ptides were sequenced by mass spectroscopy (Finnigan LCQ). Mass data were interpre ted by Bioworks software, and protein sequences were compared to multiple protein databases. Initially, six proteins were identified as Thesaurin a (42Sp50), cytoplasmic mRNA bindi ng protein p54, y-box homolog, Xp 54 (ATP dependent RNA helicase p54), Vg1 RNA binding protein variant A,
vii Zygote arrest 1(Zar1) and Poly (A) binding protein (PABP). Thesaurin a, the main component of 42S particle of previtellogeni c oocytes (stages I-III) is involved in tRNA storage and possess low tR NA transfer activity; y-box factor homolog and Xp54 are present in oocyte mRNA stor age ribonucleoprotein particles; Vg1 RBP variant A associates mVg1 RNA to microtubules in orde r to translocate to the vegetal cortex; Zar1 is involved in oocyte-to-embryo transition; and PABP initiates mRNA translation. This study is the first to characterize these oocyte specific proteins as OGlcNAc modified proteins. Overall, the presence of several OGlcNAc proteins in ooc ytes, the reduction in their levels/ OGlcNAc levels, and the variation in maturation time in the presence of HBP-flux modulators in de veloping oocyte indicates O -GlcNAc may play important roles in metabolism, cell growth and cell division of X. laevis oocytes. Therefore, identifying the remainder of these proteins and elucidating the O -GlcNAc role in their function is a worthwhile pursuit.
1 Chapter 1 Introduction 1. The use of Xenopus laevis oocyte as a model system for the cell developmental studies involving OGlcNAc modified proteins: 1.1 Statement of the Problem Olinked-N-Acetylglucosamine ( OGlcNAc) modification characterized two decades ago  has been found to be ubiquitous in metazoans and is a dynamic posttranslational modification of many nuclear and cytosolic pr oteins. Subsequent studies have found the modification on a wide variety of proteins suggesting the diversity in funtions of this modification . Importantly, the levels of OGlcNAc in different types of cells changes in response to the external s timuli, such stress , insulin signaling , glucose metabolism [5-7], and cell cycle prog ression  suggesting a potential regulatory role for OGlcNAc modified proteins in many cellular processes. Additionally, changes in OGlcNAc have been linked to diseases such as AlzheimerÂ’s , diabetes , and cancer  highlighting its poten tial clinical importance. As one approach to investigating the possible function of OGlcNAc in cell growth and division, oocytes from the South African clawed frog Xenopus laevis were selected as a model system. This oocyte system has long been used as a general model for the study of growth and differen tiation as well as analysis of cell cycle processes in eukaryotes . Interestingly, recent repor ts have demonstrated that changes of OGlcNAc
2 levels in oocytes impact the oocyte growth and maturation [11-13] although the exact function of the modification and the role of modified proteins are still unclear. For instance, the toxic effect of the galactosyl capping of OGlcNAc residues observed in maturing Xenopus laevis oocyte has suggested th e possible role of the OGlcNAc in the aster formation during meiosis . A delayed progesterone stimulated maturation of the fully grown oocytes when incubated with compounds which elevate OGlcNAc levels, and a reduction in OGlcNAc levels of developing oocyte during the stage progression (stage I-VI) have indicated that one or more OGlcNAc modified proteins may be critical for oocyte maturation and development [11,13]. In addition, the decrease in OGlcNAc levels during th e stage progression  (stage I-VI) is in correlation to the increase in OGlcNAc removal activity of OGlcNAcase. The reduction in levels that occu rs especially in high molecular weight protein (> 36 kDa) reflects the metabolic transi tion from glycolytic to gluconeogenic state . A similar phenomenon of OGlcNAc levels was observe d during the transition of cell to malignancy, indicating the fully grown oocyte (stage VI) has at least some characteristics in common with a malignant cell wher e the regulation of the modification is also altered . All these experimental fi ndings suggest that the OGlcNAc modification might play some important regulatory role in c onnecting cell growth, division and metabolism. As one step in elucidating th ese connections in a developmental system, it is important to first isolate and identify the modified oocyt e proteins that change during oocyte development. Since a relatively large fr action of stage I oocyte proteins are OGlcNAc modified, these oocytes were used as sour ce for the studies described herein .
3 1.2 Introduction to Xenopus laevis oocyte The oocyte of Xenopus laevis the South African Clawed frog (Figure 188.8.131.52) is a good model system for biochemical studies for se veral reasons. First of these is the availability of abundant l iterature on its anatomy, morphol ogy and metabolism . Second, the frogs being highly adaptable and resistant to infection can be easily maintained at low cost in the laboratory. Third, because of continuous and asynchronous oogenesis, the oocytes in all stages of development can be obtained from the ovary of an adult female frog. The ovarian tissue regenerates in two to three months and oocyte can be harvested up to four times from each frog . Finally, in particular, th e stage VI oocyte due to its large sizes can be used with ease for semiquantitative microinjection studies that have proven instrumental in understanding the cont rol of cell proliferation and the regulation of cell cycle [15, 16]. 1.2.1 Oogenesis Oogenesis is defined as the process of fo rmation of ova or unfertilized eggs from the oogonia. The process is comprised of two phases, the first phase is a growth phase and second phase is a maturation phase. Th e oogonia enter meiosis I and become arrested at prophase I, where they are termed ooc ytes. At the prophase I arrest, the oocytes begins accumulating a large store of mRNA mitochondrial DNA, and proteins required for the initial rapid cell divi sions, along with a large am ounts of yolk proteins and glycogen also required for post-fertilization. This first phase is further sub-divided into six major stages based on the morphology and anat omy of the growing oocyte  (Figure I.2.2.1). During this stage progression, the ooc yte transforms from a small and transparent stage I to large and banded stage VI oocyte that are desc ribed stage-wise in
4 Figure 1.2.1 Female Xenopus laevis (South African Clawed Frog) Female frogs larger than males. An average frog we ighs approximately 130-150 g and capable regenerating the ovarian tissu e in three-four weeks. They are maintained in tanks containing room temperature dechlorinated wa ter and approximately 1 liter of water per frog). They are fed twice to thrice a week w ith the nutrient rich frog brittle. (Nasco)
5 Figure 184.108.40.206The different stages of Xenopus laevis oocytes during oogenesis. Two phases of the oogenesis (formation of egg for fertilization) are displayed. The first phase constitutes the oocyte growth, and further subdivided into six majo r stages (I-VI) based on the features such as, diameter, pigmentati on color and the amount of yolk protein in the cytoplasm. The second phase is maturati on of fully grown oocyte (stage VI) where the oocyte undergoes germinal vesicle brea kdown (GVBD) on progesterone stimulation. Figure from the website: http://www. luc.edu/depts/biology/dev/xenoogen.htm. Stage I oocytesclear and transparent (50-300 m) Stage II oocyteswhite and opaque (300-450 m) Stage III oocyteslightly pigmented all over (450-600 m) Stage IV oocytesyolk prot ein deposition at the upper an imal hemisphere (600-1000 m) Stage V oocytesaccumulating yolk and ha ve darker pigmented color (1000-1100 m) Stage VI oocytesfully grown and have pr ogesterone receptors on their plasma membrane (1100-1300 m) Stage VI oocytes undergo meiosis when expos ed to the steroid progesterone producing a white spot at the animal pole. Stage Maturing oocyte Stage progression of oocyte
6 detail in the following section 1.2.3. In the maturation phase, the fully grown oocyte (stage VI) forms an unfertilized egg upon stimulation with the steroid hormone, progesterone . The stimulated oocyte arrested at the G2/M border resumes meiosis I, and progresses through meiosis II to become once again arrested at metaphase II. A wh ite spot, the attachment of meiotic spindle to plasma membrane was formed on the animal hemisphere of stage VI oocyte indicating the breakdown of the oocyte nucleus or germin al vesicle (GVBD) . The formation of white spot on the animal hemisphere is the most obvious external indication of oocyte maturation (Figure 220.127.116.11). Thus the stimul ated oocyte undergoes a number of morphological and biochemical changes prior to arrest at the metaphase II. The result of maturation process is a matured oocyt e or unfertilized egg that is now ready for fertilization. 1.2.2 Oocyte Development As found in the frog ovary, oocytes are ar rested at prophase I between the second growth phase (G2) and the mitotic phases (M ). They undergo a significant change from a small transparent oocyte at stage I to a larg e banded oocyte by stage VI with a distinct brown hemisphere or so called animal pole a nd green vegetal hemisphere or vegetal pole . The nucleus and majority of the metabo lic functional organelles are found in approximately 0.5 l of the animal hemisphere while the equal sized vetegal hemisphere contains mainly yolk protein and glycogen in stages IV-VI (Figure 18.104.22.168). The small stage I oocytes range from 50 to 300 m in diameter with a transparent cytoplasm and visible germinal vesicle (nucleus). The clea r cytoplasm contains a microscopic yellowish mitochondrial mass and stains intensely for RNA and lightly for pol ysaccharides. These oocytes are previtellogenic, which means th ey are not involved in active accumulation of
7 the yolk proteins. In the ovary, the interspersed oocytes from all stages of development are surrounded by three layers; the innermos t follicular epithelium, the middle theca made up of connective tissue containing blood vessels and fibroblasts and the outermost surface epithelium . The white and opaque stage II oocytes are 300-450 m in diameter, and comprises of 45% of the Stage II to VI oocyte popul ation. At this stage, the acellular vitelline envelope begins to develop. At Stage III, th e visible pigmentation and the vitellogenesis (the process of active accumulation of the yol k proteins) are initiated. These oocytes are tan or light brown in color but with no visible differentiation of the animal and vegetal poles. They range from 450-600 m in diameter and RNA synthesis p eaks at this stage. The stage IV oocytes range in size from 600-1000 m. They show the clear differenttiation of the animal and vegetal poles. The animal pole is dark brown in color containing the nucleus. Stage V grow from 1000-1200 m in diameter and develops a distinct boundary between the dark brown animal and the greenish yellow vegetal hemispheres . Stage VI oocytes are post vitellogenic. The two distinct brown animal and light greenish yellow vegetal hemispheres sepa rated by an unpigmented 0.2 mm wide equatorial band. The nucleus is eccen trically situated near the an imal pole, and a clear polarization of nuclear envelope becomes visi ble . The fully grown oocyte is 1,000,000 times the volume of typical somatic cell . Half of the volume consists of the yolk proteins while the nucleus is now 300-400 nm in diameter . These oocytes at the G2/M border arrest are responsive to the horm one, progesterone and upon stimulation undergo germinal vesicle breakdown (GVBD) into an unfertilized egg.
8 1.2.3 Protein synthesis during oogenesis The Xenopus oocytes arrested at prophase I have accumulated massive amounts of yolk proteins, lipids and ma ternal mRNA required for the se ries of cell divisions that take place during maturation, fertilization a nd embryogenesis. Therefore a fully grown oocyte (stage VI) as described above is an abnormally large cell with a bloated nucleus referred to as the germinal vesicle. The cont ents of this unusual ce ll are summarized in the table 1.5.1 . Table 1.5.1 shows that the oocyte at stage VI consists of largest amounts of yolk protein, ri bosomal protein mRNA, mitochondrial DNA and rGTP (precursors). Whereas non-yolk proteins, heat shock 70 mRNA, oocyte chromosomal DNA, and dTTP (precursors) were present in lo west amounts. The accumulation of mRNA is completed by the end of stage II of oogenesis. However, the lampbrush chromosomes that exhibit transcription rates higher than those typical for somatic cells remains active during the entire period of oogenesis [21-23] The estimated rate of poly (A) mRNA entry into cytoplasm in stag e III and VI oocytes is approximately 1.4 ng per day. Even though, this is a small percentage of the to tal maternal mRNA, the actual amount of message that enters the cytoplasm is quite hi gh due to the enormous rate of nuclear RNA synthesis . The DNA microi njection experiments show that maternal transcripts are responsible for most of the protein synthesi s during oogenesis, not the DNA injected into the oocyte nucleus . The non-yolk proteins are accumulated in the oocytes by endogenous protein synthesis and yolk deposition by micropinocytos is of vitellogenin synthesized by the adult frog in the liver [25, 26]. The rate of protein synthesis increases over 100-fold during the stage progression from stage -VI. A similar increasing trend was observed in the
9 Table 22.214.171.124Composition of a Xenopus oocyte, full grown and without follicles cells. (Total Volume, 1 l; yolk-fr ee volume 0.5 l; GV volume 40 nl) Component Weight Number of co mponents %of total in cytoplasm DNA Oocyte chromosomal 12 pg None Nucleolar (rDNA) 25 pg 2 x 106 rDNA repeats None Mitochondrial 4000 pg ~108 gemones 100% RNA 99% Ribosomal protein m-RNA 5 g 1012 ribosomes 99% 5S 60 ng 1012 99% tRNA 60 ng 1.5 x 1012 99% snRNA U1 0.07 ng 8 x 108 90% polyA+RNA 80 ng 5 x 1010 (if 2500 bases long) Ribosomal protein m-RNA 10 ng 2 x 1010 Actin mRNA ~1 ng 5 x 108 Heat-shock 70m RNA 0.004 ng 106 Protein Yolk 250 g 100% Non-yolk 25 g 5 x 1014 ( 30 K protein) 90% Histones 140 ng 5 x 1012 50% Nucleoplasmin 250 ng 5 x 1012 98% RNA polymerase I and II ~ 105 x somatic cell RNA polymerase III 5 x 105 x somatic cell Precursors dTTP 10 pmol rGTP 250 pmol Methionine 40 pmol This table is from Â‘Microinjection and Or ganelle Transplantation Techniques. Methods and ApplicationsÂ’. Edited by Celis J. E; Graessmann A; Loyter, A. (1986)
10 amount of ribosomal RNA, and approximately 2% of the ribosomal RNA was found to be engaged in protein synthesis during oogenesis . An additional two fold increase is observed when maturation is induc ed in the oocyte . Gurdon et al have demonstrated that the stage VI oocytes have spare transl ational capacity, that is injected mRNAs are translated in addition to endogenous messages . Howeve r, the latter studies have shown that the translational capacity of the ooc yte is not uniform for different classes of messages. The injected message s that are translated on the free cytosolic polysomes were expressed, whereas the ones that are translat ed on the endoplasmic reticulum resulted in the accumulation of these inject ed mRNA . This non-uniform ity in the translation of mRNAs was apparently due to the limited ava ilability of one of the translational machinery, the ribosomes on endoplasmic reticulum in the oocyte . 1.2.4 Gluconeogenic metabolism In addition to the accumulation of yolk protein, the developing oocyte synthesizes a large amount of glycogen that is used as the source of energy during embryogenesis (from gastrulation stage onwards)  Incorporation of microinjected 32P-labelled glycolytic intermediates, such as 32P-labeled phosphoenolpyruvate and glucose-6-phosphate into UDP-glucose and then presumably into gl ycogen in the stage VI oocytes, fertilized eggs, and cells of cleaving embryos has suggest ed that basic metabolism in these cells is gluconeogenic and glycogenic until the embryo r eaches the late blastula stage . Interestingly, rapid incorporation of 32P-glucose-6-phosphate into ATP, inorganic phosphate and creatine phosphate in stage II oocytes a nd not in stage VI oocyt es also indicated a metabolic transition from glycolyt ic to gluconeogenic state . Since enolase is thought to out-compete pyruvate kinase for any phos phopyruvate, and the activity of the fructose
11 1, 6-bisphosphate is low in the stage II and VI oocytes , the inorganic 32P is found in very low amounts compared to typical cells. Th is indicates that the oocyte metabolism is mainly gluconeogenic, and very little energy is derived from glycolysis. However, the (U-14C)glucose microinjection into the oocytes Stage VI) of Caudiverbera caudiverbera the Chilean frog, and Xenopus laevis (data not shown) has de monstrated glycogen synthesis by an indirect pathway involving glycolytic breakdown of glucose to lactate, which is then converted into glycogen via an appa rently connected gluconeogenic pathway . Therefore glycogen synthesis might be possible in both direct and indirect pathways in amphibian oocytes. Since there is no glycogen breakdown until the gastrulation, the amino acids especially glutamine are used as the carbon s ource for cellular energetics and macromolecular synthesis in the fertilized egg and in the oocytes . A sim ilar type of metabolism where glutamate is a major energy source has also been observed in some tumor cells [35, 33] might explain some of the decreases in OGlcNAc levels in these cells . 1.3 Introduction to Olinked-N-acetylglucosamine modification The glycosidic linkage of the -N-acetylglucosamine through the hydroxyl side chains of serine or threonine resi dues of proteins is termed as the Olinked-N-acetylglucosamine ( OGlcNAc) modification (Figure 1.3.1). The modification occurs in many nuclear and cytosolic proteins of metazoans [1, 37]. This novel post-translational modification was first discovered in murine lymphoc ytes by Torres and Hart in 1984. Since the first characterization, a myriad of proteins have been found to bear this modification  (Table1.2.1). Most of these OGlcNAc modified proteins ar e also phosphoproteins. In an analogous manner to Ophosphorylation, the modification is regulated by a unique set of
12 proteins that add and remove the modifi cation in response to cellular stimuli . Given the dynamic nature of OGlcNAc modification, and the fact that in at least in some cases it acts as an alternative to protein phosphorylation, this modification has increased the complexity of the regulatory processes in the cell [2, 9, 38-41]. The OGlcNAc modification is a dded to protein by a ubiquitous enzyme, uridine diphospho-N-acetylglucosamine: polypeptide -N-acetylglucosminyl transferase or OGlcNAc transferase (OGT). The OGT gene th at is present on the X chromosome was shown to be essential for cell viability . OGT is a soluble protein found in the cytosol but more predominantly in nucleus was first ch aracterized in rat liver extract by the Hart group . The enzyme composed of three subunits in an 2 conformation with two 110 kDa -subunits and one 78 kDa -subunit . The UDP-GlcNAc level in the cell regulates the enzyme activity of the -subunit with a low apparent Km 545 nM for UDPGlcNAc that can change with the concentr ation of UDP-GlcNAc and the other regulators . OGT contains an N-termin al tetratricopeptide repeat (TPR) domain and C-terminal catalytic domain [46, 47]. The C-terminal domain that resembles glycogen phosphorylase superfamily of glycosyl transferase hypothesi zed to contain two Rossman type folds and UDP-GlcNAc binding site . Whereas, the N-terminal TPR domain mediates proteinprotein interaction that appears to be necessary for the enzyme self-association, as well as the substrate recognition [48-52]. The crysta l structure of human OGT has shown that homodimeric TPR domain containing 11.5 TP R repeats form an elongated superhelix, and its concave surface is lined by a conserved array of asparagines. This asparagines array shows marked similarity to the array in armadillo (ARM) repeat proteins, importin and -catenin, and thus suggesting that the TP R domain of OGT uses a similar mecha
13 Figure 1.3.1Olinked -N-acetylglucosamine ( OGlcNAc) modified protein. N-acetylglucosamine group in -linkage at the hydr oxyl group of the serine, Ser residue of the protein.
