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Title: The role of N-linked oligosaccharide structure in regulation of immunoglobulin secretion and function
Author: Hashim, Onn Haji
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1987
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The functions of carbohydrate moieties of glycoproteins are subject to much controversy. Their role in the regulation of intracellular glycoprotein transport is obscured by the discovery of distinctive effects that result from treatment either with inhibitors of N-glycosylation or intracellular N-linked oligosaccharide processing. It is therefore the purpose of this study to ratify and to broaden the perspective in this area of ambiguity. The experimental approach used in the investigation generally involves biosynthetic labelling of rat hybridoma cells with various radioactive precursor and an isolation of intracellular and/or extracellular immunoglobulins by immunoprecipitation technique. Through properly-timed, pulse-chase methods, it was possible to determine the effects of various inhibitors of glycosylation or oligosaccharide processing on the kinetics of immunoglobulin export. The isolated immunoglobulins were also subjected to other analyses to identify and to confirm the structures of carbohydrate molecules that were incorporated, as well as to assess the effects of inhibition of N-glycosylation or N-linked oligosaccharide processing on the biological functions of immunoglobulins. The first part of this study involves characterization of three structural analogues of tunicamycin, TM-1, TM-2 and TM-3. Results from this investigation demonstrate that minor modifications to the structure of the antibiotic results in the loss of its biological activity (i.e., with respect to inhibition of N-glycosylation). The analogues do not inhibit N-glycosylation of immunoglobulins and have no effects on the kinetics of IgM and IgG2b secretion from rat hybridomas. The data, therefore, suggest that the selective inhibition of immunoglobulin secretion that is observed when cells are treated with tunicamycin is really due to the absence of carbohydrate moieties from the immune molecules and not a direct effect of the antibiotic. The data also demonstrate, that unlike normal tunicamycin, the three structural analogues are not cytotoxic. Their presence has no pronounced effect on the cellular uptake of tritiated-thymidine, -uridine and -leucine. This suggests that the well-recognized cytotoxic effects of tunicamycin are also a secondary effect due to the action of the drug on other cellular functions. In addition, enzymatic analysis of isolated immunoglobulins produced and secreted in the presence of the three tunicamycin analogues demonstrates that the drugs have no effect on the N-acetylglucosaminyl transferase I and II activities located in the Golgi apparatus. Susceptibility to endo H digestion is only observed in immunoglobulins isolated from within the cells. Immunoglobulins secreted in the presence of the analogues of tunicamycin display complete resistance to the enzyme. To provide a wider perspective on the current understanding of the role of N-linked carbohydrate processing in the regulation of glycoprotein transport, a study of the effects of four specific N-linked oligosaccharide processing inhibitors (i.e., CSP, dNM, dMM and SW) on the secretion of IgM and IgG2b from rat hybridoma lines was carried out. The data clearly demonstrate that inhibition of the processing of N-linked oligosaccharide at specific stages of the pathway does not lead to any significant interference with the rate of IgM and IgG2b secretion. Resolution of the reduced immunoglobulin components secreted from cells that were treated with the individual processing inhibitors on SDS-PAGE demonstrate distinctive heavy chain structures. While no apparent difference could be seen from the mu- and gamma-heavy chains that were secreted in the presence of the mannosidase Ia/b and II inhibitors (i.e., dMM and SW, respectively), treatment with glucosidase inhibitors (i.e., CSP and dNM) results in the production of mu- and gamma-heavy chains with higher Mr. Experiments were also performed by using mixtures of the carbohydrate processing inhibitors. Treatment of I1A 1.4 and 4A3 cells with selective pairs of glucosidase and mannosidase inhibitors (i.e., CSP/SW and dNM/dMM) or with all the four processing inhibitors simultaneously, also demonstrate no significant changes of the rate of immunoglobulin export. The mu- and gamma-heavy chains that were isolated in all of these cases, however, demonstrate the higher-type Mr structures. The oligosaccharide chains of IgG2b from I1A1.4 cells, under normal circumstances, although bearing the complex-type structures, do not possess terminally-linked sialic acid residues. By using neuraminidase digestion analysis, it was shown that when the processing of the carbohydrate moieties was inhibited by any of the four processing inhibitors, individually, or with all of them simultaneously, g-heavy chains become susceptible to the enzyme treatment, suggesting the presence of terminally-linked sialic acid residues. This effect may either be a result of a direct activation of intracellular sialyl transferase or due to other processing resulting in carbohydrate configurations which can act as substrates for the transferase. N-linked carbohydrate moieties of immunoglobulins have been strongly implicated to be involved in the Clq-binding interaction. Their absence from immunoglobulins have rendered the molecules less effective in activating the complement cascade. The data from experiments performed in this study are also compatible with this interpretation. A similar result was obtained when tunicamycin-treated non-N-glycosylated IgG2b from I1A 1.4 cells was subjected to complement fixation assay. In the case of immunoglobulins with high mannose structures that were secreted in the presence of oligosaccharide processing inhibitors, the data indicate otherwise. These immunoglobulins apparently possess potentiated capability in fixing complement. Complement fixation assays were also performed on normal IgG2b in the presence of free mannose, galactose and N-acetylglucosamine at 5mM concentration. The monosaccharides have no effect on the complement fixation activity. The effects of inhibition of N-glycosylation or oligosaccharide processing on Fc-binding interactions, which may involve carbohydrate moieties, have also been studied. The data indicate that neither the inhibition of N-glycosylation nor carbohydrate processing have any consequence on antibody-dependent haemagglutination by IgG2b from I1A1.4 cells. In addition, inhibition of N-glycosylation or the processing of carbohydrate moieties of IgG2b was also shown to have no effect on antigen binding capacity of the immunoglobulins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available