University of New Hampshire University of New Hampshire Scholars' Repository Doctoral Dissertations Student Scholarship Spring 2009 Modeling congenital disorders of glycosylation in Caenorhabditis elegans: Genetic influences and structural consequences of N- linked glycosylation Weston Booth Struwe University of New Hampshire, Durham Follow this and additional works at: https://scholars.unh.edu/dissertation Recommended Citation Struwe, Weston Booth, "Modeling congenital disorders of glycosylation in Caenorhabditis elegans: Genetic influences and structural consequences of N-linked glycosylation" (2009). Doctoral Dissertations. 488. https://scholars.unh.edu/dissertation/488 This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. MODELING CONGENITAL DISORDERS OF GLYCOSYLATION IN C. ELEGANS: GENETIC INFLUENCES AND STRUCTURAL CONSEQUENCES OF A/-LINKED GLYCOSYLATION BY WESTON BOOTH STRUWE B.S., UNIVERSITY OF WISCONSIN, 2002 DISSERTATION Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biochemistry May, 2009 UMI Number: 3363732 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. UMI® UMI Microform 3363732 Copyright 2009 by ProQuest LLC All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 This dissertation has been examined and approved. 'LQ_Xst*-**x_ L Dissertation Director, Vernon N. Reinhold Research Professor of Biochemistry W. Kelley Thomas, Professor of Biochemistry c*— Deena J. Small, Assistant Professor of Biochemistry David J. Asfertfne, Research Scientist Steven B. Levery, Research Associate Professor of Cellular and Molecular Medicine, University of Copenhagen 12, 2001 Date ' DEDICATION FOR MY DAD m ACKNOWLEDGEMENTS I would like to sincerely thank my advisor Vernon Reinhold for his support and guidance. I would also like to thank Andy Hanneman, David Ashline and everyone in the UNH Glycomics Center for their time, tutelage and friendship. Special thanks go out to my colleagues Will Wiswall and Justin Prien for their insightful words, discussions and collaboration in social endeavors. I would like to thank my committee members Kelley Thomas, Steve Levery and Deena Small for all their encouragement and help throughout my time at UNH. I also want to thank James Dennis and Wendy Johnston from the Mount Sinai Hospital and University of Toronto for their support. My sincerest gratitude and adoration goes to my mentor and friend Charles Warren who, through his devotion, passion and kindness, changed my life forever and showed me the beauty and astonishment of scientific research. I am grateful for the short time I spent with Charles and will never forget him. IV TABLE OF CONTENTS DEDICATION iii ACKNOWLEDGEMENTS iv LIST OF TABLES vii LIST OF FIGURES : viii. ABSTRACT xii CHAPTER PAGE 1. PERSPECTIVES OF GLYCOBIOLOGY 1 1.1 /V-linked Glycan Biosynthesis: Endoplasmic Reticulum 3 1.2 AMinked Glycan Biosynthesis: Golgi 8 1.3 Biosynthetic Complexity of /V-linked Glycans 13 1.4 A/-glycan Structure-Function Relationship 19 2. CONGENITAL DISORDERS OF GLYCOSYLATION.. 23 3. C. ELEG4NSAS A MODEL TO STUDY A/-GLYCOSYLATION 31 4. PROJECT AIMS 40 4.1 Genome-wide RNAi screen to Identify Tunicamycin Hypersensitive Loci in C. elegans 40 4.2 Comparative A/-glycan Structural Analysis of the C. elegans N2 (Bristol) and NF299 cogc-1(k179) strains 41 5. GENOME-WIDE RNAi SCREEN TO IDENTIFY TUNICAMYCIN HYPERSENSITIVE LOCI IN C. ELEGANS 43 5.1 Materials and Methods 50 5.2 Results 53 v 5.3 Discussion 63 6. COMPARATIVE A/-GLYCAN STRUCTURAL ANALYSIS OF THE C. ELEGANSN2 (BRISTOL) AND NF299 cogc-1(k179) STRAINS 71 6.1 Materials and Methods 75 6.2 Results and Dissussion 79 6.2.1 Carbohydrate Structural Analysis Through Mass Spectrometry 81 6.2.2 Native Molecular Compositional Analysis via MALDI-TOFMS 83 6.2.3 Permethylated Molecular Compositional Analysis via MALDI-TOFMS 87 6.2.4 Structural Characterization of /V-glycans via MSn 92 6.2.5 Structural Characterization of GlcNAc2Man5 93 6.2.6 Structural Characterization of GlcNAc2Man5Fuc 104 6.2.7 Structural Characterization of GlcNAc2Man5Fuc2 118 6.2.8 Structural Characterization of GlcNAc2Man5Fuc3 130 6.2.9 Structural Characterization of GlcNAc2Man5Fuc4 139 7. CONCLUSIONS 144 REFERENCES 148 APPENDICES 159 APPENDIX A TUNICAMYCIN HYPERSENSITIVE LOCI 160 APPENDIX B NATIVE AND PERMETHYLATED MALDI-TOF SPECTRA 174 APPENDIX C RELATIVE ABUNDANCE OF NATIVE A/-GLYCANS 180 APPENDIX D ADDITIONAL MSnSPECTRA 183 vi LIST OF TABLES Table 1. Common monosaccharides 2 Table 2. Congenital disorders of glycosylation by type and subtype 24-25 Table 3. Tunicamycin incudes a pleotropic condition in C. elegans 54 Table 4. RNAi of LLO genes with 2u.g/ml tunicamycin..... 