14 Table 1.3.1List of the OGlcNAc modified proteins classified based on their functions. Table from the review Â‘ Proteomic Approaches to Analyze the Dynamic Relationships between Nucleocytopl asmic Protein Glycosylation and PhosphorylationÂ’ by Whelan, Stephen A.; Hart, Gera ld W. (Circulation research, (2003), 93, 1047-58). Functional Subgroup Protein Refe rence from the above review paper Chaperones Heat shock protein 27 (HSP27) 90, 102 Heat shock cognate 70 (HSC70) 96* Heat shock protein 70 (HSP70) 104 Heat shock protein 90 (HSP90) 96* Chromatin Chromatin associated proteins 105 Cytoskeleton Actin-based Ankyrin G 106 Cofilin 96* E-cadherin 107* Myosin 108* Protein band 4.1 109 Synapsin 110 Talin 111 Intermediate Filaments Keritins 8, 13, 18 112, 113 Neurofilaments H, M, L 114, 115 Microtuble-based -tubulin 104* Dynein LC1 96* Microtubule associated proteins 2 & 4 (MAP 2 & 4) 116 Tau 16 Other Adenovirus type 2 & 5 fiber proteins 117, 118 Assembly protein 3 & 180 (AP-3 & AP-180) 119, 120 -Amyloid precursor protein ( -APP) 23 -Synuclein 121 Piccolo 96* Plakoglobin 122 Kinases and Adaptor Proteins Casein Kinase II (CKII) 63 Glycogen synthase kinase-3 (GSK-3 ) 63 Insulin receptor substrate 1 & 2 (IRS-1 & -2) 27, 28, 67 PI3-kinase (p85) 28 Metabolic Enzymes Enolase 96* Endothelial nitric oxide synthase (eNOS) 72 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 96*
15Glycogen synthase (GS) 70 Phosphoglycerate kinase (PGK) 96* Pyruvate kinase (PK) 96* UDP-glucose pyrophosphrylase (UGP) 96* Nuclear Hormone Receptors Estrogen receptor& (ER& ) 17, 123, 124 Verb A 125* Nuclear Pore Proteins (NUP) Nup 62 126 Nup 153, 214, 358 127 Nup 180 128 Nup 54, 155 96* Phosphatases Nuclear ty rosine phosphatase p65 129 Phosphatase-2a inhibitor (i2pp2a) 96* Polymerases RNA Pol II 15 Prot Ooncogenes c-Myc 14 RNA binding proteins 40S ribosomal protein S24 (40SrpS24) 96* Elongation factor 1(EF-1) 96* Eukaryotic initiation factor 4A1 (EIF 4A1) 96* Ewing-sarcoma RNA-binding protein (EWS) 96, 130 RNA binding protein G (hnRNP G; La-antigen) 131 Transcription factors AP-1 (c-fos and c-jun) 132* -catenin 107 CAAT box transcription factor (CTF, NF-1) 132* Cyclic AMP response element-binding protein (CREB) 95 ELF-1 (Ets transcription factor) 26 Enhancer factor 2D (EF-2D) 96* Hepatocyte Nuclear Factor 1 (HNF-1) 133 KIAA0144, Oct1 96* NFB 134 OGT interacting protein 106 (OIP-106) 38 p53 135 Pancreatic/duodenal homeobox-1 protein (PDX-1, IPF-1, STF-1) 136 PAX-6 137 Pancrease-specific tran scription factor (PTF-1) 138* Human C1 transcription factor (HCF) 96* Serum Response Factor (SRF) 91 Sp1 132 Ying Yang 1 (YY1) 139 Tumor suppressors Retinoblastoma protein (Rb) unpublished Viral Proteins Baculovirus gp41 tegument protein 140 HCMV UL32 (BPP) tegument protein 141 NS26 Rotavirus Protein 142
16SV-40 large T-antigen unpublished Virion basic phosphoprotein 143 Other Annexin 1 96* Collapsin response mediator protein-2 (CRMP-2) 121 Elongation initiation factor-2 associated 67 kDa (EIF2 p67) 144 Gaba-receptor interacting protein-1 (GRIF-1) & Splice variants 38 Glut-1 & 4 145 Nucleophosmin 96* Peptidyl prolylisomerase (PPI) 96* Proteosome component C2 96* OGlcNAc transferase (OGT) 29 Q04323, UCH homolog 96* Sec23, human homolog (hhSec23) 96* Ran 96* Rho GDP-dissociation inhibitor 1 (Rh OGDI ) 96* Ubiquitin carboxy hydrolase (UCH) 121 *These identifications are still considered putative as supporting structural work has not been published. nism of protein-protein interaction . A group of pr oteins known as OGlcNAc interacting proteins (OIP) exists that medi ate the interactions be tween the tetratricopeptide repeats of enzyme and the substrates . Even though, OGT is found in all tissues examined, the levels of expression differ among tissues. In addition, the levels of expr ession are not correlated to the activity, implying that the enzyme is regulated post-tra nslationally [43, 46]. For example, OGT is modified by both tyrosine phosphorylation and OGlcNAcylation , although the role of these modifications on the localization and the activity of en zyme are unknown. At the substrate level, both the Km and Vmax for a variety of substrat es is altered by the UDPGlcNAc levels. The enzyme has apparent Km for UDP-GlcNAc ranges from 0.05 M-4.8 mM in cell extracts. The UDP-GlcNAc levels ar e in turn depends on extracellular signals, the state of nutrition, developmen t and differentiation [51, 54-59]. OGT remains active across the physiological range of UDP-GlcNAc in the absen ce of UDP, a potent inhibitor (Ki 200 nM) [45, 60].
17 The removal of OGlcNAc group is accomplished by a specific enzyme, -NAcetylglucosaminidase ( OGlcNAcase) with a neutral pH optimum that is predominantly localized to the cytosol. Th e enzyme is characterized from both rat spleen and human brain. The ubiquitous enzyme is abundantly found in the brain, placenta and pancreas [61, 62]. OGlcNAcase has molecular weight of 106 kDa, and exists as a heterodimer with a 54 kDa -subunit and 51 kDa -subunit. The enzyme is found in both cytoplasm and nucleus. Unlike, the more general specif icity of lysosomal hexoaminidases with acidic pH optima, the neutral enzyme is not i nhibited by GalNAc or its analogs, and shows no other glycosidase activity. In addition, the enzyme does not cross -react with the antibodies against the lysosomal hexoaminidase s [61,62]. However, similar to lysosomal hexoaminidases, OGlcNAcase has a Km = 1.1 mM for a general glycosidase substrate paranitrophenyl-GlcNAc . In contrast, streptozotoc in (STZ) a glucosamine nitrosourea specifically inhibits OGlcNAcase and not other hexoaminidases . Another strong inhibitor of OGlcNAcase is O(2-acetamido-2-deoxy-Dglucopyranosylidene) amino-N-phenylcarbamate (Ki =54 nM PUGNAc) [65, 66]. Interestingly, OGlcNAcase retains the enzymatic activity upon cleavage by caspase-3 rel easing the regulatory domain from the catalytic moiet y, indicating that removal of OGlcNAc may play a role in cell death process . Many sol uble nuclear and cytosolic proteins as shown in table 1.3.1 are the targets of OGT and OGlcNAcase. Many of the proteins characterized so far, such as Estrogen Receptor (ER ), tau, SV-40 large T antigen, c-Myc oncogene, eNOS, RNA polymerase II and -crystallin show that OGlcNAc and Ophosphate groups compete for the same site [67-72]. However, in some proteins, there is a s ynergistic interplay of OGlcNAc and Ophosphate , and in all likelihood an antagonist and/or even no connec-
18 Figure 1.3.2Schematic diagram of the he xosamine biosynthetic pathway and the dynamic processing of OGlcNAc modification by OGlcNAc transferase and OGlcNAcase. The diagram represents the substrat es, enzymes and inhibitors of the hexosamine biosynthetic pathway (H BP) and during the regulation of OGlcNAc modification. OH Glc Glc-6-PF-6-P 23 4 GlcN-6-P Protein-O-GlcNAc Protein 1. Glutamine:Fructose-6-phosphateamino transferase(GFAT) 2. Glucosaminephosphate acetyltransferase 3. Phosphoacetylglucosamine pyrophosphorylase 4. UDP-acetylglucosamine pyrophosphorylase 5. UDP-acetylglucosaminyltransferase (OGT) 6. -N-acetylglucosaminidase ( OGlcNAcase) a. 6-diazo-5-oxo-norleucine (DON) b. O -2-acetamido-2-deoxy-D-gluconosylidene (PUGNAc) c. Streptozotocin (STZ)O 6 5 UDP-GlcNAc b,c 1 GlnGlu OH + GlcNAcProtein a
19 tive effect may well be formed as more proteins are analyzed. The galactosyl capping studies using -D-1-4-galactosyltransferase in Xenopus maturing oocyte have demonstrated the deleterious e ffect of the blocking the addition and removal of OGlcNAc, thus suggesting this post-t ranslational modification may play a significant role in cellular regu lation [12, 74]. However, struct ural changes may also have played a role in these outcomes. In another study, the elevation of OGlcNAc using PUGNAc has shown no effect on the cell grow th rate . The contradicting results might be due to the rapid turnover of the PUGNAc and the replacement of it every 48 hours. As mentioned earlier, the OGT deleti on studies in ES cells, embryonic fibroblast or tissue using Cre-lox technology  is lethal, suggesting th at OGT is essential for life at single cell level [42, 76]. Since the subs trate specificity of OGT changes at different concentrations of UDP-GlcNAc, the OGlcNAc levels on key re gulatory proteins, including OGT can be modulated by altering the extracellular glucos e levels through the hexosamine biosynthetic pathway (as shown in the Figure 1.3.3) . The change in OGlcNAc levels to different signals based on its nutritiona l state modulating the overall behavior of the cell suggests that OGlcNAc is a nutritional sensor [41, 63, 82, 88, 89]. For instance, the glucose star vation and forskolin treatmen t led to decreased glycosylation and increased degradation of Sp1, transc ription factor and s ynthetic peptide through the ATPases in 19S regulatory subunits [ 90]. Apart from being a nutritional sensor, OGlcNAc is also a stress sensor . In many other parallel studies, an increase in the OGlcNAc levels due to increase in glucose flux into the cells is obser ved in response to the stress such as heat shock, UV, hypoxia, re ductive, oxidative and osmotic stress . Interestingly, the rapid induction of heat s hock proteins (HSP) such as HSP70 and HSP40
20 with the increase in OGlcNAc shows that OGlcNAc mediates stress tolerance . However, the studies on diabetes have asso ciated hyperglycemia with the increased cell death in several systems that might be due to the down-regulation of AKT activation . The paradox may be due to the differences in the sensitivities of tissues/ cells to insulin, the dependence on AKT signaling or the basal cell death rate to that of induced cell death rate . 1.4 Earlier findings of the studies on OGlcNAc modification in oocytes of Xenopus laevis Recent reports have demonstrated that changes of OGlcNAc levels in oocytes are related to the oocyte growth and maturation [11, 95], the exact function of the modification and the role of the modified proteins are still unclear. Slawson et. al demonstrated delayed progesterone stimulated maturation of the fully grown oocyte when incubated with compounds such as glucose, glucosam ine and PUGNAc before progesterone stimulation . Additionally, when the oocytes were inc ubated with the glutamine-fructose-6phosphate amino trasferase (GFAT) inhibito r, DON (6-diazonorbenzene) that reduces UDP-GlcNAc synthesis, nullified the glucose effect on the maturation. While the total cellular OGlcNAc content apparently does not ch ange during progesterone stimulation , another study has showed an a pproximate 4.5-fold increase in the OGlcNAc content, mainly on two cytoplasmic prot eins, one of 97 kDa, identified as -catenin and another unidentified 66 kDa protein . Micr oinjection of GlcNAc has delayed progesterone-induced maturation in Xenopus oocytes without any change in OGlcNAc content suggesting that the modification could regulat e protein-protein interactions required for the cell cycle kinetic . Li ke Sp1 transcription factor, -catenin is also stabilized by O-
21 GlcNAcylation [83, 13]. In addition, the microi njection of galactosyl transferase (GalT) into the progesterone-stimulated oocyte was sh own to be toxic . This GalT toxicity was reported due to the galactosyl capping of the OGlcNAc residues that appear to disrupt aster formation during the meiosis or causes any other cellula r effects. Thus, the OGlcNAc modification might be facilitating the proteinÂ–protein interactions necessary for the maturation process of the oocyte. Thus, s howing one or more modified proteins might be critical for oocyte maturation. In addition to the maturation of oocyte, th e development of oocytes has also been associated with changes in OGlcNAc levels . A gradual decrease in the OGlcNAc levels was observed as during the oocyte prog ression from the stage I to stage VI along with a concommitant increase in the activity of OGlcNAcase. Analysis of the oocyte proteins of all the stag es I-VI has clearly demonstrated the reduction of OGlcNAc levels in the high molecular weight proteins (>36 kDa) and herein, this thesis . This reducetion in turn correlates with the metabolic tr ansition of oocyte from glycolytic to gluconeogenic state . In a ddition, no incorporation of 3H-glucosamine into proteins in the stage VI suggested very low levels of new OGlcNAc modification in this stage . Interestingly, a similar phenomenon of change in OGlcNAc levels was observed during the transition of cell to malignancy, indica ting the fully grown oocyte (stage VI) may have more characteristics in common with a malignant cell where the regulation of OGlcNAc modification is disrupted .
22 Chapter 2 Materials and Methods 2.1 Materials: 2.1.1 Reagents: Reagents used in immun oprecipitation and polyacryl amide gel electrophoresis were obtained from Biorad (Richmond, CA), Fi sher (Atlanta, GA), Pierce (Rockford, IL) and Sigma (St. Louis, MO). Anti-mouse RL-2 antibody was purchased from affinity Bioreagents (Golden, CO), and CTD110.6 was a gene rous gift from Dr. Gerald Hart and laboratory at Johns Hopkins University, Ba ltimore MD. The Immobilized pH Gradient (IPG) strips and IPG buffer, both in the ra nge of pH 3-10 and pH 6-11 were obtained from Amershams Biosciences (Piscataway, NJ). Collagenase, Anti-mouse IgM conjugated agarose beads, and streptozotocin were purchased from Sigma (St. Lousie, MO). Secondary antibodies anti-mouse and anti-rabb it IgG were obtained from Biorad (Hercules, CA). The affinity column material, agarose wheat germ agglutinin beads were obtained from Vector Laborat ories (Burlingame, CA). Nano pure water is used in the preparation of the buffers. Spectra dialys is tubing of MW 10,000 cut-off and Amicon centricons of MW 3,000-30,000 cut-off was purch ased from Fisher Scientific. The protein standards used were Biorad High ra nge molecular weight and Invitrogen Benchmark prestained protein ladder. All the othe r reagents used were as per the ACS quality.
23 2.1.2 Equipment: Mini-gel cassette and Genie Electroblotter were obtained from Idea Scientific (Minneapolis, MN). Conductivity meter and pow er supply for the electrotransfer were obtained from Biorad, while UV/ visible spectrophotometer from Pharmacia Biotech. Amershams EttanTM IPGphorTM Isoelectric focusing system was borrowed from Biochemsitry Department, USF and 6.0 Dexon 3/8 Circle Reserve Cutting surgical needle along with absorbable suture or non-absorbabl e suture from Davis and Geck, Inc. (Pearl River, NY) and Ethicon (Somerville, NJ), were obtained. OptimaTM TL-100 Ultracentrifuge obtained from Beckman In struments, Inc., (Palo Alto, CA) 2.1.3 Animals: Female Xenopus laevis frogs were purchased from Nasco (Fort Atkinson, WI) and maintained at room temperature (20-22 oC) and fed with Frog brittle from Nasco two to three times a week 2.1.4 Buffers and Solutions: The oocyte medium called Oocyte Ringers solution 2 (OR-2) (Wallace et al ., 1973) was composed of 82.5 mM NaCl, 2.5 mM KCl, 1.0 mM MgCl2, 5 mM HEPES pH 7.8, 2 mM sodium pyruvate, 10,000 units pe nicillin, and 10 mg streptomycin. The oocyte homogenization buffer (Ten ) was comprised of 50 mM Tr isÂ–HCl (pH 7.4) containing 5 mM EDTA, the phosphatase inhi bitors, 100 mM NaF and 25 mM -glycerol phosphate; and in addition, the protease inhibitor 2 mM PMSF, and the OGlcNAcase inhibitor 5 mM streptozotocin are added before us e. Note: The stock solution of 200mM PMSF was prepared in isopropanol and stored.
