CALIFORNIA STATE UNIVERSITY, NORTHRIDGE PRODUCTION OF SIALIC ACID AFFECTED BY GNE GENE MUTATIONS A thesis submitted in partial fulfillment of the requirements For the degree of Master of Science In Biology By Atefeh Rajaei August, 2012 © 2012 Atefeh Rajaei ALL RIGHTS RESERVED ii The thesis of Atefeh Rajaei is approved: Dr. Aida Metzenberg Date Dr. Daniel Darvish Date Dr. Yadira Valles-Ayoub Date Dr. Stan Metzenberg, Chair Date California State University, Northridge iii Acknowledgements Though it is ceremonious to write an acknowledgment, this is not merely to formalize the custom but to express my cordial gratitude to all those who have directly or indirectly helped me through the course of this work as it is the fruit of the outcome of a set of seemingly unrelated yet intertwined and dependent factors that by His grace worked in my favour. Any accomplishment requires the efforts of many people and this work of Thesis project is no different. I have been fortunate enough to get the help and guidance from many people. It is a pleasure to acknowledge them, though it is still inadequate appreciation for their contribution. I shall forever be highly grateful to Dr. Stan Metzenberg. He unravelled each and every riddle in my project with his prodigious knowledge. I would also like to express my sincere gratitude towards Dr. Aida Metzenberg for extending her support throughout my project and in widening my knowledge. I express my sincerest gratitude and thanks to Dr. Yadira Valles. Under her brilliant untiring guidance I have completed the project successfully on time. Dr. Daniel Darvish who has helped me in simplifying the problem involved in the work. His meticulous attention and invaluable suggestions are admirable. I also convey my cordial thanks to Zeshan Khokher, Rosangela Carbajo and other members of HIBM research group for their support and help. Special mention of Ishita Shah, Bansari Shah, and other members of Dr. Stan Metzenberg’s and Dr. Aida Metzenberg’s lab for their constant enthusiastic encouragement and valuable suggestions without which this would not have been successfully completed. iv TABLE OF CONTENTS Copyright page ii Signature page iii Acknowledgements iv List of Figure viii List of Tables ix Abstract x CHAPTER 1: INTRODUCTION Distal Myopathy Overview and History 1 Clinical Description of HIBM 3 Diagnosis of HIBM 3 Molecular Basis of HIBM 4 Structure of GNE enzyme 6 Sialic acid 9 Sialuria 19 GNE Mutations Examined in this Study 20 Purpose of the Study 21 Hypothesis 21 CHAPTER 2: MATERIALS AND METHODS Materials 22 Sample: GNE/Topoblunt II plasmid 22 Site Directed Mutagenesis 23 Generation of the Mutated Gene 26 v Agarose Gel Electrophoresis 28 Gel Extraction 28 Digestion of GNE DNA insert after PCR 29 Digestion of Vector 30 Ligation 31 Transformation 31 Clone Confirmation 32 Sequencing of GNE gene 33 Plasmid DNA Isolation 33 Cell Culture 34 CHO Cells Transfection 35 Cell Viability Assay 35 DNA Extraction Assay 36 PCR for CMV Promoter Detection 36 RNA Extraction Assay 37 Reverse Transcription PCR 38 Protein Extraction Assay 39 Sialic Acid Assay 40 CHAPTER 3: RESULTS Site Directed Mutagenesis of the GNE Gene 41 Sequencing of GNE Gene 44 CHO Cells Transfection 46 Cell Viability Assay 47 vi CMV Promoter Presence in Cells Post-Transfection 48 Confirmation of Transcription of GNE Post-Transfection 49 Products of GNE Enzyme Post-Transfection 50 CHAPTER 4: DISCUSSION Expression of Mutated GNE Genes in CHO Cells 52 Significance of the Study 54 Future Directions 55 REFERENCES 57 APPENDIX Appendix I: Homosapiens GNE gene sequence cDNA (variant I) 64 Appendix II: Homosapiens GNE gene sequence cDNA(variant II) 66 Appendix III: Homosapiens GNE protein sequence isoform1 68 Appendix IV: Homosapiens GNE protein sequence isoform 2 69 vii LIST OF FIGURES Figure 1: Diagram of Human Chromosome 9 4 Figure 2: Secondary structure of GNE enzyme 7 Figure 3: Tertiary structure of the active site of epimerase domain of GNE/MNK enzyme 8 Figure 4: The N-acetylmannosamine kinase (MNK) domain of the human GNE/MNK enzyme 9 Figure 5: Schematic diagram of the sialic acid biosynthetic pathway 10 Figure 6: pUMVC3 Mammalian expression plasmid 30 Figure 7: Two segments of mutated GNE gene amplified 42 Figure 8: Amplified GNE gene with N-Term and C-Term primers 43 Figure 9: Screening of supercoiled DNA sizes for clone Candidates 44 Figure 10: Electropherograms of mutated region in each GNE constructs 44 Figure 11: CHO cells 0 hour post-transfection 46 Figure 12: CHO cells 48 hours post-transfection 47 Figure 13: CHO Cell Viability Post-Transfection 48 Figure 14: CMV Promoter Detection 49 Figure 15: Detection of transcription of GNE gene 50 Figure 17: Bar graph of sialic acid amount produced in CHO cells 51 viii LIST OF TABLES Table 1: Reported patients from different origins with HIBM phenotype 12 Table 2: Reported patients with GNE gene mutations 17 Table 3: Primers for amplification of GNE D176V