57 Table 5. RNAi of "maturation" genes with 2|ig/ml tunicamycin 59 Table 6. Tunicamycin treatment does not alter RNAi effectiveness in C. elegans 60 Table 7. Relative quantities of native O-methylated A/-linked glycans 86 Table 8. Relative quantities of permethylated AMinked glycans 89 Table 9. Relative intensity of MS/MS B-ion fragments from GlcNAc2Man5Fuc 117 Table 10. Relative intensity of MS/MS B-ion fragments from GlcNAc2Man5Fuc2 129 Table 11. Relative intensity of MS/MS B-ion fragments from GlcNAc2Man5Fuc3 138 Table A1. RNAi screen results 162 Table C1. Relative abundance of native A/-glycans 182 vn LIST OF FIGURES Figure 1. Synthesis of the lipid-linked oliosaccharide precursor in the endoplasmic reticulum 4 Figure 2. The calnexin/calreticulin cycle 6 Figure 3. Common A/-linked glycan types 9 Figure 4. Processing of the precursor LLO 12 Figure 5. Anterograde vesicular transport and cisternal maturation: two models of Golgi morphology 15 Figure 6. CDG location and type 28 Figure 7. C. elegans A/-glycans 33 Figure 8. MS profiles of C. elegans A/-glycans released with PNGase Fand hydrazine 35 Figure 9. Biosynthetic glycosylation pathway in C. elegans 37 Figure 10. Lipid-linked oligosaccharide pathway in the endoplasmic reticulum 45 Figure 11. A/-glycosylation pathway from synthesis to localization 48 Figure 12. Tunicamycin induces a dose-dependent lethality in the N2 C. elegans strain 55 Figure 13. Tunicamycin effects postembryonic development among C. elegans strains 56 Figure 14. RNAi phenotype penetrance and expressivity 62 Figure 15. Eukaryotic Orthologous Gene (KOG) gene assignemtns 66 Figure 16. MALDI-TOF spectra of native A/-linked glycans (1 of 3) 84 Figure 17. MALDI-TOF spectra of permethylated A/-glycans 88 Figure 18. Prevalence of A/-glycans by subtype 90 viii Figure 19. Comparative analysis of fucosylated /V-glycans organized by subtype 91 Figure 20. GlcNAc2Man5 and the chitobiose core 94 3 Figure 21. MS of m/z 1302 from GlcNAc2Man5 (m/z 1595) 96 Figure 22. GlcNAc2Man5 Structural Isomer #2 97 4 Figure 23. MS of m/z880 ion from Man5 Isomer #2 98 Figure 24. MS5 spectra and fragmentation assignments of m/z 667 from GlcNAc2Man5 100 5 Figure 25. MS of m/z 866 from GlcNAc2Man5 (m/z 1595) 102 6 Figure 26. MS of m/z 648 from GlcNAc2Man5 (m/z 1595) 103 Figure 27. Structural Isomers of GlcNAc2Man5 in N2 and NF299 103 2 2+ Figure 28. MS of GlcNAc2Man5Fuc(m/z896 ) 105 4 2+ Figure 29. MS of m/z 880 from GlcNAc2Man5Fuc (m/z 896 ) 107 5 2+ Figure 30. MS of m/z662 from GlcNAc2Man5Fuc (m/z896 ) 109 3 2+ Figure 31. MS of m/z 694 from GlcNAc2Hex5Fuc (m/z 896 ) 110 5 2+ Figure 32. MS of m/z 866 from GlcNAc2Man5Fuc (m/z 896 ) 112 Figure 33. MS5 spectra and fragmentation assignments of m/z 667 from Man5Fuc 113 Figure 34. MS3 spectra and fragmentation assignments of m/z 1476 from GlcNAc2Man5Fuc... 114 Figure 35. Structural Isomers of GlcNAc2Man5Fuc 116 2 2+ Figure 36. MS of GlcNAc2Man5Fuc2(m/z983 ) 119 3 Figure 37. MS of m/z894from GlcNAc2Man5Fuc2 121 n Figure 38. MS of m/z 1098 from GlcNAc2Man5Fuc2 123 n Figure 39. MS of m/z 1302 from GlcNAc2Man5Fuc2 124 ix 3 Figure 40. MS of m/z1272 from GlcNAc2Man5Fuc2 126 3 Figure 41. MS of m/z 1476 from GlcNAc2Man5Fuc2 128 2 2+ Figure 42. MS of GlcNAc2Man5Fuc3 (m/z 1070 ) 131 3 Figure 43. MS of m/z 1272 from GlcNAc2Man5Fuc2.. 132 Figure 44. MS4 spectra and fragmentation assignments of m/z 667 from GlcNAc2Man5Fuc3 133 4 Figure 45. MS of m/z 1054 from GlcNAc2Man5Fuc2 (10702+-*1272^1054) 134 3 Figure 46. MS of m/z 1476 from GlcNAc2Man5Fuc3 135 3 Figure 47. MS of m/z 1446 from GlcNAc2Man5Fuc3 (10702+-^1446)inN2 136 3 Figure 48. MS of m/z 1650 from GlcNAc2Man5Fuc3 (10702+^1650)inN2 137 2 2+ Figure 49. MS of GlcNAc2Man5Fuc4 (m/z 1157 ) 140 n Figure 50. MS of m/z 1446 from GlcNAc2Man5Fuc4 140 3 Figure 51. MS of m/z 868 from GlcNAc2Man5Fuc4 141 Figure 52. MS3 of m/z 490 and m/z 690 from 11572+ 142 Figure 53. MS3 of m/z 894 from 11572+ 143 Figure B1. MALDI-TOF spectra of native AMinked glycans released by hydrazinolysis (2 of 3) 175 Figure B2. MALDI-TOF spectra of native AMinked glycans released by hydrazinolysis (3 of 3) 176 Figure B3.