24 For immunoprecipitation with RL-2 antibody the immunocomplexed beads were washed with Wash Buffer 1 containing 150 mM NaCl, 10 mM HEPES pH 7.4, 1% Triton X-100, 0.1% SDS and Wash Buffer 2 with the same composition as Wash Buffer 1 without salt. For immunoaffinity purification with CTD110.6 antibody required the washing buffer, radioimmunoprecipitation assay (RIP A) buffer containing TBS (136.9 mM NaCl, 2.7 mM KCl, and 24.8 mMTris-HCl, pH 7.6) along with detergents such as 1% IGEPAL, 0.5% deoxycholate and 0.1% SDS was used to wash the immunoaffinity column before elution with 1M GlcNAc in TBS, pH 7.6. In affinity chromatography the binding buffer used for equilibrating the agarose wheat germ agglutinin beads contains 10 mM HEPES, pH 7.8. The used beads were stored in the storage buffer containing 10 mM HEPES, pH 7.5, 0.15 M NaCl, 20 mM GlcNAc and 0.08% sodium azide. The proteins of interest were eluted off the column using a buffer containing 10 mM HEPES, pH 7.8 with 0.3 M NaCl and 0.5 M GlcNAc. The column can be regenerated with 0.1% acetic acid buffer, pH 3.0 with 1 M NaCl. In one-dimensional (1D) gel electrophoresis the samples were diluted with protein solubilizing mixture (PSM) in the ra tio 1:1. PSM is a 50 mM Tris-HCl buffered solution (pH 7.5) containing 2.5% (w/v) SDS, 25% (v/v) sucrose, 0.25 mg/ml pyronin Y, 25 mM Tris-HCl /2.5 mM EDTA and 1.5% -mercaptoethanol. The resolving gel required 41.5% stock acrylamide solution (5.6 M acrylamide and 97mM bis-acrylamide), resolving buffer (2 M Tris pH 8.9), 20% SDS 10% ammonium persulfate and TEMED (N, N, NÂ’, NÂ’-tetra-methyl-ethylenediamine). For the stacking gel, same materials except for 4% acrylamide and stacking buffer (0. 5 M Tris pH 6.7) was required. The SDS
25 electrophoresis buffer containing 25 mM Tr is-base pH 8.3, 195 mM glycine and 0.1% (w/v) SDS was used to run the SDSPAGE. In two-dimensional ( 2D) gel electrophoresis the samples were solubilized in the IEF sample buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 60 mM DTT, 0.2% (v/v) IPG buffer (pH 6-11 or pH 310), and 0.002%(w/v) bromophenol blue. The IPG strips were reswelled in rehydration so lution containing 8 M urea, 2% (w/v) CHAPS, 0.2% (w/v) DTT, 0.5% (v/v) IPG buffer (p H 6-11 or pH 3-10) and 0.02% bromophenol blue. The 2nd dimension equilibration buffer contai ned 2% (w/v) SDS, 50 mM Tris-HCl pH 8.8, 6 M urea, 30% (v/v) glycerol, 0.002% bromophenol blue and 100 mg of DTT or 250 mg iodoacetamide. The 0.5% agarose se aling solution containing 0.002% bromophenol blue was used to seal the IPG strip on SDS gel. All other solutions and buffers used were similar to that of 1D-gel electrophoresis. The molecular weight markers used for molecular weight comparison were used in either the unstained form or prestained ( dye modified) form. The prestaining (modification with dye) of these proteins alters th e molecular weight and relative migration (Mr) through the gel, the relative molecular wei ghts of both forms (normal/prestained) are presented. The protein standards included carbonic anhydrase (Mr 31 000/ 37 000), ovalbumin (Mr 45 00/50 000), serum albumin (Mr 66 200/ 75 000), phosphorylase b (Mr 97400/ 100 000), -galactosidase (Mr 116 250/ 150 000) and myosin (Mr 200000/ 250000) respectively for unstained or stained. The gel staining solution Coomassie blue staining solu tion contains 80 % stock solution and 20% methanol (stock solution cont ains 1 g of Brilliant blue G250, 11.6 ml of 85% H3PO4 acid and 100 g (NH4)2SO4 diluted to one liter).
26 For electroblotting cold (4oC) transfer buffer cont aining 50 mM Tris, 192 mM glycine (pH 8.3) and 20% methanol was used. (Note: The pH of the solution is not measured) For CTD110.6 immunoblotting the buffers used were TBS-HT (136.9 mM NaCl, 2.7 mM KCl, and 24.8 mMTris-HCl pH 8.0/ 0.3% Tween-20) and TBSD (136.9 mM NaCl, 2.7 mM KCl, and 24.8 mMTris-HCl pH 8.0/ 0.25% deoxycholate/ 1% Triton X 100/ 0.1% SDS). The CTD110.6 antibody, monoclona l mouse IgM was stored at 0.2ug/ul in TBST (136.9 mM NaCl, 2.7 mM KCl, and 24.8 mMTris-HCl pH 8.0, with 0.05% Tween20)/ 3% BSA in -70 C freezer until needed. A stock d ilution of antibody (1:500) was prepared in TBST/ 3% BSA/ 0.01% sodium azide stored at 4 C and used for two weeks to one month. The RL-2 immunoblot requires PBST (136.9 M NaCl, 2.7 mM KCl, 5 mM Na2HPO4, and 2 mM KH2PO4 pH 7.2/ 0.05% Tween-20) and hi gh salt PBST (HSPBST) (479 mM NaCl, 3 mM KCl, 5 mM Na2HPO4, 2 mM KH2PO4 pH 7.2, 0.05% Tween-20). The RL-2 antibody, monoclonal mouse IgG1 was aliquot ed in PBST (136.9 M NaCl, 2.7 mM KCl, 5 mM Na2HPO4, and 2 mM KH2PO4 pH 7.2, with 0.05% Tween-20) and 3% BSA and stored at -70 C. A stock antibody dilution of 1: 5 00 in PBST/ 3% BSA/ 0.01% sodium azide stored at 4 C lasts for a month. The membrane staining solutions are 1% India ink in TB ST (0.3% Tween) and 0.2% Ponceau S stain (stock solution contains 2% Ponceau S in 5% acetic acid) for nitrocellulose, and 0.1% (w/v) Coomassie blue R in 50% methanol and 10% acetic acid is used for PVDF membranes.
27 The gel staining solution Coomassie blue staining solu tion contains 80 % stock solution and 20% methanol (stock solution cont ains 1 g of Brilliant blue G250, 11.6 ml of 85% H3PO4 acid and 100 g (NH4)2SO4 diluted to one liter). 2.2 Methods 2.2.1 Oocyte harvesting and isolation: Mature Xenopus frogs were maintained in the tanks filled with dechlorinated tap water at room temperature. The animals were fed with Nasco vitamin fortified frog brittle two or three times per week. To obtain the ooc ytes, first a healthy frog without surgical scars or with clearly healed prior incisions was anesthe tized by immersing in NaHCO3 buffered solution of MS 222 (500 mg of MS222 and 10 mEq of NaHCO3 in one liter deionized water) in a tank for 10-20 minutes. Late r it was placed on its ba ck on a bed of ice, and the ovarian tissue was surgically rem oved through incision on the ventral surface. The muscle incision was sutured with absorbab le suture and the skin with non-absorbable suture. An injection of Xylazine hydrochlor ide (10 mg/kg) was given intracoelomically. The animal was transferred to an empty reco very tank and allowed to recover at room temperature. Once the frog flipped on its stomach, room temperature water was added until it covered the animal and monitored for 30 minutes. The frog was kept under observation for twoÂ–three days, and if apparently normal was transferred back to one of the common holding tanks until the ovarian sacs were regenerated . The excised ovarian tissue was cut into small sections (~ 1 cm2) and washed with OR-2 solution to remove all the debris. Then ovarian tissue in fresh OR-2 solution was incubated in collagenase (1 mg/ml) for appr oximately 4 hours to free the oocytes from collagen. Later the turbid supe rnatant of oocytes freed fr om ovarian tissue framework
28 was carefully decanted without disturbing the sedimented oocytes to remove the unreacted collagenase and fresh OR-2 containing Ca+2 was added. Being smaller in size and lighter than the other stages, the stage I and II oocytes do not sediment quickly. Therefore, these oocytes in the fresh OR-2 solution can be separated from th e rest by a series of swirling followed by a quick decantation of the supernatant containing mostly stage I and II oocytes. Oocytes in this supern atant were sorted into stage I and II in a petridish under a microscope. Typically 1000-1500 stage I oocy tes were isolated from a healthy frog. 2.2.2 Homogenization of the oocytes The sorted oocytes were transferred to a test tube (200 l) an d the liquid carefully removed with a pipet. Oocytes were then wash ed twice with two volum es of ice cold Ten that was removed by 20 l pipette, and then followed by the addition of fresh ice cold Ten approximately 1ul per stage I oocyte, and 10 l per stage VI oocyte was added. Oocytes were then homogenized on ice in Ten by rapidly drawing and expelling them from the 200 l pipette tip. For stage VI oocyt es an equivalent volume (1:1) of ice cold Freon (1, 1, 2-trichloro-trifluor o-ethane) was added to remove yolk protein and lipid , and the solution mixed thoroughly. The so lution is centrifuged at 12,000 x g at 4 C for ten minutes to remove the yolk proteins and membrane materials. The aqueous supernatant was carefully collected w ithout disturbing the pellet at the Freon/buffer interface and stored at -20 C. Previous work has demonstrated that the Freon does not change the protein profile in stage VI oocyte . Sa me procedure was used to homogenize the stage IV and V oocytes using 5 l of Ten per oocyte to lyse the oocytes, and followed by Freon extraction to remove the yolk. While, the stage II and III were homogenized in the same manner as the stage I, excep t the stage III requires 3 l of Ten per oocyte.
29 2.2.3 Protein Estimation: The protein concentration of the homogena te was estimated using Bradford micro protein assay (Biorad). Pure bovine IgG (B iocompare, CA) (stored as 1 mg/ml in H2O at -20 C) was used to obtain the standard curve using protein concentrations from 2 ug to 20 ug. First, six dilutions of protein standard were prepared by thorough mixing with 200 ul of reagent and nanopure water. After incuba tion at room temperature for 5 minutes, the absorbance was read at 595 nm on a spectr ophotometer. In the same way, the sample solution was prepared in trip licate and absorbances were re corded at 595 nm. The protein amount in the samples was calcula ted from the standard curve. 2.2.4 Immunoprecipitation with RL-2 antibody: The OGlcNAc modified proteins were immunoprecipitated using mouse monoclonal RL-2 antibody, monoclonal mouse IgG1 to form an immunocomplex with the Anti-rabbit IgG preabsorbed Protein A trisacryl beads . All the steps of the procedure were performed at 4 C. Each experiment was performed with three samples. The immunoprecipitation was carried ou t using the homogenate of the stage I and a few stage II (<10%) oocytes, with the final concentr ation of protein 12 g/ l using Ten The homogenate was incubated with RL -2 antibody (5 g for every 1mg of protein) and protein A trisacryl beads (50 l) that were preabsorbed by overnight incubation with Rabbit Anti-mouse IgG (50 g) and left overnight on the rotating mixer. Later the immunocomplex with the beads was ha rvested by centrifugation at 10,000 x g for 10 minutes to collect the pellet. This procedure was repeated after each washing step using Wash Buffer 1 and Wash Buffer 2. Then the pellet was solubilized with PSM as mention-
30 ed in the above procedure. All the fractions the supernatant, washes and pellet were analyzed by silver staining the gel and CTD110.6 antibody immunoblotting. 2.2.5 Immunoaffinity purification using CTD110.6 antibody This method employed CTD110.6 antibody, mouse monoclonal IgM that specifically binds to the modified proteins to the Anti-mouse IgM conjugated agarose column . All the steps of the procedure were performed at 4 C. Each experiment was performed with three separate samples. The immunoprecipitation was carried out using the homogenate of the stage I and a few stage II (<10%) oocytes, with th e final concentration of protein 1-2 g/l using Ten First to preclear so as to reduce nonspecific binding, the homogenate was incubated with the Anti-mouse IgM conjugated agarose beads for one hour on the rotating mixer, and centrifuged at 10,000 x g for 10 minutes to collect the supernatant. The precleared homogenate was then incubated with CTD110.6 antibody (5 g for every 1mg of protein) and Anti-mouse IgM conjugated Ag arose beads (50 l) for 3 hours on the rotating mixer. Next the slurry was transferred in to the column (1.5 cm x 6.5 cm) and the flow through was collected. The column was then wa shed twice with five volumes of RIPA buffer and once with two volumes of TBS. After washing the column, the proteins were eluted three times, each with two volume of 1 M GlcNAc in TBS after 20 minutes incubation. The eluted fractions were precipitat ed by overnight incubation with ten volumes of cold methanol at -20 C. Samples were centrifuged at 10,000 x g for 15 minutes and the pellets were solubilized in PSM by vor texing for 1-2 minutes and boiling for two
31 minutes. All fractions, the flow through, the wa shes and the elutions were analyzed by silver staining and RL-2 immunoblotting. 2.2.6 Affinity chromatography: The affinity chromatography was based on agarose wheat germ agglutinin column that specifically binds to all the terminal N-acetyl glucosamine residues on proteins , and thus enriching the samp le with proteins of interest Initially, the affinity purification was performed using binding buffe r containing 10 mM HEPES, pH 7.8 and 0.15 mM NaCl (as per the maufacturerÂ’s instructions ). Later, the low salt conditions using the same binding buffer without NaCl were employe d for affinity purification of the sample, in order to enhance binding to the wheat germ agglutinin . Homogenate (typically 1 mg) with a final concentration of approximate ly 1-2 g/l was incubated with 100 l of agarose wheat germ agglutinin (WGA) beads fo r two hours on the rotating mixer. Before incubation with the protein mixture, the bead s were washed with binding buffer to remove salt and N-acetylglucosamine (GlcNAc) pr esent in the storage medium and were resuspended in the binding buffer. The incuba ted mixture was poured into a column (1.5 cm x 6.5 cm). Next the affinity column was wa shed five times with five column volumes of binding buffer. Elutions were performed with two column volumes of the elution buffer containing 0.5 M GlcNAc. Then, the eluted beads were regenerated using regeneration buffer at pH 3.0. Once regenerated, the be ads were stored in the storage buffer. The eluted fractions were concentrated and pooled using ultra-centrifugation device with10,000 MW cut-off. The concentrated sa mples were solubilized with 2x PSM.
32 2.2.7 Differential sedimentation for enrichment of OGlcNAc modified proteins: Stage I oocytes were first homogenized in ten as mentioned in the previous sections, and centrifuged at 1,000 x g for 10 mi nutes to remove cell debris and unlysed cells. The collected supernatant was then cen trifuged at 10,000 x g for 10 minutes and the pellet was stored at -70 oC for further analysis. Later, the supernatant at 10,000 x g was further centrifuged at 100,000 x g for one hour and the fractions were stored at -70 oC. All the above fractions were analyz ed by 1D/ 2D gel electrophoresis. 2.2.8 One-dimensional gel electrophoresis: Samples were further separated and an alyzed on either 8-10% SDS-polyacrylamide minigel (9.5 cm x 6.5 cm) (SDS-PAGE) th at was made following the protocol of Laemmili . The resolving gel was made fr om appropriate dilutions of 41.5% stock acrylamide solution (5.6 M acrylamide and 97m M Bis-acrylamide) with resolving buffer (2 M Tris pH 8.9), nanopure water, 20% SD S 10% ammonium persulfate and TEMED (N, N, NÂ’, NÂ’-tetra-methyl-ethylenediamine ). The mixture was degassed for a minute before adding ammonium persulfate and TEM ED. The stacking gel was made in a same manner except the final percent acrylamide wa s 4%, and stacking buffer (0.5 M Tris pH 6.7) was used instead of resolving buffer. Gels were run at room temperature at 15 mamps for approximately 1-2 hours, usually 40 -50 minutes after the dye has completely left the gel.
33 2.2.9 Two-Dimensional Gel Electrophoresis: For higher resolution, the samples were subjected to two-dimensional (2D) Gel Electrophoresis. The method has two discrete step s. In the first step (first-dimension), the proteins were separated base d on their isoelectric points us ing 7cm Immobilized pH gradient (IPG) strips of pH 6-11 or pH 3-10 NL from amershams. The proteins focused at their characteristic pI on the IPG strip were further analyzed based on their molecular weight in the second step (sec ond-dimension) using SDS-PAGE. IEF was performed in four main steps, the sample preparation, the rehydration of the IPG strips, the sample loading and the isoe lectric focusing. First, the sample was prepared by dialyzing against low concentrati on Tris buffer, pH 7.4 (5 mM Tris, 0.1% IGEPAL and 1 mM DTT) using dialysis tubing with a 10,000 M.W. cut off to reduce the concentration of salts including Tris that interferes with IEF. The dialyzed sample was solubilized with IEF buffer in a ratio of 1: 6. The pH range of IEF buffers depends on the pH range of the used strip. Note: If the sa mples are too diluted (<3 g/l) following dialysis, should be concentrated by ultra filtra tion or acetone precipitation . The second step of IEF involved rehydration of IPG stri p as per supplierÂ’s instructions. The dry IPG strip was soaked overni ght with the gel-side down in re hydration solution containing the appropriate IPG buffer with the overlay of DryStrip Cover Fluid. A 7cm IPG strip requires 125 l of rehydration solution . Sample loading constitutes the third step in isoelectric focusing (IEF). For the IEF, the IPG strip was first positioned on the Ettan IPGphor Cup Loading Strip Holder as per the manufacturerÂ’s instructions. The sample was applied to the rehydrated strip by the
34 sample cup application method. For a basic IP G strip, the sample was applied at the anodic end and at the center for a whole pH range IPG strip to avoid significant extremes of pH that can lead to loss of O-GlcNAc modi fication and/or protein precipitation. The final step comprises of isoelectric focusing (IEF) of the samples by using Ettan IPGphor Isoelectric Focusing System (Amershams). Accord ing to the instructions given in the manual, Â‘2D-Gel Electrophoresis using immobilized pH gradients, Principles and MethodsÂ’, the IEF was performed on the rehydrated stri p at constant ampera ge of 50 A at 20oC, and ramping the voltage initially in a grad ient mode to 500 V for 1 m and 4000 V for 1h 30 m, and later in a step and hold mode to 5000 V for 45 m. After the IEF, the IPG strip was transf erred to 10ml screw capped tube and was either stored at -70 oC or analyzed by SDSPAGE in th e second step. The second dimension consists of a 1 mm thick, 10% laem mili SDSPAGE polymerized in between two glass plates with one plate pr otruding out, to position the IPG strip; and 4% stacking gel with two small wells and a larger well. Prior to positioning the strip on gel, the strip was incubated in equilibration buffer for first 15 minutes with 100 mg of DTT. This was followed by 15 minutes incubation in equilibra tion buffer with 250 mg of Iodoacetamide. Subsequently, the equilibrated strip was briefly immersed in the SDS electrophoresis buffer to lubricate, and later the plastic ends of the strip were caref ully trimmed to adjust its length according to the size of 2nd dimension. The lubricated strip was then loaded on to the prestacked second dimension gel with the plastic side agai nst one of the glass plates and sealed into place using the agarose sealing soluti on described before. With the protein standards and sample loaded at le ft of the gel, the gel electrophoresis was
35 performed at amperage of 10 mA for 15 minut es and continued for 40 minutes after the dye runs off. 2.2.10 Gel Staining methods The staining was performed usually to obtai n overall protein prof ile of the sample resolved on SDS PAGE. Generally, the gel was first incubated in the fixing solution to enhance the staining. The met hod of staining completely de pends on the requirement of the experiment. 126.96.36.199 Coomassie Blue staining The gel was fixed in a solution containi ng 20% methanol, 10% Acetic acid with constant shaking for a total of 20 minutes changing solutions once after 10 minutes. The gel was rehydrated by soaking in nanopure wa ter for 15 minutes with constant shaking. The rehydrated gel was then incubated overn ight in the G-250 coomassie blue staining with 20% methanol with constant shaking. Th e stained gel was destained for two hours in deionized water to get rid of the background staining . 188.8.131.52 Silver Staining The proteins separated by SDSPAGE were stained by using Biorad Silver stain kit (as per the instructions given by the manufactur er) The gel, after el ectrophoresis was incubated in fixing solution containing 40% methanol and 10% acetic acid for 30 minutes minimum. The fixed gel was immersed in oxidizing solution (K2Cr207) for five minutes. The gel was then washed with nanopure wate r for a maximum of 15 minutes, changing 67 times especially in the first 5 minutes. Af ter washing the excess oxidizer, the gel was
36 immediately transferred to the Silver soluti on containing 8% silver reagent and incubated for 20 minutes. Next, the gel was quickly ri nsed in nanopure water for a maximum of 30 seconds and immediately the developer (6.4 g/ 200 ml) was added to the gel. The developing solution is changed once brown or sm oky precipitate appear s. This step was repeated until a stain of desira ble intensity was obtained. Then use 5% acetic acid to stop the staining for 15 minutes. All st eps were performed on a shaker. 2.2.11 Gel Drying Drying of the gel was done by various methods. In one of the method, the gel was dried using Idea scientific Gel drying frame. (as per the instructi ons of manufacturer). The gel was first incubated in 10% ethanol and 5% glycerol for 30 minutes. The gel was then sandwiched between the wet gel drying me mbranes, and left encased in the gel drying plastic frame to dry. In another method, the gel was thoroughly rinsed with nanopure water and placed on a wet filter paper covere d with the wet cellophane paper. The gel was left in the heated gel drye r (Biorad) for one hour to dry. 2.2.12 Immunoblotting The analysis of the samples on the SD SPAGE was followed by transfer of the proteins to nitrocellulose or PVDF membranes.  Both the hydrophobic membranes were first equilibrated in transfer buffer for 15 minutes. The PVDF membrane being highly hydrophobic was initially wetted by meth anol (5 seconds) and followed immersions in nanopure water (5 mi nutes) with constant shaking. The Genie transfer apparatus (Idea Scientific) was used to transfer the prot eins for 2-3 hours at a constant voltage of 12 volts at 4oC in the cold (4oC) transfer buffer.
