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Proquest Dissertations structure and expression of the fi1,4-galactosyltransferase gene in reiation to igG giycosyiation by Parvaneh AiipourJeddi Department of Immunology University College London Medical School A thesis submitted for the degree of Doctor of Philosophy University of London 1996 ProQuest Number: 10046107 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10046107 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT Rheumatoid arthritis (RA) is associated with an increase in the level of serum IgG glycoforms lacking terminal galactose residues (i.e., agalactosyl IgG). The agalactosyl IgG shows altered effector functions and there is evidence that it may be pathogenic. Furthermore, levels of agalactosyl IgG have been shown to have a predictive value in RA. There is evidence that the decreased galactosylation of IgG occurs as a pre-secretory event and there are several reports relating this defect to aberrant control of the enzyme p1,4- galactosyltransferase (pi,4-GalTase). This project aimed to examine the structure and expression of the pi,4-GalTase gene in human RA and also in a murine model of arthritis, MRUMp-lpr/lpr (MRL Ipr/lpr), which shows the same defect in IgG galactosylation. No gross structural alteration of the gene was observed in human RA nor in the MRL Ipr/lpr mice, using restriction fragment length polymorphism analysis. An RNase protection assay established that there are similar levels of pi ,4- GalTase gene expression in 0019"^ cells isolated from peripheral blood of RA patients and normal healthy individuals. IgG-expressing lymphocytes isolated from spleens and lymph nodes of MRL Ipr/lpr and CBA/Ca (which exhibit normally galactosylated IgG) mice also showed comparable levels of pi,4- GalTase mRNA. The known pregnancy associated increase in IgG galactosylation was examined in the Balb/c mice. Although the p1,4-GalTase transcription was highly upregulated in the mammary gland in the third trimester of pregnancy and into lactation, no changes in the mRNA and enzyme levels were observed in the lymphocytes isolated from spleens of these mice. The cytokines IL -6 and TNF-a are proposed as giycosyiation regulating factors. In addition, IL -6 has been shown to be associated with increased agalactosyl IgG. Therefore, the level of p1,4-GalTase gene expression was measured in IL -6 and TNF-a transgenic mice in relation to the IgG galactosylation level. In these studies, comparable levels of pi,4-GalTase mRNA were observed in the transgenics and their littermates in both cases. Peripheral blood lymphocytes stimulated in vitro with the mitogens PHA, phorbol ester and pokeweed, with the cytokines IL -6 and TNF-a, with the calcium ionophore ionomycin and with the cAMP-inducer forskolin, did not show altered levels of pi,4-GalTase mRNA. However, the addition of prolactin to peripheral blood B cells cultured in the presence of anti-IgM plus IL-2 resulted in a small increase in mRNA levels but with no concomitant increase in IgG galactose. In conclusion, these studies indicate that IgG galactosylation is not regulated at the level of p1,4-GalTase gene expression. CONTENTS ABSTRACT...............................................................................................2 TABLE OF CONTENTS........................................................................... 4 LIST OF FIGURES................................................................................... 12 LIST OF TABLES..................................................................................... 16 ABBREVIATIONS.................................................................................... 17 ACKNOWLEDGMENTS........................................................................... 21 REFERENCES......................................................................................... 210 PUBLICATIONS....................................................................................... 244 TABLE OF CONTENTS CHAPTER 1: GENERAL INTRODUCTION 1.1 RHEUMATOiD ARTHRITiS AND IgG................................................23 1.1.1 Rheumatoid arthritis ..............................................................23 1.1.1.1 Human rheumatoid arthritis ......................................................23 1.1.1.2 Animal models of arthritis .........................................................26 a) Experimentally-induced arthritis ...............................................27 b) Spontaneous arthritis ...............................................................27 1.1.2 immunogiobuiins and rheumatoid arthritis ........................ 29 1.2 igG STRUCTURE AND GLYCOSYLATiON.......................................31 1.2.1 igG structure ........................................................................... 31 1.2.2 Biochemistry of giycosyiation..............................................34 1.2.2.1 Classification of protein-linked oligosaccharides..................... 34 1.2.2.2 N-linked oligosaccharides........................................................34 1.2.2.3 Regulation of oligosaccharide synthesis................................. 36 1.2.2.4 Biological roles of oligosaccharides..........................................37 1.2.3 Abnormalities in igG oiigosaccharides ............................... 40 1.2.3.1 Agalactosyl IgG ........................................................................ 40 a) Human agalactosyl IgG ............................................................40 b) Murine agalactosyl IgG ............................................................43 1.2.3.2 Other changes in IgG carbohydrates .......................................44 1.2.4 The effect of igG giycosyiation changes on igG function ................................................................................... 45 1.3 pi,4-GALTASE ENZYME ...................................................................48 1.3.1 General enzymoiogy and background ................................ 48 1.3.2 pi,4-GaiTase activity in RA ...................................................57 1.3.2.1 Human RA ................................................................................ 57 1.3.2.2 Murine models of arthritis .........................................................57 1.3.3 p1,4-GaiTase associated protein kinase ............................. 58 1.4 GENETIC DISORDERS AFFECTING GLYCOSYLATiON ................ 59 1.4.1 Lysosomal storage diseases ................................................59 1.4.2 Tn syndrome ........................................................................... 60 1.4.3 HEM PAS .................................................................................. 60 1.4.4 Carbohydrate-deficient glycoprotein syndromes .............. 61 1.4.5 Tumours .................................................................................. 62 1.4.6 immunodeficiency ..................................................................63 1.4. 7 Thyroid autoimmunity...........................................................63 CHAPTER 2: MATERIALS AND METHODS 2.1 CLONING IN PLASMID VECTORS.................................................. 66 2.1.1 Déphosphorylation of plasmid DNA.................................... 66 2.1.2 Ligation and transformation.................................................67 2.1.3 Screening colonies by hybridisation .................................. 68 2.1.4 Preparation of competent cells .............................................68 2.1.5 Growth In liquid media ..........................................................69 2.1.6 Mlnlpreps of plasmid DNA ....................................................69 2.1.7 Maxi preps of plasmid DNA................................................... 70 2.1.8 DNA purification using low melting temperature agarose gel .............................................................................................71 2.2 ISOLATION AND ANALYSIS OF DNA..............................................71 2.2.1 Isolation of genomic DNA .....................................................71 2.2.1.1 Source of human DNA .............................................................71 2.2.1.2 Source of murine DNA .............................................................72 2.2.1.3 Extraction of DNA ..................................................................... 72 2.2.2 Southern blotting assays......................................................73 2.2.2.1 Restriction enzyme digestion ...................................................73 2.2.2 2 Agarose gel electrophoresis ....................................................73 2.2.2 3 Transfer of DNA onto nylon membrane ................................... 74 2.2.2.4 Hybridisation and washing the membrane .............................. 74 2.2.3 Isolation of cDNA probes ......................................................75 2.2.3.1 Murine pi,4-GalTase ...............................................................75 2.2.3 2 Murine ..........................................................................
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