segments 23 Table 4: Primers for amplification of GNE R263L segments 24 Table 5: Primers for amplification of GNE M265T segments 24 Table 6: Primers for amplification of GNE V572L segments 25 Table 7: PCR reaction mix for GNE gene amplification 25 Table 8: PCR conditions, temperature and time for each step 26 Table 9: Primers for amplification of GNE gene 26 Table 10: PCR reaction mix for GNE gene amplification 27 Table 11: PCR conditions, temperature and time for each step 27 Table 12: Restriction enzyme digestion reaction mix 29 Table 13: Ligation reaction mix 31 Table 14: Primers used for sequencing 33 Table 15: PCR reaction mix for CMV promoter presence 36 Table 16: PCR conditions for CMV promoter presence 37 Table 17: RT PCR reagent mix 38 Table 18: RT PCR conditions, temperatures, times and cycles 39 ix Abstract Production of Sialic Acid Affected by GNE Gene Mutations. By Atefeh Rajaei Masters of Science Biology Hereditary Inclusion Body Myopathy (HIBM) is an autosomal recessive disorder characterized by adult onset muscle-wasting, affecting both proximal and distal muscles. HIBM is caused by different mutations in the GNE gene including the common Middle Eastern founder allele, p.M712T. The GNE gene is located on chromosome 9p13.3 of humans. The GNE gene encodes a bifunctional rate limiting enzyme UDP N-acetyl glucosamine 2-epimerase/N-acetyl mannosamine kinase (UDP-GNE/MNK). This enzyme catalyzes the first two steps in the sialic acid biosynthetic pathway. Mutations in the GNE gene may lead to, either decreased sialic acid biosynthesis and reduced sialylation of a variety of proteins like alpha-dystroglycan, Neprilysin and NCAM, (Huizing et al., Broccolini et al., Ricci et al.,) or to increased sialic acid production. Glycosylation defects have recently become recognized as an important cause of muscular dystrophy (Muntoni et al.). The functional capacity of sialic acid production can be restored by introduction and expression of the wild-type GNE gene. In order to demonstrate this, I used lectin resistant Chinese Hamster Ovary (CHO) cells (Lec3), which lack GNE/MNK activity. The CHO cells were transfected with pUMVC3-GNE recombinant constructs expressing either a wild-type GNE, D176V, R263L, M265T, V572L, M712T or R266Q mutant insert. CHO cells transfected with the R263L and R266Q GNE expression plasmid had an increase in sialic acid production. Those transfected with the D176V, M265T, V572L, and M712T GNE expression plasmid x showed significantly lower amounts of sialic acid production. We intend to use these data to construct a model for gene therapy in mice and investigate its safety and effectiveness in the alleviation of muscle degeneration manifested in HIBM. xi Chapter 1: Introduction Distal Myopathy or Distal Muscular Dystrophy is a type of disease that affects the muscles of the extremities such as the hands, feet, lower arms, or lower legs. It is challenging to determine the cause of this dystrophy in each patient because it can be a mutation in any of several genes, all of which are not yet known. These mutations can be inherited from one parent, autosomal dominant, or from both parents, autosomal recessive. Distal Myopathy Overview and History: The first case of a distal myopathy was reported by Growers in 1902. In 1951 Welander reported a large group of patients in Sweden with a hereditary form of distal myopathy which was a late onset familial form, with an autosomal dominant inheritance pattern (Welander L, 1951). Through this landmark publication, this group of disorders became firmly established. Other forms of myopathies, inherited dominantly, recessively, or with a mitochondrial pattern of inheritance, have since been recognized and reported from different populations. Inclusion-Body Myopathy (IBM) is a term used by Yunis and Samaha in 1971 for a slowly progressive myopathy that clinically mimicked a chronic polymyositis (Kagen L.J., 2009). The first case of Inclusion Body Myositis was reported by Chou in 1967. Light and electron microscopic pathological observations were reported by him (Chou S.M., 1967). In 1971, a patient was reported with Hereditary Inclusion Body Myopathy (HIBM) due to mutations in a gene on chromosome 9 (Yunis et al. 1971). This patient had onset of leg weakening at the age of 18. 1 The most common form of HIBM was first described in 1984 by Professor Zohar Argov from the Department of Neurology of the Hebrew University-Hadassah Medical School in Jerusalem. HIBM was first recognized in individuals of Iranian-Jewish decent, and nearly 104 affected individuals from 47 Middle Eastern families were found to have the same mutation in a homozygous state in the gene that encodes the enzyme (UDP-N-acetyl) glucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) (Eisenberg et al., 2001). Affected individuals in families of other ethnic origins were found to be compound heterozygotes for other distinct mutations in the GNE gene.