37 184.108.40.206 CTD110.6 immunoblotting The blot after the transfer was immediately incubate d in the blocking solution, TBS-HT for 2-3 hours at room temperature w ith constant shaking. Typically all incubations were carried out in car efully cleaned parafilm boats containing 20-50 ml solution. Next the blot was transferred to the 1o antibody (CTD110.6 antibody) in TBS-HT at a dilution of 1:5,000 and incubated overnight at 4 oC with constant shak ing. The incubated blot was then washed 2 x 10 minutes with TBS-D and later 3 x 10 minutes each with TBS-HT. Next the blot was incubated in the 2o antibody (Goat anti-mouse IgM) at a dilution 1: 15,000 in TBS-HT for 1 hour at room temperature with constant shaking. Followed by the washing step as mentioned above a nd then the dried blot was treated with the Super Signal chemiluminescent substrate (P ierce) for 5 minutes again following manufacturer directions. Immediat ely the blot was dried and tr ansferred to the cassette and exposed to film for 30 seconds or longer. To demonstrate the specifi city of the CTD110.6 antibody, a duplicate blot was produced using the same protocol as above, except the primary antibody solution contained 15 mM NAcetylglucosamine as a competing sugar. 220.127.116.11 RL-2 immunoblotting Nitrocellulose immunob lots using RL-2 antibody were allowed to dry after transfer to improve recovery of antigenic sites. Th e dried blot was first soaked in the PBS at 70 oC in water bath for one hour with constant shaking. The wet blot was then blocked using high salt PBST (HSPBST) with 3% BSA at room temperature for two hours with the constant shaking. Typically all incubations were carried ou t in carefully cleaned parafilm boats containing 30-50 ml solution. Followi ng blocking the blot was transferred into
38 a solution containing the primary antibody, RL-2 in HSPSBT with 3% BSA at a dilution of 1: 5,000 and incubated with modest shaki ng for two hours. The incubated blot was then washed 5 x 10 minutes with HSPBST w ith 0.01% BSA. The washed blot was transferred to the secondary antibody, goat anti-mous e IgG in PBST with 3% BSA at a dilution of 1: 20,000 at room temperature for one hour. The blot was then washed 8 x 10 minutes with PBST and the dried membrane was treated with Super signal chemiluminescence for 5 minutes. The blot was quickly dr ied and placed in the cassette and exposed to the film for half a minute and longer . 2.2.13 Wheat germ agglutinin affinity blotting The proteins resolved by the SDSPAGE were transferred to PVDF (Polyvinyl difluoride) membrane. The bl ot was blocked in a TBST (0.05% Tween 20) solution containing 5% BSA for two hours at room te mperature with constant shaking. Once again, all incubations were carried out in ca refully cleaned parafilm boats containing 3050 ml solution. After blocking, the blot was rinsed with TBST, and incubated in HSTBST (1 M NaCl) with the WGA-HRP at 1: 10, 000 dilution  for 2 h at room temperature. The blot was then washed 3 x 10 mi nutes with HS-TBST and then 5 x 10 minutes TBST. The washed blot was developed at r oom temperature using freshly prepared 3mM DAB (diaminobenzidine) and 3% peroxide in 50 mM Tris-HCl (pH 7.6). Typically developing times were from 10 mi nutes to 20 minutes. The developed membrane was then washed with PBS and dried.
39 2.2.14 Membrane staining methods Once again, the staining method was used to visualize the overa ll protein profile of the sample transferred on to the membra ne, and the method of staining was chosen based on the requirement of experiment. 18.104.22.168 India ink staining: The nitrocellulose/PVDF membrane was fi rst soaked in PBST with 0.05% tween 20 for 10 minutes. If the membrane is nitro cellulose, it was then treated with 1% KOH solution for 5 minutes to enhance India ink staining. The blot was incubated in PBS for 30 minutes with one change of solution after 15 minutes. Then the blot was incubated for one and half an hour at 37 oC and one hour at room temperature with PBST, 0.3% tween 20 with changes of the solution every 30 mi nutes. On completion of washing, the blot was incubated overnight in 1% India ink staini ng solution. The blot that was washed with PBST twice for a few seconds gives a permanently stained membrane . Note: The membrane was first stripped with a glycine so lution if it had been previously immunoblotted. This staining method is more sensit ive than other methods with high level of detection (100 ng). 22.214.171.124 Ponceau S staining Ponceau S stain can be used for nitrocellulose membranes with a detection limit of 1ug protein. First, the membranes were in cubated in 2% Ponceau S for 20-30 minutes until the stained bands appear. Then, the st ained membrane was destained in nanopure water to remove the background. This stain is a reversible.
40 126.96.36.199 Coomassie staining This method is used for staining PVDF membranes with detection limit of 1.5 g of protein. The membrane is incubated in 0.1% Coamassie stain for 5-10 minutes. Once the stained bands appear, it is destained w ith a 50% methanol/10% acetic acid (v/v) solution to remove the background. 2.2.15 Membrane stripping methods The immunoblot was incubated in 20 0mM glycine, pH 3.0 for 20 minutes at room temperature with constant shaking. The blot was then washed 3 x 10 minutes with PBST . For affinity bolt, -mercaptoethanol method was used. First the membrane was incubated in the stripping buffer cont aining 62.5 mM Tris pH 6.8, 2% (w/v) SDS, and 100 mM -mercaptoethanol for 30 minutes at 60 oC. Next, the stripped blot was washed 3 x 10 minutes in TBST 2.2.16 Identification of the protein bands of interest Once 2D-gels of oocyte (stage I) proteins were sufficiently stained with coomassie so that identification was possible, the protein bands of intere st immediately were excised for protein sequencing. Being sure to wear gloves and taki ng care not to introduce any contaminants, protein material ( ie. ke ratin). In order to id entify these bands, the 2D-pattern of proteins in coomassie st ain was compared to the 2D-pattern of O -GlcNAc modified proteins in the CTD110.6 immunoblot The bands that matched the ones in the immunoblot by the distances traveled in both th e dimensions were selected to be sequenced. The selected protein bands were cut as close to the stained portions to get rid of
41 extra gel using a razor. Each of these gel pi eces containing the protein was then placed separately into a 1.5 ml eppendroff tube that was previously washed with aceton itrile to remove dust and keratin, and stored at -20oC. 2.2.17 Mass Spectrometric peptide sequencin g and identification of proteins The protein bands that were identified as OGlcNAc modified by using the CTD110.6 immunoblot All the protein bands of in terest were excised from the coomassie stained gel and sent to John Hopkins Th e peptide sequencing and identification was performed by the Dr. Stephen A Whelan at John Hopkins Univers ity using the MS/MS peptide sequencing coupled with database search as descri bed in the following three steps. 1. In gel digest: The excised protein bands of the G-250 commassie stained 2D-gel were first chopped into small pieces. Then the gel pie ces were destained by washing three times with 100 mM NH4HCO3 for 10 minutes each and dehydrat ing with aceton itrile for another 10 minutes. The dehydrated gel pieces were vacuum dried and rehydrated in 20 l of 10 ng/l trypsin (Promega, mass spectrometry grade) in 50 mM NH4HCO3 on ice for 45 minutes. Once the gel pieces were hydrated, excess trypsin was removed and gel pieces covered with 50 mM NH4HCO3, pH 8.0 were left overnight at 37 oC for digestion. Once digested, the peptides were extracted with 50 l of 50 mM NH4HCO3 and then, followed by three washes with 50 l of 5% formic acid/ 50% acetonitrile solution for 20 minutes each. The extractions were pool ed and vacuum dried. The ex tracted peptides were suspended in 0.1% Trifluoro acetic acid (TFA) and passed through Vydac C18 silica Micro-
42 spin columns (The Nest Group Inc.) and el uted with 75% aceton itrile and 0.09% TFA. The purified peptide mixture was once again vacuum dried. 2. LC-MS/MS Analysis: The vacuum dried and purified peptides were resuspended in 1% acetic acid and loaded on a 10 cm x 0.075 mm column packed with 5 m diameter C18 beads using N2 pressure. The column was then washed with 1% acetic acid and then the peptides were separated by a 75 minute gradient of increasi ng methanol at a flow rate of 190 nl/minute directing effluent into the source (Finnigan LCQ). The LCQ was operated in an automatic mode collecting a MS scan (2 x 500 ms) of the peptide mixture, followed by two MS/MS scans (3 x 750 ms) of the two high intensity peptides with a dynamic exclusion set at 2 with a mass gate of 2.0 Da. 3. Mass data analysis: In order to identify the proteins, the MS /MS data was interpreted using Bioworks software. The MS/MS spectra of the peptides were searched against several protein database including Xenopus laevis database (downloaded from NCl, National Institutes of Health at Frederick, MD) using the bioworks software. The MS/MS protein or peptides spectra were identified based on Â“Best hitsÂ” in the Xenopus laevis database search. The Â“Best hitsÂ” were those showing high degree of confidence that is the Xcorr > 2.5 and the dX-corr for the second entry > 0.1 . At the same time, the data was also manually inspected for accuracy.
43 Chapter 3 Results and Discussion 3.1 Confirmation of the presence of high OGlcNAc modified proteins in the stage I oocytes compared to the stage VI oocytes per unit mass: Slawson et. al.  have shown the presence of high levels of OGlcNAc in stage I oocytes, and a dramatic reduction in the le vels during stage progression (I-VI) of oogenesis. As a first step in el ucidating the po ssible role of OGlcNAc modification in developing oocytes, the initial efforts were focu sed on isolation and identification of these modified proteins. Prior to beginning the pur ification process, a series of experiments were performed to confirm and reexamine the m odified proteins of interest, and to examine the specificity of the proteins to CTD110.6 antibody (Fig ure 3.1.1 & 3.1.2 respectively). Every experiment was repeated three sepa rate samples to confirm the results. Several immunostained bands above 30 kDa we re reduced in the intensity while progressing from stage I to VI. Figure 3.1.1 shows that the protein bands # 1-14 at approximately 29, 48, 52, 63, 66, 70, 76, 84, 90, 101, 106, 116, 128, and140 kDa respectively were of initial interest. Competition for antigen binding with 15mM/30mM Nacetylglucosamine for CTD110.6 antibody demonstr ated the specificity of all the above mentioned protein bands. (Figure 3.1.2) For co mparison and as a control, the cytosolic fraction of a glioma cell line (contributed by Aaron Mathews) that is rich in the OGlcNAc modified proteins also clearly demonstrated the sp ecificity, but interestingly
44 Figure 3.1.1CTD110.6 immunoblot of one-di mensional gel separation of proteins from oocytes at stages I, II, III and VI. Approximately 20 g of proteins each of oocytes at stages I, II, III and VI were individually separated on the 8% gel and immunoblotted with CTD110.6 antibody as described in Methods. The figure represents an autoradiogram of the blot showing the pattern of OGlcNAc modified proteins in oocytes at different stages and the decreasing trend on OGlcNAc levels during stage progression of oogenesis. The arrows on the left-hand side sh ow the protein bands of interest # 1-14 at approximately 29, 48, 52, 63, 66, 70, 76, 84, 90, 101, 106, 116, 128 and140 kDa respectively. 150 100 75 50 37 25 kDa 1 2 3 4 5stage I II III VI 6 7, 8, 9 10,11 12 1314
45 Homogenate (stage I) Homogenate (stage I) Homogenate (stage I)No GlcNAc 15mM GlcNAc 30mM GlcNAc 200 150 50 37 25 100 75Cytosol (Glioma cells 3h) Cytosol (Glioma cells 3h) Cytosol (Glioma cells 3h) Cytosol (Glioma cells 9h) Cytosol (Glioma cells 9h) Cytosol (Glioma cells 9h)Figure 3.1.2Competition with 15mM N-Acetyl glucosamine to show specificity of OGlcNAc modified to CTD110.6 antibody. Approximately 20 g each of proteins from the cytosolic fractions from glioma cell line treated with 8 mM GlcNAc for 3 hours and 9 hours and the oocytes at stage I were individually separa ted, and immunoblotted with CTD110.6 antibody in absence and presence of 15mM/ 30mM GlcNAc. The figures represent the autoradiograms of the blots (a) without Glc NAc, (b) with 15 mM GlcNAc, and (c) with 30 mM GlcNAc while incuba ting the membrane with CTD110.6 antibody. The complete or partial disappearance of the bands in (b) and (c) indicates the specificity of the O-GlcNAc modified pr otein bands to CTD110.6 antibody. (a) (b) (c)
46 Figure 3.1.3RL-2 immunoblot of one-dimens ional gel electrophoretic separation of proteins from oocytes at stage I. Approximately 20 g of pr otein was separated on the 10% gel and immunoblotted with RL-2 as descri bed in Methods. The figure represents an autoradiogram of the blot showing a pattern of OGlcNAc modified proteins in stage I oocytes similar to that shown by CTD110.6 anti body. The difference is in the intensity of specific bands especially the bands around 50 kDa. The positions of molecular weight marker are shown with arrows on left. Stage I 25 37 50 75 100 150 200 kDa MW
47 shows quite a different pattern of modified pr oteins. A similar pattern of oocyte modified proteins was observed using RL-2, another an tibody recognizing with slightly different affinity but overlapping speci ficity to CTD110.6 antibody. Since stage I oocytes contained the largest amounts of these proteins per uni t mass, these oocytes were used to isolate the O -GlcNAc modified proteins of interest A few stage II oocytes (15% by number) that show a highly similar pattern of the m odification were included to increase the amount of starting material so as to improve yi elds. Even though, the stage I oocytes are relatively abundant in the OGlcNAc modified proteins, the to tal amount of these pro-teins in an oocyte is still small. Therefore, the stra tegy was to use a minimum number of purification steps to enhance overall recovery of proteins. First step of the scheme was to examin e the two-dimensional (2D) gel electrophoresis ability to directly se parate the proteins of intere st in quantities sufficient for sequencing. In addition, the level of detection of coomassie that used to stain the protein bands to be sequenced is low (1 ug). Dependi ng on the success of this approach, the next step was to partially purify or enrich using various affinity technologies coupled with 2Dgel electrophoresis. The enrichment techniqu es attempted were immunoaffinity methods using the OGlcNAc specific antibodies such as CTD110.6 and RL-2, affinity chromatography using the lectin, wheat germ agglutinin that specifically binds to the terminal Nacetylglucosamine residues; and differential sedimentation based on association of this protein with complex high molecula r weight aggregates in the cell. 3.2 Two-dimensional gel analysis of the stage I and VI oocytes: To examine and compare the 2D-pattern of the total proteins in stage I and VI oocytes, the whole cell homogenates were i ndividually analyzed by 2D-gel electropho-
48 resis and immunoblotted with CTD110.6 antibody. Both samples were separately resolved by using two IPG strips with different pH ranges while performing IEF. In order to view all the oocyte modified proteins an IP G strip of full pH range 3-10 (Non-linear) was used. The non-linear range was initially selected since it gives a bett er resolution of the proteins within the range of pH 5-8, the ra nge where generally the majority of cellular proteins are focused. Surprisingly, the majority of the modified proteins seen on onedimensional SDS PAGE were not present on the final blot. The 2D analysis of stage I at this pH range 3-10 showed only three faint m odified protein bands # 1-3 at approximately 29, 65 and 69 kDa respectively across an approximate pH range 5-8 on probing with CTD110.6 antibody. (Figure 3.2.1 a) However, the India ink total protein stain of same blot stripped using the glycine method show ed the presence of several other proteins bands on the membrane. (Figure 3.2.1 b) Sim ilar results were obtained with three separate samples. This suggests that the absence of the other bands of interests might be due to the unlikely hydrolysis and loss of the m odification at the mode rately low pH range. Alternately, and more likely the proteins might have focused off strip due to the steep rise in the pH near the end of non-lin ear pH strip, and as we all se e later the very basic nature of the majority of proteins of interest. In addition to above reasons, the proteins get focused slightly at different regions due to the ch anges in the pH of the IPG buffer used during the IEF that might also explain the absence of the proteins that focus in pH range 8-10 in the later 2D-gel electrophoresis . In the same manner, the proteins of st age VI oocytes were analyzed by 2D-gel electrophoresis and compared to the 2D-patterns of total proteins and OGlcNAc modified proteins of the stage I oocytes. Simila r results were obtained with three separate
49 Figure 3.2.1Two-dimensional gel electrophoresi s of oocyte proteins (stage I) using isoelectric focusing with pH range 3-10 in the horizontal dimension and SDS-PAGE (10%) in the vertical dimension. Approximately 100 g of prot ein from oocytes at stage I was separated and immunoblotted with CTD1 10.6 and later the blot was glycine stripped and stained with India ink as described in Methods. Figures (a) Autoradiogram showing three faint bands at 29, 65 and 69 kDa re spectively, and (b) I ndia ink stain showing the overall 2D-pattern of the proteins of oocyte st age I. On the left hand side of the gel, 20 ug of the stage I was separated on one-dimension acts as reference. (a) (b) 45 66.2 97.4116.25200 Homogenate Stage I oocytepH 3 10 kDa2 1 3 Homogenate Stage I oocytepH 3 10 45 66.2 97.4 116.25 200 kDa
50 Figure 3.2.2Two dimensional gel electrophore sis of oocyte proteins (stage VI) by isoelectric focusing with pH range 3-10 in the horizontal dimension and SDS-PAGE (10%) in the vertical dimension. Approximately 100 g of prot ein from oocytes at stage VI was separated and immunoblotted with CTD110.6 and later the blot was glycine stripped and stained with I ndia ink as described in Met hods. Figures (a) Autoradiogram showing four bands # 1-4 at approx. 32, 35, 45 and 66 kDa respectively, and (b) India ink stain showing the overall 2D-pattern of the proteins of oocyte stage VI. On the left hand side of the gel, 20 ug of the stage VI was separated on one-dimension acts as reference. (a) (b) Homogenate Stage VI oocytepH 3 10 45 66.2 97.4 116.25 200 kDa 3 1 4 2 116.25 Homogenate Stage VI oocytepH 3 10 45 66.2 97.4 200 kDa
51 samples. Once again, the CTD110.6 immunoblot of stage VI oocyte proteins on the 2Dgel with pH range of 3-10 showed four small bands at approximately 32, 35, 45 and 66 kDa across an approximate pH range 5-8. (Figure 3.2.2 a) While the India ink stain of the stripped membrane gave the overall 2D-picture of the stage VI ooc yte proteins. (Figure 3.2.2 b) In comparison to 2D-pattern of stage I oocyte proteins, the 2D-pattern of stage VI was once again quite differe nt as clearly shown in Indi a ink stain (Fig 3.2.2 b). Particularly, the protein bands of stage I oocytes th at are focused at the extreme right of the membrane (around pH 10) were completely absent in stage VI oocytes. Careful examination of the India ink stains of the gl ycine stripped blots of stage I and stage VI oocytes have showed a large fraction of the stage I oocyte proteins, unlike that of stage VI were focused mainly at cathodic end of the IPG strip indicating that majority of stage I oocyte proteins are basic in nature. Assuming that most of the OGlcNAc modified proteins of interest in stage I were basic in nature and thus, suggesting that the proteins were focused off the IPG strip, the more basic range of pH 6-11 was selected for IEF. The stage I analyzed within this pH range showed the majority of the modified proteins resolved into 12 discrete protein bands within the pH range of 8-10. (Figur e 3.2.3 a) These protein bands were numbered as 1-2, 3a, 3b, 7, 8a, 8b, 8c, 9a, 9b, and 10 as in dicated in figure were approximately at 28, 35, 37, 49, 55, 58, 62, 67, 91 and 98 kDa respectively. Among these bands, the bands # 1-3 are clustered together (a s seen in Figure 3.2.3 b) origin ated from the big band # 1 at 29 kDa in 1D-gel, and the bands # 4-10 corr esponds to bands # 2-6 and 9-11 respectively in the 1D-gel. However, the high mol ecular weight bands (> 100 kDa) do not appear due to proteolytic degrada tion or precipitation of these proteins during IEF. This is
52 Figure 3.2.3Two-dimensional gel electrophore sis of oocyte protei ns (stage I) by isoelectric focusing with pH range 6-11 in the horizontal dimension and SDS-PAGE (10%) in the vertical dimension. Approximately 100 g of protein from oocytes at stage I was separated and immunoblotted with CTD110.6 and later the blot was glycine stripped and stained with India i nk as described in Methods. Figures (a) Autoradiogram showed the modified proteins mainly focuse d at approx. pH 8-10, and 12 distinct bands # 1-2, 3a, 3b, 7, 8a, 8b, 8c, 9a, 9b and 10 were identified at 28, 35, 37, 49, 55, 58, 62, 67, 91 and 98 kDa respectively, and (b) India ink stain showing the overall 2D-pattern of the proteins from stage I oocyte. On the left-hand side of gel, 20 g of protein of stage I was separated on one-dimension acts as reference. (a) (b) pH 6 11 3b 2 1 4 6 5 8c 8b 7 9b 9a 10 45 66.2 97.4 116.25 kDaHomogenate Stage I oocytes8a 3a Homogenate Stage I oocytepH 6 11 45 66.2 116.25 97.4 kDa
53 Figure 3.2.4Two-dimensional gel electrophore sis of oocyte proteins (stage VI) by isoelectric focusing with pH range 6-11 in the horizontal dimension and SDS-PAGE (10%) in the vertical dimension. Approximately 100 g of protein from oocytes at stage VI was separated on 10% two-di mensional gel and analyzed by CTD110.6 immunoblotting and later the blot was glycin e stripped and stained with India ink as described in Methods. Figures (a) Autoradiogram showed the modified proteins mainly focused at approx. pH 7-10, and showing ba nd # 1, 2, 3a. 3b. 4a-c, 5, and 6 at around 22, 25, 26, 43, 49, 63 and 65 kDa respectively with pI 7.8, 8.8, 8.2, 8.4, 7.1, 7.9, 8.5, 9.1, 6.9 and 10 resp.; and (b) India ink stain showing the overall 2D-pattern of the proteins of oocyte stage I. On the left-hand side of the ge l 20 g of protein of stage VI was separated on one-dimension acts as reference. (a) (b) Homogenate Stage VI oocytepH 6 11 45 66.2 97.4 116.25 200 kDa 1 2 3b 3a 4a 4b4c 5 7 6 45 66.2 97.4 116.25 kDa 6 11 Homogenate Stage I oocyte
54 the limitation of the 2D-techniqu e that was used. The India i nk stain of the stage I have shown that most of the basic proteins were O -GlcNAc modified prot eins. (Figure 3.2.3 b) Thus, the results of the 2D separation with pH range 6-11 substa ntiated the earlier findings, and indicated the majority of the oocyte proteins from a stage I oocyte were basic in nature, like most of the ribosomal proteins . While the stage VI oocyte proteins in pH range 6-11 were resolved into a very different pattern to that of stag e I. In stage VI, the majority of proteins focused within pH range of 7-10, with bands that were number ed as 1, 2, 3a, 3b, 4a-c, 5, 6 and 7 at approximately 22, 25, 26, 43, 49 63 and 65 kDa respectively with approximate pI 7.8, 8.8, 8.2, 8.4, 7.1, 7.9, 8.5, 9.1, 6.9 and 10 respectively. (Figur e 3.2.4 a) Once again, the India ink stain of the stage VI oocytes have showed quite a different 2D-patterns of whole oocyte proteins from that of the stage I oocytes. (F igure 3.2.4 b) In addition to the differences in isoelectric points (pIs) of the proteins, there were fewer OGlcNAc modified proteins of stage VI oocytes were apparent. 3.3 Immunoprecipitation with RL-2 antibody Since, the proteins separated from th e whole homogenate were not enough and sufficient to see by the coomassie staining, we felt the need to enrich the samples with OGlcNAc modified proteins. In itially, the enrichment of OGlcNAc modified proteins was attempted by the immunoprecipitation with OGlcNAc specific antibodies such as RL-2 antibody. The immunoprecipitations with RL-2 antibody was successful in enriching at least some of O-GlcNAc modified proteins of interest as shown by results in the CTD110.6 immunoblot (Figures 3.3.1 a). Approxim ately 20 g of protein from oocytes at stage I in lane 1, one tenth of the total im munoprecipitate in lane 2, and approximately 20
55 g of supernatant in lane 3 (this amount was estimated taking into account the dilution and the concentration of orig inal sample) were separated on 8% SDS PAGE and immunoblotted with CTD110.6 antibody. The sec ond lane containing immunoprecipitate showed a few of the protein bands of inte rest namely those at 37, 54 and 62 kDa. The high intensity bands at 50 and 75 kDa in the sa me lane were of dena tured antibodies. The RL-2 immunoprecipitation performe d without the sample (the negative control) clearly showed the presence of these bands (Figur e 3.3.1 c). Thus, the presence of foreign proteins such as heavy chains of the denatured antibodies, which get resolved at nearly the same regions as proteins of interest in one -dimensional gel electropho resis interfered with the detection of the OGlcNAc modified proteins. In addition, huge difference in the band intensities of imm unoprecipitate and supernatant lanes in the silver stai n clearly indicated large portions of OGlcNAc modified proteins remained in the supernatan t. (Figure 3.3.1 b) The huge protein band at around 60 kDa in supernatant of the silver stain (shown by an arrow in Figure 3.3.1.b) appeared to be Â‘Protein AÂ’ conjugated to trisacryl beads used in RL-2 immunoprecipition. This band appears as a large non-illuminated ar ea in the same region of supernatant lane on immunoblotting with CTD110.6 antibody (Fig ure 3.3.1.a). Since all the protein bands of interest in that region were completely concealed by this large Protein A band, it becomes difficult to estimate the OGlcNAc modified proteins re maining in supernatant. In order to reduce the contamination with th e Protein A, immunopurification by column method was attempted. But also gave poor resu lts (Data not shown). Increasing the RL-2 concentrations in immunoprecipitation also did not significantly improve the yields, but increased the level of interf erence with immunoblot detectio n. (Data not shown) Thus,
56 Figure 3.3.1One-dimensional gel electrophore sis of the RL-2 immunoprecipitate of stage I oocytes. Approximately 20 g of protein from oocytes at stage I in lane 1, one tenth of the total immunoprecipitate in lane 2, and approximately 20 g of supernatant in lane 3 (this amount is estimated taking into account the dilution and the concentration of original sample) were separated on 8% SDS PAGE and immunobl otted with CTD110.6 antibody and stained with silver as described in Methods. (a) Autoradiogram of CTD110.6 immunoblot and (b) silver stain showing few prot ein bands of interest approximately at 37, 59 and 62 kDa in the RL-2 immunocomplex; and ( c) Autoradiogram of CTD110.6 immunoblot with RL-2 immunocompl ex and RL-2 immunocomplex without oocyte proteins (representing a negative control) were loaded in lane 1 and 2 respectively, showing the bands at 50, 75 and 100 kD a might be the bands of antibody. a. CTD110.6 immunoblot b. Silver stain c. Negative control 250 150 100 75 50 25 37 kDaHomogenate (stage I) Immunoprecipitate Supernatant Wash 1 Wash 2kDa Homogenate (stage I) Immunoprecipitate Supernatant Wash 1 Wash 2250 150 100 75 25 37 5035 66 70 Protein A 50 75 100 150 250 kDaRL-2 immunoprecipitate RL-2 immunoprecitate Without homogenate
57 another approach was investigated. 3.4 Immunoaffinity purification using CTD110.6 antibody To reduce the contamination of the samples with the foreign proteins (antibodies), the separation was performed using an an ti-mouse IgM-agarose immunoaffinity column, where the antibody is covalently linked to the agarose bead wh ile retaining its ability to specifically bind to CTD110.6 antibody (IgM sub-t ype). The proteins were eluted with a solution of TBS buffer containing 1 M N-acet ylglucosamine and concentrated by methanol precipitation that also removes the majo rity of N-acetylglucosamine. All the collected column fractions and the precipitated elutions were analyzed by RL-2 immunoblotting and silver staining (Figure 3.2.2 a and b). Approximately 20 ug of stage I oocyte proteins (original sample) in lane 1, one half of the eluted proteins in lanes 2, 3 and 4, and approximately 20 ug of flow through (this amount wa s estimated taking into account the dilution and the concentration of orig inal sample) in lane 5, one tenth of RIPA washes in lane 6 and 7, were separated on 8% SDS PAGE and analyzed by RL-2 immunoblotting. The analysis showed most of the OGlcNAc modified proteins we re in the flow through and washes (Figure 3.4.1.a). Thus indicating poor binding of OGlcNAc modified proteins to the column. Once again, the band at 60 kDa in both the flow through and washes that concealed the protein bands of interest. While some of this appears to be due to leaching of antibody from the column, it also repres ents the presence of BSA. BSA used as stabilizer in the stock solution of the CTD110.6 antibody used in the immunoprecipitation. Since so much of the prot eins of interest were found in the flow through, this material was incubated with a new set of beads to see if the yield could be improved. On the analyzing the collected fracti ons, once again the elutions of the immunoaffinity puri-
58 Figure 3.4.1One-dimensiona l gel electrophoresis of the CTD110.6 immunoaffinity purified proteins of stage I oocytes. Approximately 20 ug of stage I oocyte proteins (original sample) in lane 1, one half of the eluted proteins in lanes 2, 3 and 4, and approximately 20 ug of flow through (this amount was estimated taking into account the dilution and the con centration of original sample ) in lane 5, one tenth of RIPA washes in lane 6 and 7, were sepa rated on 8% SDS PAGE and analyzed by RL2 immunoblotting and silver staine d as described in Methods. ( a ) Autoradiogram of RL-2 immunoblot (8% gel) s howing few protein bands approximately between 50-60 kDa in the immunopurified sample and most of the modified proteins in the flow through and washes; ( b ) silver stain of 10% SDS Gel showing the presence of majority of proteins in the flow through. (c) Autoradiogram of RL-2 immunoblot (8% gel) whose lanes representing fr actions of repeated immunopr ecipitation of flow through. The faint bands around 50-60 kDa indicated no improvement in the yield using immunoaffinity purificati on of the flow through. (a) RL-2 immunoblots (b) Silver Stai n (c) RL-2 immunoblot (Normal Immunoprecip itation) (Repeated Imm unoprecipitation) 150Homogenate (stage 1) Flow through Immunoprecipitate RIPA Wash 1 RIPA Wash 5 250 100 75 50 37 25 kDa kDa Homogenate (stage I) Elution 1 Elution 2 Elution 3 Flow through RIPA wash 1 RIPA wash 2 250 150 75 100 50 37 25 20(r) = repeated immunoprecipitationElution 3 (r) Elution 2 (r) Elution 1 (r) Flow through (r) RIPA wash 1(r) RIPA wash 2 (r) 250 150 75 100 50 37 25 20kDa
59 fication showed very weak bands at 50 kDa, a nd with majority of the proteins again in the flow through and washes. (Figure 3.4.1 c) Th e results was also confirmed with silver staining that demonstrated very low protein content in the el utions compared to the flow through. (Figure 3.4.1 b) In an attempt to improve the yield, the batch-wise incubation method was adopted as a way of exposing more of the column mate rial to the proteins for longer periods of time. Most of the protein bands of intere st were detected on analyzing CTD110.6 immunoaffinity eluants following the same proce dure of RL-2 immunoblotting. Again some of the bands were concealed by the heavy chains of antibodies at 60 and 100 kDa. These bands are confirmed as bands of the antibodies because sim ilar bands at region of same molecular weight appeared when the anti -mouse IgM-agarose beads were resolved by SDS PAGE, and immunoblotted with RL-2 antibody (Figure 3.4.2 a). In addition, the presence of considerable amounts of OGlcNAc modified protei ns in the flow through indicated that signifi cant amounts of proteins were still not being captured with this techique (Figure 3.4.2 a). Further purification of the immunopurified material by 2D-gel electrophoresis showed that the OGlcNAc modified proteins we re not in most cases sufficiently separated from contaminating i mmunoglobulin and BSA, which was clearly visible in the RL-2 immunobl ot as shown in the Figure 3.4.2 b. Three protein bands on the extreme right of the membrane which are indicated by the arrows in the Figure 3.4.2 b appeared to be well separated in the immunoblot. However, the I ndia ink stain of this blot after glycine stripping showed a huge smear in that region indicating the presence of additional protein and would t hus, confound sequencing of the OGlcNAc modified proteins (Data not shown). These streaking a nd smear that were also observed in the
60 Figure 3.4.2 One-dimensional and two-dimensional gel electrophoresis of CTD110.6 immunoprecipitate (Batch-wise incubation method) of stage I oocytes Approximately 20 g of protein from oocytes at stage I, a quarter of the total immunoprecipitate collected, and a volume of supernat ant representing 20 g compared to Homogenate (stage I) were loaded in the first thr ee lanes of 10%1D-Gel; a nd nearly half of the immunocomplex was separated on 10% 2D-gel al ong with 20 ug of proteins of oocytes at stage I on left hand side of the gel as a re ference and immunoblotted with RL-2 antibody as described in Methods. Autoradiograms of (a) 1D-gel showing most of the protein bands of interest in imm unocomplex along with the bands of foreign proteins, and (b) 2D-gel showing vertical and horizontal streakin g of the foreign protei ns that overlaps the protein bands of interest pointed out using arrows. ( a) 1DGel Electrophoresis ( b ) 2DGel Electrophoresis 250 150 100 75 50 37 25 kDaHomogenate (stage 1) Immunoprecipitate Supernatant Ten Wash 1 Homogenate (stage I)pH 6-----------------------------------11 200 116.25 97.4 66.2 45 kDa Note: Blue arrows represent the O-GlcNAc modified proteins Anti-mouse IgM agarosebeads
61 immunoblot might be due the presence of the agarose bead s conjugated anti-mouse IgM in the immunoprecipitate. Even though, kits are available to remove some of the contaminating proteins, typically not all immunoglobu lin and albumin were removed. Therefore, it was decided not to pursue the method, because of already low yields and potential loss of sample in additional steps. 3.5 Affinity chromatography To overcome the problems of low yields and contamination with the foreign proteins mainly antibody, lectin affinity chromatography using agarose wheat germ agglutinin was used in an atte mpt to enrich the samples with OGlcNAc modified proteins. This procedure has been used with some success in nonXenopus somatic cell systems . To first assess the potential effectiveness of this tool for our Xenopus system, oocytes stage I and stage VI were an alyzed by the wheat germ agglutinin (WGA) affinity blotting. WGA binds to all glycoprot eins with terminal N-Acetylglucosamine residues and thus, can bind to both OGlcNAc modified proteins and glycoproteins with terminal GlcNAc on N-linked oligosaccharide chains. As expected the pattern of modified proteins from the affinity blot was sim ilar but not identical to that observed in the immunoblots with CTD110.6 and RL-2 antibod ies (Figure 3.1.1 and 3.1.3). For instance, the bands at 50 and 97 kDa of stage VI oocyt es shown by arrows in Figure 3.5.1 appeared in the affinity blot, but did not show up in the previous shown immunoblots. While appearance was at a similar molecular weight on 1D-analysis, the proteins seen at 50 and 97 kDA in stage VI are likely not the same as those in stage I samples. This is likely due to the affinity of WGA for all the carbohydrate containing proteins, with terminal Nacetylglucosamine residues on some Nlinked carbohydrate containing proteins.
62 Figure 3.5.1WGA Affinity blot of the oocytes at stages I and VI. Approximately 20 g of proteins from oocytes at stage I a nd stage VI were separated on the 10% SDS gel and probed with WGA-HRP. The blot show ed all the bands that appeared on the immunoblots with CTD110.6 and RL-2 in compar atively lower intensities due to the low sensitivity of colorimetric detection. The bands of stage VI shown by arrows are absent in the CTD110.6 immunoblot of the same system. Stages I VI 200 97.4 45 kDa 66.2 116.25 50 kDa 97 kDa
63 Moreover, only the most prominent bands were visible on the affinity blot due to lower sensitivity of the colorimetric detecti on method suggested for use by manufacturer. Attempts to use the chemiluminescent detec tion resulted in very high backgrounds that could not be sufficiently reduced with the st andard blocking agents such as BSA, tween 20. (data not shown) Note non fat dry milk a popular blocking agent cannot be used with CTD110.6 and RL-2 antibodies, since one or more carbohydrates in the solution reduces the antibody signal and gives high background. Once the affinity of WGA for the proteins of interest was verified, the affinity purification was initially performed at a pproximately 0.15 M NaCl as per the manufacturerÂ’s instructions, and late r the concentration of salt wa s reduced to less than 0.1 M NaCl as suggested by a published study , in order to maintain the weak intermolecular interactions in protein complexes a nd improve yields. The results of both the experiments as shown in Figure 3.5.2 a & b demonstrated that low salt conditions improved yields of affinity purification. In both experiments, the proteins bound to the column were eluted using the binding bu ffer containing 0.15 M Na Cl and 0.5 M GlcNAc. The collected fractions were pooled, desalted and concentrated by ultra-filtration using centricon-10 in order to proceed to 2D-gel electrophoresis, the ne xt purification step. Once again, all the collected fractions we re analyzed by CTD1 10.6 immunoblotting are shown in Figures 3.5.2 a and b represents the affinity purification at low salt (> 0.1 M NaCl) and high salt (0.15 M NaCl) conditions respectively. The second lane in the Figure 3.5.2 a shows the sample from the pooled elu tion contained all of the target bands, whereas the second lane in the figure 3.5.2 b did not show the bands at 54, 57 and 82 kDa. Thus, indicating that low salt binding conditions maximized both the yield and
64 Figure 3.5.2One-dimensional ge l electrophoresis of affini ty purified proteins from oocytes at stage I. Approximately 20 g of proteins from oocytes at stage I, a quarter of the pooled elution of the affinity purified prot eins at two different conditions, 0.1 M NaCl and 0.15 M NaCl, estimated volume containing 20 g calculated using the concentration of the homogenate (stage I) were separated in the first three lanes of the 10% gel and immunoblotted with CTD110.6 antibody as descri bed in Methods. Autoradiograms of the affinity purification (a) at low salt conditi ons (< 0.1 M) showing al most all the protein bands of interest in elutions and (b) high sa lt conditions (0.15M) sa lt showing bands at 35, 59, 69 and 97 kDa only. (a) Affinity purification (b) Affinity purification in lower salt (< 0.1 M NaCl) in higher salt (0.15M NaCl) Homogenate (stage I) pooled Elution Wash 1 Flow through 116.25 97.4 66.2 45 kDa Homogenate (stage I) Pooled Elution Flow through 45 66.2 97.4 116.25 kDa
65 number of OGlcNAc modified proteins. Therefore, the enrichment was qualitatively successful in concentrating all the modified pr oteins of interest. However, the yield was only 20% apparently due to th e loss of proteins by aggreg ation and precipitation during the concentration process. Hence, in order to acquire sufficient amounts of protein for sequencing, with this technique we would need to start with a larger amount of the oocyte material which unfortunately is a limiting factor in our process of purification. 3.6 Differential Sedimentation To overcome the problem of low initial proteins available from stage I oocytes, an another method was sought that minimized sa mple loss while still achieving enrichment of OGlcNAc modified proteins. To this end, it was noted that the proteins of interest tended to precipitate in stored homogenates s uggesting that the protei ns tend to form high molecular weight complexes. Based on th is hypothesis, the enrichment of the OGlcNAc modified proteins was attempted by simple differential sedimentation. First, centrifugation of the lysed oocytes at 1,000 x g for 10 mi nutes was performed in order to sediment any unlysed cells and large cell debris. The supernatant-1 (that is collected after 1,000 x g) was then centrifuged at 10,000 x g for anot her 10 minutes, to further fractionate the homogenate (stage I oocyte) into supernat ant-10 and pellet-10. The supernatant-10 was fractionated again by ultra-cen trifugation at 100,000 x g for one hour into supernatant100 and pellet-100. All the fractions were re solved on 10% SDS PAGE and analyzed by CTD110.6 immunoblotting (Figure 3.5.1 a). Later, this blot was stripped with glycine solution, and stained with I ndia ink (Figure 3.5.1 b). All th e lanes contained approximately 20 g of oocyte proteins. The OGlcNAc protein pattern in the supernatant-1 and supernatant-10 was similar to the earlier patterns of stage I. But the pellet-10 in the lane 2
66 Figure 3.6.1One-dimensional gel electrophoresis of proteins from oocytes at stage I fractionated by Differential Sedimentation. The homogenate of the stage I oocytes (after 1,000 x g) was fractionated by th e centrifugation at 10,000 x g and 100,000 x g, and 20ug each of the collected supernatants and pellets were sepa rated on 10% ID-Gel electrophoresis and analyzed by CTD110.6 i mmunoblotting and later glycine stripped CTD110.6 blot was stained by India ink as described in Methods. (a) Autoradiogram showing few O-GlcNAc modified proteins in the pellet at 10,000 x g and the distribution of protein bands of interest in both the supernatant and th e pellet collected at 100,000 x g where majority of O-GlcNAc modified proteins were found in pellet; (b) India ink stain of the glycine of the blot show ing the concentration of the pr oteins bands of interest in the pellet at 100,000 x g. a. CTD110.6 immunoblot b. India ink stain of glycine stripped membrane Homogenate(1,000 x g) Pellet (10,000 x g) Supernatant(100,000 x g) Pellet (100,000 x g) Supernatant(10,000 x g) 116.25 66.2 45 kDa 97.4 Homogenate(1,000 x g) Pellet (10,000 x g) Supernatant(10,000 x g) Supernatant(100,000 x g) Pellet (100,000 x g) 200 116.25 97.4 66.2 45 kDa
67 of India ink stain (Figure 3.5.1 b) showed the presence of large amounts of unmodified proteins indicating the centrif ugation at 10,000 x g concentrated th e proteins of interest in the supernatant-10. Thus, the centriguation of lysed oocytes at 10,000 x g can be performed to remove the unlysed oocytes and debris without signif icant loss of desired proteins. In contrast, the u ltra-centrifugation of supernatant-10 at 100,000 x g for 1 hour had sedimented the majority of modified prot eins as shown in the Figure 3.6.1 a. Interestingly, the India ink stain (Figure 3.6.1 b) reve aled the fact that the majority of the oocyteÂ’s unmodified proteins remained in the supernatant-100. Th erefore, this method not only enriched the target proteins, but al so minimized the loss of these proteins. The enriched samples analyzed by 2D-G el Electrophoresis across the pH range 611, as expected showed the same 2Dpattern of the modified proteins as that of whole homogenate of stage I oocytes (Figures 3.6.2 a and 3.2.3 a). In addition, the majority of the proteins were basic in na ture and were better resolved on the IPG strip, pH 6-11 as shown in Figure 3.6.2 a. Once again, the modifi ed proteins were mainly focused in the pH range 8-10. Interestingly, these protein ba nds appeared at slightly lower molecular weight than that of the whole homogenate. The high electrophoretic mobility of the proteins might be due to the presence of few non-modified proteins in the sample. The estimated molecular weights of these prot ein bands # 1-7, 8a, 8b, 9a, 9b, 10, 11a and 11b in Figure 3.6.1a were 21, 23, 36, 46, 47, 53, 52, 54, 83, 87 and 108 respectively. In addition, the intensity of protein bands of the pellet-100 (figure 3.6.2 a) is higher than that of the homogenate (Figure 3.2.3) sh owing successful enrichment of OGlcNAc modified proteins. Competition experiments with 15 mM GlcNAc demonstrated specificity through the reduction in the intensit y of all the bands as seen in Figure 3.6.2 b, especially in
68 Figure 3.6.2Two-dimensional gel electrophore sis of pellet at 100,000 x g from stage I oocytes by isoelectric focusing with pH 6-11 in the horizontal dimension and SDSPAGE (10%) in the vertical dimension. Approximately 100 g of protein from oocytes at stage I was separated a nd analyzed by CTD110.6 immunobl otting without and with 15 mM GlcNAc as described in Methods. (a ) Autoradiogram of immunoblotting in the absence of GlcNAc showed the modified pr oteins mainly focused at approx. pH 8-10, and 10 distinct bands # 1-4, 5a, 5b, 6, 7, 8a 8b, 9a, 9b,10, 11a and 11b were identified at 21, 23, 36, 46, 47, 53, 52, 54, 83, 87 and 108 kDa respectively. On the left-hand side of gel, 20 g of protein of stage I was separa ted on one-dimension acts as reference. (b) Autoradiogram of immunoblotting in the presence of 15mM GlcNAc showed the modified proteins mainly focused at appr ox. pH 8-10, showing all bands nearly reduced in the intensity. (a) Without GlcNAc (b) With 15mM GlcNAc 45 66.2 97.4 116.25 kDa4 1 2 3 6 7 8b 8a 5a 10 9b 9a 11a 11b 5b6 11 Homogenate Stage I oocytes 45 66.2 97.4 kDaHomogenate Stage I ooc y tes6 11 116.25
69 Figure 3.6.3Two-dimensional gel electrophore sis of pellet at 100,000 x g from stage I oocytes by isoelectric focusing with pH 3-10(NL) in the horizontal dimension and SDS-PAGE (10%) in the vertical dimension. Approximately 100 g of protein from oocytes at stage I was separated and anal yzed by CTD110.6 immunoblotting as described in Methods. (a) Autoradiogram of immunoblot showed all the bands focused at the extreme right due to the non-linear ity of the IPG strip. On the left-hand side of gel, 20 g of protein of stage I was separate d on one-dimension acts as reference. 3 10NL Homogenate Stage I oocytes 45 66.2 97.4 116.25 200 kDa
70 the case of bands # 5a, 5b, 11a, 11b and 4. In addition, the 2D-Gel El ectrophoresis using a full range IPG strip, pH 3-10 (non-linear) again showed the majority of th e proteins bands of interest focused across the pH range 8-10 substantiating that most of the modified proteins are basic in nature. (Figure 3.6.3) However, the protein bands of interest are not well separated due to the sharp rise in the pH of the non-linear IPG st rip (with an extended region across pH 5-8). Since the extended region on the IPG strip wa s pH 5-8, some of the neutral and weakly acidic proteins did separate (H ighlighted by an arrow in th e Figure 3.6.3). Especially the band 3a at pH 6-7 spotted in 2D-gel analys is of the homogenate (stage I) showed up once again in this 2D-gel (as shown with an arrow in Figure 3.2.3 a). 3.7 Identification and selection of the modified protein bands for sequencing Once the scheme of purification was fina lized as shown in the flow diagram, Figure 3.7.1, the proteins were isolated by di fferential sedimentation coupled with 2Dgel electrophoresis and coom assie stained for peptide sequencing by mass spectrometry. The 2D-separated proteins from stage I oocyt es that are sufficiently stained with Coomassie nearly showed all the fifteen protein ba nds of interest. (Figur e 3.7.2) Since the stained protein pattern was very much similar to the 2D-patterns observed earlier, the protein bands of interest could be easily identif ied by superimposing the CTD110.6 immunoblot of the stage I oocyte and by th eir positions on the gel. The identified protein bands shown in the Figure 3.7.2 by using arrows were carefu lly excised wearing gloves to avoid contamination.
71 Figure 3.7.1Scheme for isolation and identification of the O -GlcNAc modified proteins Separation of the oocytes by collagenase treatment Differential sedimentation of the homogenate at 10,000 x g for one hour Homogenization of oocytes stage I in Ten Silver Staining Isolation of stage I oocytes under microscope Resolution of the Pellet (at 100,000 x g) by 2D-gel Electrophoresis Separation of the oocytes by collagenase treatment Extraction of ovarian tissue Differential sedimentation of the homogenate at 100,000 x g for one hour G-250 Coomassie Staining Excising the protein bands of interest that are identified using the CTD110.6 immunoblot Destaining of the excised pr otein bands using 100 mM NH4HCO3 In gel digestion of the destained bands using trpsin LC-MS/MS analysis CTD110.6 immunblotting Protein identification by Xenopus laevis and other protein databases search of MS/MS spectra us ing Bioworks software
72 Figure 3.7.2 Two-dimensional gel electrophoresis of pellet at 100,000 x g from stage I oocytes by isoelectric focusing with pH 6-11 in the horizontal dimension and SDSPAGE (10%) in the vertical dimension. Approximately 100 g of protein from oocytes at stage I was separated on 10% two-dimensi onal gel, and stained by coomassie as described in Methods. The stain showed all the fifteen distinct bands # 1-4, 5a, 5b, 6, 7, 8a, 8b, 9a, 9b,10, 11a and 11b were identified at 21, 23, 36, 46, 47, 53, 52, 54, 83, 87 and 108 kDa respectively, mainly focused at approx. pH 8-10. On the left-hand side of gel, 20 g of proteins of stage I was sepa rated on one-dimension acts as reference. Homogenate Stage I oocytes pH 6 11 1 7 3 6 5a 5b 4 8b 28a 9b 9a 10 11a 11b 2 200 116.25 97.4 66.2 45
73 3.8 Mass Spectroscopy and database search Gel containing proteins were tr eated with trypsin and extract ed as described in the methods. The in-gel digested peptides of four protein bands were analyzed by LC-MS/MS and the MS/MS was interpreted using Biowor ks software. All the MS/MS spectra identifying proteins or peptides reported in the a ppendix C were Â“best hitsÂ” or Â“best matchesÂ” in a Xenopus database search with an Xcorr > 2. 5. The spectral data were also manually inspected for accuracy. First, the mass fingerprint of trypsin digested peptides was obtained, and then 5 to16 peptides that are easil y ionizable were fragmented further. The fragmentation of peptides resu lted in two types of peptide fragment ions, the N-terminal and the C-terminal ions. These fragment i ons were labeled based on the type of bond cleavage as a, b and c for C-terminal ions a nd x, y and z for N-terminal ions as shown in the Figure 3.8.1. These peptide fragment ions were fed into the mass spectroscopy instrument to obtain a CID (Collision induced dissociation) spectrum of each peptide. The spectrum of each peptide revealed the p eaks of all the fragments, from a molecular ion to a final product ion form ed during fragmentation. Moreove r, this disso ciation into the peptide fragment ions helps to differentia te the isomers of amino acids or amino acids with nearly the same molecular masses, for example isoleucine from leucine and glutamine from lysine respectively. In order to identify a protein, we need bot h the mass fingerprint of the protein (by MS scan) and the spectra of few peptide spect ra for a given protein (by MS/MS scan). Figures 3.8.2 & 3.8.2, and Tables 3.8.1 & Tabl e 3.8.2 provides an example of mass data analysis of one of the protein identifi ed as Vg1 RNA binding protein variant A (Vg1RBP). The spectral data for rest of the identified proteins are given in Appendix C.
74#1401-1401 RT:51.61-51.61 NL: 3.45E6 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance b1-12 1317.8 a1-12 1289.7 y1-10 1220.6 b1-11 1189.4 b1-10 1118.5 y1-9 1083.5 b1-9 1005.3 a1-9 977.5 y1-8 936.4 b1-8 877.6 a1-8 849.4 b1-7 790.2 y1-7 773.3 b1-6 719.3 y1-5 615.2 b1-5 556.2 a1-5 528.1 y1-4 487.7 b1-4 409.0 a1-4 381.1 y1-3 374.1 y1-2 302.9 a1-3 244.1 b1-2 215.1 b1-1 114.1 a1-1 86.1 Figure 3.8.1General fragmenta tion pattern of peptide and sequence nomenclature for mass ladder Figure 3.8.2MS/MS spectrum of a prot ein band # 9a identified as Vg1 RNA binding protein variant A. CID spectrum/ product ion spectrum of a peptide ITGHFYASQLAQR of the Vg1 RBP showing the peaks of all the peptide fragment ion in the order of their molecular masses.
75 Table 3.8.1Mass data of a peptide of Vg1 RBP variant A. Table lists out the masses of all the peptide fragme nt ions of the peptide ITGHFYASQLAQR Table 3.8.2Peptide sequences of Vg1 RBP Sequences of sixteen peptides of Vg1 RBP along with their ionic masses, positions, mass percentages and amino acids percentages were given. The total protein coverage of 20.62% by mass is a decent % to obtained the sequence of the protein. AAM+nHAA*AoBB*BoCXYY*Yo I 115.1086.1069.0768.09114.0997.0796.08131.121491.771474.741473.76 T 216.15187.14170.12169.13215.14198.11197.13232.171404.671378.691361.661360.68 G 273.17244.17227.14226.16272.16255.13254.15289.191303.621277.641260.611259.63 H 410.23381.23364.20363.21409.22392.19391.21426.251246.601220.621203.591202.61 F 557.30528.29511.27510.28556.29539.26538.28573.311109.541083.561066.531065.55 Y 720.36691.36674.33673.35719.35702.33701.34736.38962.47936.49919.46918.48 A 791.40762.39745.37744.38790.39773.36772.38807.42799.41773.43756.40755.42 S 878.43849.43832.40831.42877.42860.39859.41894.45728.37702.39685.36684.38 Q 1006.49977.48960.46959.471005.48988.45987.471022.51641.34615.36598.33597.35 L 1119.571090.571073.541072.561118.561101.541100.551135.59513.28487.30470.27469.29 A 1190.611161.611144.581143.601189.601172.571171.591206.63400.19374.22357.19356.20 Q 1318.671289.661272.641271.651317.661300.631299.651334.69329.16303.18286.15285.17 R 1474.771445.771428.741427.751473.761456.731455.75201.10175.12158.09157.11 SequenceMH+%by MassPosition%byAAÂ’sESKIPFTGQFLVK1493.842.2924 -362.19 IPFTGQFLVK1149.671.7627 -361.69 AIDTLSGK804.451.2353 -601.35 VIEVEHSVPK1136.631.7467 -761.69 PQSEVPLR925.511.42201 -2081.35 FTEEIPLK976.541.49282 -2891.35 ILAHNNFVGR1140.631.75290 -2991.69 FAGASIK693.391.06446 -4521.18 IAPAEGPDAK968.511.48453 -4621.69 MVIITGPPEAQFK1430.772.19465 -4772.19 LKEENFFGPK1208.631.85486 -4951.69 EENFFGPK967.451.48488 -4951.35 VPSYAAGR820.431.26506 -5131.35 DQTPDENDQVVVK1486.702.28538 -5502.19 ITGHFYASQLAQR1491.772.28551 -5632.19 IQEILAQVR1069.641.64565 -5731.52Protein Coverage By Mass13475.2 % by Mass20.62 By Position 122 % by AAÂ’s 20.57
76 Figure 3.8.3Sequence of Vg1 RN A binding protein variant A Sequnce is downloaded from http://www.expasy.org/sprot/ and sequences in red are the ones analyzed by mass spectrometry to sequence the peptide and identify the protein band # 9a Vg1 RNA binding protein variant A gi|2801766|gb|AAB97457.1| KH domain-containing transcription factor B3 [Xenopus laevis]gi|3172447|gb|AAC18597.1| Vg1 RNA binding protein variant A [Xenopus laevis]gi|35505483|gb|AAH57700.1| MGC68429 MNKLYIGNLSENVSPPDLESLFKESKIPFTGQFLVKSGYAFVDCPDETWAMKAIDTLSGKVELHGKVIEVEHSVPKRQRSRKLQIRNIPPHLQWEVLDSLLAQ YGTVENCEQVNTDSETAVVNVTYANKEHARQGLEKLNGYQLENYSLKVTYIPD EMATPQSPSQQLQQPQQQHPQGRRGFGQRGPARQGSPGAAARPKPQSEVPLRML VPTQFVGAIIGKEGATIRNITKQTQSKIDIHRKENAGAAEKPITIHSTPEGCSAACKI IMEIMQKEAQDTKFTEEIPLKILAHNNFVGRLIGKEGRNLKKIEQDTDTKIT ISPLQDLTLYNPERTITVKGSIETCAKAEEEVMKKIRESYENDIAAMNLQAHLIPGL NLNALGLFPPSSSGMPPPSAGVSSPTTS ASYPPFGQQPESETVHLFIPALAVGAIIGK QGQHIKQLSRFAGASIKIAPAEGPDAKLRMVIITGPPEAQFKAQGRIYGKLKEENFFGPKEEVKLEAHIKVPSYAAGRVIGKGGKTVNELQNLTSAEVVVP RDQTPDENDQVVVKITGHFYASQLAQRKIQEILAQVRRQQQQQQKT AQSGQPQPRRK
77 Figure 3.8.2.showed all the peaks of the peptid e fragment ions obtained from a peptide ITGHFYASQLAQR of Vg1RBP. The mass data given in the table 3.8.1 for the spectrum of peptide provides the sequence and mo lecular masses of the fragment ions. These overlapping fragment ions we re aligned on the basis of their masses to identify the sequence of the peptide. All th ese operations were carried out by software programmed to operate mass spectroscopy instrument, Biowor ks software was used for Finnigan LCQ in this study. Table 3.8.2 one of the outputs of this program that summarizes the number of peptides sequenced for one protein and protei n coverage (by the por tions of protein sequenced) percentages by mass and number of amino acids for protein identification. The sequenced peptides were then matched agains t one or more databases, and the protein with highest hits (matching the most peptides) in Xenopus laevis database with Xcorr > 2.5 was selected. The Xcorr >2.5 is the value us ed to determine efficacy of match . The Figure 3.8.2 shows the sequence of the iden tified protein that was downloaded from http://www.expasy.org/sprot/. Figure 3.8.2 shows some portions of sequence highlighted in red were the sequenced peptides used in the protein identification. The seven mass-analyzed protein bands were identified as following: Band # 3 as Zygote arrest 1 protein, Band # 4 as an oocyt e specific form of el ongation factor-1 alpha (42Sp50/thesaurin a), Bands # 7, 8a and 8b as Cytoplasmic mRNA bi nding protein p54 (y box factor homolog), Band # 9a as Vg1 RNA bi nding protein variant A, and Band # 10 as poly (A) binding protein. One peptide out of the protein bands around 50 kDa was identified as Xp54, RNA helicase with very high Xc orr. Most probably, the signals of other peptides of this protein might be lost or submerged among the strong signals of peptides from other proteins during mass analysis. Th e identified proteins are well characterized
78 proteins of Xenopus laevis oocytes. However, none of thes e proteins have previously been shown to be modified with OGlcNAc. As oocyte specific proteins, they have been shown to play significant roles in the regul ation of translation or intracellular mRNA translocation during the oogenesis. While, one of the proteins has been shown to play an important role in the oocyt e-to-embryo transition. Zygote arrest 1 ( Zar1 ), protein band # 3 is an ovary-s pecific maternal factor that plays an important role duri ng the transition of oocyte to embryo . This 34 kDa protein is 295 amino acids long and found in multiple tissues including lung, muscle and ovary. Zar 1 mRNA is specifically synthesized in oocytes. Zar 1, localized predominantly in cytoplasm of oocytes, rapidly disappears at the two-cell stage of embryogenesis, suggesting a critical role in th e oocyte-to-embryo transition [ 116]. Presence of an atypical eight cysteine Plant Homeo Do main (PHD) motif at C-termi nus, suggests that the protein might act as a translational activator, represso r or cofactor, and/ or form complexes that modulate chromatin. In addition, Zar 1 is found to be essential for female fertility in mice . The gradual disappearance of OGlcNAc of protein or reduction in their levels suggests a role for O -GlcNAc in regulating the function of this protein. Thesaurin a (42Sp50), the protein band # 4 was th e first to be identified, and is homologous to eukaryotic EF-1 and prokaryotic EF-Tu that recruits aminoacyl tRNA to the A-site of ribosome . There ar e three forms of elongation factor-1 in Xenopus laevis two of them are oocyte specific forms, Thesaurin a and EF-1 and third one is somatic form implying its absence in the ooc yte, and present in embryo and adult cells only. Thesaurin a, consistent with our identif ication, is an early oocyte form, and unlike the other oocyte specific EF-1 O, thesaurin a is exclus ively and abundantly found in
79 previtellogenic oocytes (s tage I, II and III) of Xenopus laevis . In addition, thesaurin a is uniformly distributed throughout the cyto plasm with a one-order of magnitude lower activity than that of the EF-1 O that is concentrated in the mitochondrial mass (Balbiani body) . Thesaurin a is a major component of th e 42S ribonucleoprotein (RNP) particle that in addition includes of thesaurin b (40 kDa protein), tRNA, and 5S RNA.  The main function of the 42S particle in oocyte s is long-term storage of tRNA and 5S RNA. Thesaurin a show specific binding to tRNA, wher eas thesaurin b specif ically binds to 5S RNA. Even though, thesaurin a binds to tRNA, GTP and GDP like EF-1 O, it has relatively low affinity for these ligands and low tRNA transfer activity. However, inspite of its low activity, the thesaurin a can presumably function as a substitute for EF-I due to its high concentration in pr evitellogenic oocytes. Theusa rin a differs from EF-1 by its binding properties. Thesaurin a binds more strongly to GTP than GDP, and charged tRNA than uncharged tRNA, whereas the opposite is the case for EF-1 Since thesaurin a does not binds GDP weakly, the replacement of GDP can occur in the absence of the GDP/GTP exchange factor, EF-1 Thus, 42S particle contai ning thesaurin a can fully function as a substitute for both EF-1 and EF-1 in the previtellogenic oocytes, that is replaced by the EF-1 O at the beginning of vi tellogenesis, and which in turn is replaced by the somatic form of EF-1 at the beginning of the embr yogenesis  However, the presence of thesaurin a only in the earlier st ages of oogenesis and its existence as a main component of the 42S particle with one orde r of magnitude low tRNA transfer activity also suggests the major function of thesaurin a is to store tRNA for later use during oogenesis and perhaps, early embryogenesis.
80 Even though, phosphorylation has not yet b een demonstrated in thesaurin a, a genetically distant re lated form of EF-1 from rabbit reticulocytes was reported to be phosphorylated . Interestingly, this prot ein when phosphorylated dissociates from complexes of monoand polyribosomes thus affecting the rate of polypeptide chain elongation, suggesting a regulatory role in translation for this post-translational modification. In another study, EF-1 was immunoprecipitated with CTD110.6 antibody from rat liver extract suggesting that it might be OGlcNAc modified or associated with proteins that are modified [1 19]. Now that thesaurin a ha s been identified as an OGlcNAc containing protein, the role of th is modification can be investig ated. It is partially attracttive to speculate that the disappearance of th esaurin a at the beginning of vitellogenesis, might be due to the removal of OGlcNAc group from the protein, and thus, may lead to its proteasomal degradation as reported in the case of tran scription factor, Sp1 . While tRNA and 5SRNA stored along with thesaurin in complexes referred as thesaurisomes, oocyte maternal mRNA storage is also stored in a nontranslated ribonucleoprotein (RNP) complexes for the rapid translational recruitment during embryonic development, after mid-blastula stage [ 120]. The protein bands # 7, 8a and 8b were identified as Cytoplasmic mRNA binding prot ein p54, y box factor homolog is one of the nonribosomal proteins associated with mRNAs in the RNP complexes. It can inhibit translation in vitro, and also has been considered as a putative translati onal regulator in vivo. This protein is found in two states in cells, a heterodime r form with p56 (another y-box factor) found free in cytoplasm or complex form with ribonucleopr otein particles. By stage II of oogenesis, the highe st concentration of p54 is ach ieved and the levels of the
81 proteins were maintained throughout oogenesi s and into early embryogenesis, but after gastrulation it is no longer found in soluble form. This p54 is a basic protein with a high content of arginine residues (11%) with predicted to pI > 9 that is very near to our experimental value of pI 8.7. The proteinÂ’s RNA binding site containing four arginine rich Â“basic/ aromatic islandsÂ” that are similar to the RNA-binding domain of bacteriophage mRNA antiterminator proteins and tat protein of human immunodeficiency virus . In addition, the C terminal domain of the protein is homologous to the small E. coli co ld-shock proteins These proteins are highly expressed at very low temper ature and may protec t the cells from damage due to freezing, by inhibiting translational in itiation. By inference, from the cold shock proteins, the p54 may be involved in the partial blocking of translational initia tion processes in an adaptive measure to store the untranslated mRNA for later us e in early embryogenesis. In addition, the in-vitro studies have dem onstrated that phosphorylation activates RNA binding . The maintenance of p54 levels thoroughout the oogenesis in contrast to our observations where OGlcNAc modified p54 is reduced might be due to the deglycosylation of these proteins in the later st ages. Alternately, since our observations are based on the changes per unit mass, if total amount of protein is fixed while the oocyte enlarges and accumulates other proteins its relative amount would be reduced. Since the protein bands # 8a and 8b were also identif ied as y-box factor ho molog, p54 suggests that the protein has different pI. Th is pI difference might be due to the different levels of phosphorylation of this protei n. Highly phosphorylated protein is more acidic than less phosphorylated forms. Thereby, indicating that O -phosphorylation might be alternating with O -GlcNAcylation in this proteins.
82 Similar to y-box proteins, Xp54 (ATP dependent RNA helicase p54) is abundant and an integral component of stored mRNP particles . This protein belongs to the family of DEAD-box RNA helicases that regulat e RNA secondary structure in translation initiation, splicing and ribosome biosynthesis. It possesses ATP-de pendent RNA helicase activity  High levels of Xp54 are expres sed during early oogenesi s, and are totally missing in adult tissue. Earlier studies sugge sted that the Xp54 might represent expression of an oocyte specific gene. High levels of Xp54 transcripts in small oocytes correlates to maximum production of stored mRNP par ticles, the levels is later on reduced may reflect relatively its low net pr oduction of mRNP part icles in later stages of oogenesis and embryogenesis. Xp54 has multiple potential Ca esin kinase 2 phosphorylation sites, with four out of five of these site s located near the C-terminus. It has been suggested the helicase activity might be regulated in two possible ways. One is by the addition or removal of phosphate groups, and the other by the availab ility of cofactor at appropriate stages of development. Since levels of Xp54 maximal remains fairly constant throughout ogenesis up to blastula, the decrease OGlcNAc form seen in the stages suggests deglycosylation of the protein during the oogenesis. In addition to the above proteins involv ed in storage of the tRNA and mRNA, the band # 9a was identified as an OGlcNAc modified protein, Vg1 RNA binding protein (RBP) variant A. This protein associates Vg1RNA to the microtubules in order to translocate the RNA to the vegetal cortex  is identified as the O-GlcNAc modified proteins. The translocation of Vg1RNA requires intact microt ubules and a 3Â’ untranslated region (UTR) cis-acting element (termed vegetal localization element, VLE). This Vg1RBP has five domains, four K homo logy (KH) and one RNA recognition motif
83 (RRM) domains. The KH domains of Xenopus Vg1RBP appeared to mediate cytoplasmic trafficking, RNA binding and self associati on to homodimer . However, the RRM does not have any role in the above men tioned processes although the sequence suggests potential for RNA binding . This protein specifically binds to the VLE and is homologous to the micro-filament-binding zipcode binding protein (ZBP-1) that is involved in -actin mRNA localization. The Vg1RBP appe ars to bind independently at two distinct sites in the VLE, and these binding sites re present a cis-acting element required for localization via the microtubule dependent pathway that oocurs in late stage III-early IV oocytes. Interestingly, OGlcNAc modified protein disappe ars during the stage progresssion, indicating the modification may have some role to play in activating the protein after previtellogenesis (at stage III), perh aps through modulation of protein-protein associations. The protein band # 10 has been identified as Poly (A) binding protein (PABP). This cytoplasmic 71 kDa protein is 633 ami no acid long, binds to Poly (A) tail of adenylated mRNA and acts as positive regulator of translation . PABP contains four RNA recognition motif (RRM) domains, three of whic h can act independently of each other in RNA binding, whereas the fourth one in the N-terminal region shows no detectable RNA binding activity . Even though, Western blot analyses did not detect the Poly (A) binding proteins in oocytes or early embryos but whole mount immunocytochemistry of oocytes (unpublished data) and direct anal ysis of oocyte messe nger ribonucleoprotein particles  reveal the pres ence of this protein. Overall, these Northern and Western analyses suggests that the expression of PABP is not constitutive, but is instead
84 modulated in oocytes and the developing embryos . This protein is localized in the cell types and subcellular domai ns that are most active in protein synthesis . In immature oocytes, the cytoplasmic polyadenylation element (CPE) in 3Â’ untranslated region (UTR) of maternal mRNA is bound by CPEB, which in turn bound by Maskin, which in turn bound by eIF4E [ 127, 128]. The progesterone stimulation induces CPEB phosphorylation and polyade nylation. Polyadenyl ation, through PABP destabilizes the Maskin-eIF4E complex, and l eads to the binding of eIF4G with eIF4E that stimulates translation. Interestingly, the cyclin B which associates with protein kinase cdk1 to form MPF in Xenopus oocytes was found to be translated by this polyadenylation binding (PAB)-mediated stimulation  Thus, the protein involved in the regulation of cyclin B1 mRNA tran slation that is esse ntial for the embryonic cell cycle . Since these cytosolic proteins are found in association with RNA and/ or other proteins, the characteristics of the proteins in these high molecular weight particles likely explains all the difficulties encountered duri ng the initial purifica tion attempt. Immunoaffinity and affinity methods depend on the availability of OGlcNAc binding sites on the proteins. These sites may not be accessible in the particles due to steric constraints in the native associated state. Thus explaining the low yields fr om these technologies. However, the presence of these proteins in the high molecular weight complexes provided an opportunity to both enrich samples with OGlcNAc modified prot eins and achieve high yields using differential sedimentation cen trifugation. The complex formation had no effect on the ability to detect the OGlcNAc modification by immunoblotting. Since this technique involves SDS-PAGE analysis prio r to blotting. The denaturation of the com-
85 plexes during this process makes the OGlcNAc available for antibody interaction and thus detection. Apart from the proteins isolat ed thus far, there are some neutral proteins that need to isolated and identified in order to get the overall picture of the oocyte proteins showing the changes in their levels or the O -GlcNAc levels during the stage progression. In addition to the proteins associated to high molecu lar weight particles, there are a few soluble proteins left behind in the supernatant-100. These proteins which also show changes in the OGlcNAc levels during oocyte development mi ght play an important role in regulating the cellular processes of the oocyte. Sin ce these proteins constitute a small percentage of total oocyte proteins, they need to be enriched by WGA affinity chromatography at low salt conditions, and late r resolved by 2D-gel electrop horesis for protein sequencing and identification.
86 Chapter Four Conclusion The proteins that are identified th us far are among the most abundant, OGlcNAc modified cytosolic proteins of Xenopus laevis oocytes (stage I) and largely basic in nature. Thesaurin a, y-box factor homol og, and Xp 54 (ATP dependent RNA helicase p54) that store tRNA and mRNA respectivel y as the ribonucleoprotein (RNP) complexes are found in maximum levels at early oogenesi s. In addition, thesaurin a is an oocyte specific EF-1 form that is present only in previt ellogenic oocytes. Earlier studies have characterized these oocytes proteins as regul ators of protein transl ation or as masking (repressing translation) protei ns for mRNA or as involved in RNA translocation during oogenesis. The regulation of translation is cr ucial during the oogenesis, since the oocytes store mRNA and tRNA for the post-fertilization se ries of cell divisions until mid-blastula, a period where transcription is severely li mited. In the same manner, the intracellular mRNA localization that leads to asymmetric pr otein synthesis is necessary for the pattern formation during early embryogenesis. Interestingly, the zygote arrest 1 that is ovary specific protein has been suggested to play a critical role during the oocyte-to -embryo transition. Additionally, the presence of PHD motif also indicate a possible role as transcriptional activat or, repressor or cofactors that are essential during the developmental process. On the other hand, the Poly (A) binding protein has a role in in itiating the protein translati on. Importantly, the cyclin B1 mRNA of Xenopus oocytes was shown to be translat ed by this polyadenylation binding.
87 Thus, indicating a role in re gulating the cell cycle. To su mmarize, all the identified proteins play crucial roles in the devel opmental process, and the changes in the OGlcNAc levels might be modulating their activities like the Ophosphorylation. Finally, the high content of ar ginine residues in these prot eins explains their basic nature, and these arginine rich regions facilitate the possibl e interactions with RNA. The O -GlcNAc on the proteins involved in the pa ckaging, translocation and translation of RNA suggest that O -GlcNAc may play a role in the m odulating the intera ctions. In addition, the sudden disappearance of OGlcNAc modified thesaurin a that is at the stage III of oogenesis might be due to the deglycosyl ation leading to proteosomal degradation. Therefore, in order to unde rstand the putat ive role of OGlcNAc modification on this oocyte specific proteins, these proteins need to further stud ied. In addition, there are some more modified oocyte proteins that needs to be identified in or der understand the significance of these proteins a nd the potential function of OGlcNAc in the developmental process of oocyte. The findings of this invest igation could be a signi ficant contribution to the biochemistry of oocyte deve lopment and more generally to OGlcNAc mediated cellular processes. In addition, it adds severa l more proteins to the growing list of O -GlcNAc modified proteins.
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101 Appendix A: Abbreviations OGlcNAc N-acetylglucosamine attached to a serine or threonine GlcNAc N-acetylglucosamine OGlcNAcase N-acetyl-glucosaminidase mRNA Messenger RNA DNA Deoxy ribonucleic acid GVBD Germinal vesicle breakdown tRNA Transfer RNA snRNA Small nuclear RNA rGTP Guanosine triphosphate dTTP Deoxyribothymidine 5Â’ Â–triphosphate UDP-glucose Uridine Diphosphoglucose HSP Heat shock protein OGT OGlcNAc Transferase PUGNAc O2-acetamido-2-deoxy-D-glucopranosylidene STZ Streptozotocin WGA Wheat germ agglutinin GFAT Glutamine: Fructose-6 -phosphate amidotranferase DON 6-diazo-5-oxonorleucine OR-2 Oocyte ringers buffer 2 HEPES 4-2-hydroxymethyl-1-piperazine ethanesulfonic acid BSA Bovine serum albumin Tris Tris-hydroxymethyl-aminomethane
102 IgG Immunoglobulin type G IgM Immunoglobulin type M UDP Uridine diphosphate SDS Sodium dodecyl sulfate PAGE Polyacrylamide gel electrophoresis TEMED N, N, NÂ’, NÂ’-tetra-methyl-ethylenediamine PVDF Polyvinyl difluoride PBS Phosphate buffer saline RL-2 Monoclonal Antibody against O-GlcNAc CDT110.6 Monoclonal Antibody against O-GlcNAc PBST Phosphate buffered saline with Tween 20 HSPBS High salt phosphate bu ffered saline with Tween 20 CTD C-terminal domain TBST Tris buffered saline with Tween 20 TBS-HT Tris buffered sa line with high Tween 20 TBS-D Tris buffered salin e with high detergent DAB Diaminobenzidine ATP Adenosine triphosphate Glc Glucose Gal Galaactose PMSF Phenylmethyl-sulfonyl fluoride EDTA Ethylene-diamine tetracetic acid HRP Horse radish peroxidase
103 1D-gel One-dimensional gel 2D-gel Two-dimensional gel RIPA Radioimmunoprecipitation assay IPG Immobilized pH gradient IEF Isoelectric focusing NL Non-linear DTT Dithiothretol CHAPS 3-[(3-cholamidopropyl) dimet hylammonio]-1-propanesulfonate MS 222 Tricaine methyl sulfonate PSM Protein solubilizing mixture RBP RNA binding protein Vg 1 RBP Vegetal 1 RNA binding protein VLE Vetegal localization element EF-1 Elongation factor-1 alpha RNP Ribonucleoparticle EF-1 O Elongation factor alpha (oocyte specific form) CK 2 Casein kinase 2 UTR Untranslated region KH K Homolog ZBP-1 Zipcode binding protein HBP Hexosamine biosynthetic pathway Zar 1 Zygote arrest 1 Xp54 ATP dependent RNA helicase p54
104 PABP Poly (A) binding protein CPE Cytoplasmic polyadenylation element CPEB Cytoplasmic polyadenyla tion element binding protein eIF Elongation initiation factor pI Isoelectric point
105 Appendix B: Structures of some of the discussed and used sugars and inhibitors Glucosamine Glucose N-Acetylglucosamine Galactose
106 Guanosine 5Â’-triphosphate Uridine diphosphate-N-acetylglucosamine
107 Inhibitors Streptozotocin 6-Diazo-oxonorleucine PUGNAc
108 Appendix C: Mass data analysis of Th esaurin a, p54 (y-box factor homolog), Xp54 RNA helicase, Zygote arrest 1 and Poly (A) binding protein Figure 1-Mass spectrum of a peptide of El ongation factor (Thesaurin a)(42Sp50) Elongation factor 1-alpha (EF-1-alpha) (42Sp50)gi|416929|sp|P17506|EF11_XENLA Elongation factor 1-alpha (EF-1-alpha) (42Sp50) (ThesaurinA)gi|64489|emb|CAA79605.1| 42Sp50 [Xe nopuslaevis] MTDKAPQKTHLNIVIIGHVDSGKSTTTGHLIYKCGGFDPRALEKVEAAAAQLGKSSFKFAWILDKLKAERERGITIDISLWKFQTNRFTITIIDAPGHRDFIKN MITGTSQADVALLVVSAATGEFEAGVSRNGQTREHALLAYTMGVKQLIVCVNKMDLTDPPYSHK RFDEVVR NVMVYLKKIGYNPATIPFVPVSGWTGENISSPS QKMGWFKGWKVKRKDGFTKGQSLLEVLDALVPPVRPANKPLRLPLQDVYK IGGIGTVPVGK IETGILKPGMTISFAPSGFSAEVKSIEMHHEPLQMAFPGFNIGFN VKNIAVKSLKRGNVAGNSKSDPPTEASSFTAQVIILNHPGFIKAGYSPVIDCHTAHITCQFAELQEKIDRRTGK KLEDNPGLLK SGDAAIITLKPIKPFCVER FFDYPP LGR FAARDLKQTVAVGVVKSVEHKAGAAARRQVQKPVLVKSequenceMH+% by Mass Position% by AAÂ’sVEAAAAQLGK 957.541.8945 -542.16 FTITIIDAPGHR 1340.732.6588 -992.59 RFDEVVR 920.501.82169 -1751.51 FDEVVR 764.391.51170 -1751.30 IGGIGTVPVGK 997.601.97259 -2692.38 KLEDNPGLLK 1126.652.22389 -3982.16 FFDYPPLGR 1111.562.19418 -4261.94Protein coverage TotalsBy mass 6359.5 % by mass 12.56 By position 59 % by AAÂ’s 12.74 #934-934 RT:41.27-41.27 NL: 5.08E6 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance y1-10 957.5 a1-10 911.5 y1-9 858.4 b1-9 811.2 a1-9 783.4 y1-8 729.4 a1-8 726.1 y1-7 658.4 b1-7 641.3 a1-7 613.0 y1-6 587.3 y1-5 516.3 b1-6 513.2 y1-4 445.3 b1-5 442.1 a1-5 414.3 b1-4 371.0 a1-4 343.0 y1-3 317.1 b1-3 300.0 a1-3 272.0 b1-2 228.9 y1-2 204.1 y1-1 147.1 b1-1 100.1 a1-1 72.1
109 Figure 2Mass data analysis of a pept ide Xp54, RNA Helicase (only one peptide was found with High Xcorr). #1753-1753 RT:68.05-68.05 NL: 6.63E5 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance b1-14 1474.8 y1-13 1380.3 b1-13 1319.0 y1-12 1264.5 b1-11 1164.3 a1-11 1136.8 b1-10 1063.5 a1-10 1035.6 y1-9 949.4 b1-8 879.4 a1-8 851.0 y1-8 812.5 b1-7 780.2 a1-7 752.3 y1-7 713.3 b1-6 681.3 a1-6 653.3 y1-6 614.3 b1-5 544.0 y1-5 501.2 b1-4 445.4 y1-4 430.2 a1-4 417.2 b1-3 344.1 y1-3 329.1 a1-3 316.1 y1-2 232.3 a1-2 201.1 b1-1 114.1
110 Figure 3Mass data analysis of a peptide of p54, y-box factor homolog #1736-1736 RT:72.90-72.90 NL: 1.31E6 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance b1-16 1650.3 y1-15 1610.0 y1-14 1552.4 a1-15 1492.7 b1-14 1463.7 y1-13 1437.7 a1-14 1435.6 b1-13 1334.3 y1-11 1251.5 b1-12 1235.0 y1-10 1150.4 b1-11 1136.3 a1-11 1108.5 y1-9 1051.4 b1-10 1020.9 y1-8 922.2 b1-9 874.3 y1-7 775.4 a1-8 717.2 y1-6 660.0 a1-7 618.3 y1-5 561.1 b1-6 545.0 a1-6 517.2 y1-4 462.0 b1-5 415.9 b1-4 359.1 y1-3 333.1 a1-4 331.2 y1-2 276.5 a1-3 216.1 a1-2 159.1 b1-1 88.0 AAM+nHAA*AoBB*BoCXYY*Yo S 89.0560.0443.0242.0388.0471.0170.031795.821778.801777.81 V 188.12159.11142.09141.10187.11170.08169.101708.791691.771690.78 G 245.14216.13199.11198.12244.13227.10226.121609.721592.701591.71 D 360.16331.16314.14313.15359.16342.13341.151552.701535.681534.69 G 417.19388.18371.16370.17416.18399.15398.171437.671420.651419.66 E 546.23517.23500.20499.22545.22528.19527.211380.651363.631362.64 T 647.28618.27601.25600.26646.27629.24628.261251.611234.581233.60 V 746.34717.34700.32699.33745.34728.31727.331150.561133.541132.55 E 875.39846.38829.36828.37874.38857.35856.371051.491034.471033.48 F 1022.46993.45976.43975.441021.451004.421003.44922.45905.43904.44 D 1137.481108.481091.451090.471136.471119.451118.46775.38758.36757.37 V 1236.551207.551190.521189.541235.541218.521217.53660.36643.33642.35 V 1335.621306.621289.591288.611334.611317.591316.60561.29544.26543.28 E 1464.661435.661418.631417.651463.651446.631445.64462.22445.19444.21 G 1521.681492.681475.651474.671520.681503.651502.67333.18316.15315.17 E 1650.731621.721604.701603.711649.721632.691631.71276.16259.13258.15 K 1778.821749.821732.791731.811777.811760.791759.80147.11130.09129.10 MSSEVETQQQQPDALEGKAGQEPAATVGDKKVIATKVLGTVKWFNVRNGYGFINRNDTKEDVFVHQTAIKKNNPRKYLRSVGDGETVEFDVVEGEKGAEAANVT GPEGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNYQNNESGEKAEENESAPEGDDSNQQRPYHRRRFPPYYTRRPYGRRPQYSNAPVQGEEAEGADSQGTDEQG RPARQNMYRGFRPRFRRGPPRQRQPREEGNEEDKENQGDETQSQPPPQRRYRRNFNYRRRRPENPKSQDGKETKAAETSAENTSTPEAEQGGAE SequenceMH+ % by MassPosition% by AAÂ’s MSSEVETQQQQPDALEGK2004.925.791 -185.94 EDVFVHQTAIK1286.673.7260 -703.63 SVGDGETVEFDVVEGEK1795.825.1980 -965.61 FPPYYTRRPYGR1572.814.54172 -1833.96
111 Figure 4Mass data analysis of a peptide of Zygote arrest 1, Zar 1 Sequence of Zar 1 MYPAYNPYSYRYLNPRNKGMSWRQKNYLASYGDTGDYCDNYQRAQL KAILSQVNPNLTPR LCRANTRDVGVQVNPRQDASVQCSLGPRTLLRRRPGALRKPPPEQGSPASPTKTVRFPRT IAVYSPVAAGR LAPFQDEGVNLEEK GEAVRSEGSEGGRQEGKQGDGEIKEQMKMDKTDEE EAAPAQTRPK FQFLEQK YGYYHCKDCNIRWESAYVWCVQETNKVYFKQFCRTCQKSYNPY RVEDIMCQSCKQTRCACPVKLRHVDPKRPHRQDLCGRCKGKRLSCDSTFSFKYII 157.11 158.09 175.12 1386.79 1387.77 1404.80 1358.79 1359.77 1376.80 1405.80 R 254.16 255.15 272.17 1230.68 1231.67 1248.70 1202.69 1203.67 1220.70 1249.70 P 355.21 356.19 373.22 1133.63 1134.62 1151.64 1105.64 1106.62 1123.65 1152.65 T 468.29 469.28 486.30 1032.58 1033.57 1050.59 1004.59 1005.57 1022.60 1051.60 L 582.34 583.32 600.35 919.50 920.48 937.51 891.51 892.49 909.52 938.52 N 679.39 680.37 697.40 805.46 806.44 823.47 777.46 778.45 795.47 824.48 P 793.43 794.42 811.44 708.40 709.39 726.42 680.41 681.39 698.42 727.42 N 892.50 893.48 910.51 594.36 595.35 612.37 566.37 567.35 584.38 613.38 V 1020.56 1021.54 1038.57 495.29 496.28 513.30 467.30 468.28 485.31 514.31 Q 1107.59 1108.581125.60 367.23 368.22 385.25 339.24 340.22 357.25 386.25 S 1220.68 1221.66 1238.69 280.20 281.19 298.21 252.21 253.19 270.22 299.22 L 1333.76 1334.74 1351.77 167.12 168.10 185.13 139.12 140.11 157.13 186.14 I 1404.80 1405.78 1422.81 54.03 55.02 72.04 26.04 27.02 44.05 73.05 A Yo Y* Y Bo B* B Ao A* A M+nH AA 0 100 200 300 400 500 600 700 800 900 1000 1100 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance b1-11 1151.3 y1-10 1125.7 b1-10 1050.3 y1-9 1038.3 a1-10 1022.2 b1-9 938.2 y1-8 910.6 a1-9 909.5 b1-8 823.5 y1-7 811.3 a1-8 795.5 b1-7 726.2 a1-7 698.4 b1-6 612.2 y1-5 600.7 b1-5 513.4 a1-5 485.4 b1-4 385.7 y1-3 373.2 a1-4 357.2 b1-3 298.2 y1-2 272.2 a1-3 270.2 b1-2 185.1 b1-1 72.0 a1-1 44.0
112 Figure 4Mass data analysis of a pept ide of Ploy (A) binding protein (PABP) Sequence of PABP QPADAERALDTMNFDVIKGRPVRIMWSQRDPSLRKSGVGNIFIKNLDKSIDNKALYDTFS AFGNILSCKVVCDENGSKGYGFVHFETQEAAERAIDKMNGMLLNDRKVFVGRFKSRKERE AELGARAKEFTNVYIKNFGDDMNDERLKEMFGKYGPALSVKVMTDDNGKSKGFGFVSFER HEDAQKAVDEMYGKDMNGKSMFVGRAQKKVERQTELKRKFEQMNQDRITR YQGVNLYVK N LDDGIDDERLRKEFLPFGTITSAKVMMEGGRSKGFGFVCFSSPEEATKAVTEMNGRIVAT KPLYVALAQRKEER QAHLTNQYMQR MASVRVPNPVINPYQPPPSSYFMAAIPPAQNRAAY YPPGQIAQLRPSPRWTAQGARPHPFQNMPGAIRPTAPRPPTFSTMRPASNQVPRVMSAQR VANTSTQTMGPRPTTAAAAAASAVRAVPQYKYAAGVRNQQHLNTQPQVAMQQPAVHVQGQ EPLTASMLAAAPPQEQKQMLGERLFPLIQAMHPTLAGKITGMLLEIDNSELLHMLESPES LRLKVDEAVAVLQAHQAKEAAQKVVNATGV PTA #1098-1100 RT:46.92-47.00 NL: 9.80E5 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Relative Abundance b1-8 956.3 y1-7 940.4 a1-8 928.5 y1-6 840.1 b1-7 792.9 a1-7 765.4 y1-5 725.3 b1-6 665.1 a1-6 636.6 y1-4 597.4 b1-5 551.3 a1-5 523.3 b1-4 450.2 y1-3 434.2 a1-4 422.4 157.11 158.09 175.12 1353.65 1354.63 1371.66 1325.65 1326.64 1343.66 1372.67 285.17 286.15 303.18 1197.55 1198.53 1215.56 1169.55 1170.54 1187.56 1216.57 416.21 417.19 434.22 1069.49 1070.47 1087.50 1041.49 1042.48 1059.50 1088.51 579.27 580.26 597.28 938.45 939.43 956.46 910.45 911.44 928.46 957.47 707.33 708.31 725.34 775.39 776.37 793.40 747.39 748.37 765.40 794.40 821.37 822.36 839.38 647.33 648.31 665.34 619.33 620.32 637.34 666.34 922.42 923.40 940.43 533.28 534.27 551.29 505.29 506.27 523.30 552.30 1035.50 1036.49 1053.52 432.24 433.22 450.25 404.24 405.23 422.25 451.25 1172.56 1173.55 1190.57 319.15 320.14 337.16 291.16 292.14 309.17 338.17 1243.60 1244.58 1261.61 182.09 183.08 200.10 154.10 155.08 172.11 201.11 1371.661372.64 1389.67 111.06 112.04 129.07 83.06 84.05 101.07 130.07 Yo Y* Y Bo B* B Ao A* A M+nH