Characterisation of human B-lymphocyte specific antigens and receptor immunoglobulins

Considerable attention has recently been focused on the mechanisms which allow B-lymphocytes to effect simultaneous biosynthesis and expression of receptor and effector forms of the immunoglobulin molecule as mandated by the Clonal Selection Hypothesis. The genetic mechanisms responsible for the above phenomenon have now been established for the biosynthesis of IgM in murine lymphocytes. Also of great interest is the possible regulatory influence exerted by carbohydrate moieties on the secretion and membrane deposition of immunoglobulin molecules. The objectives of the work described in this thesis were to attempt to ascertain whether or not human B-lymphocytes expressing IgG, IgA and IgE adopted similar genetic strategies to allow simultaneous expression of structurally distinct membrane and secretory heavy chain polypeptides. The role of N-linked oligosaccharides in secretion and membrane deposition of immunoglobulin molecules was also studied and compared with data from similar investigations on the biosynthesis of human transplantation antigens in the hope of revealing possible carbohydrate-influenced structural signals necessary for transport and expression of immunoglobulins. The antibiotic tunicamycin was used to inhibit N-glycosy1ation of proteins in cells actively engaged in protein biosynthesis. SDS-PAGE analysis of specifically immunoprecipitated immunoglobulins of different classes synthesised in cells treated with tunicamycin revealed that four-chain immunoglobulin molecules were readily formed in all cells. It is concluded that immunoglobulin assembly occurs independently of N-glycosylation of the newly-synthesised immunoglobulin chains. HLA-A,-B,-C/beta2 microglobulin complexes were also efficiently assembled in the presence of tunicamycin. Evidence is presented to support the hypothesis that 19S IgM and 11S IgA polymers are formed in cells treated with tunicamycin. No conclusive evidence was obtained with respect to incorporation of J-chain into nonglycosylated polymeric immunoglobulins. The presence of N-linked oligosaccharides on the immunoglobulin heavy chain appeared to be mandatory for the efficient secretion of IgM and IgA from human cells synthesising these classes of immunoglobulin. By contrast non-N-glycosylated IgG and IgE molecules were efficiently secreted from IgG and IgE producing human B-lymphocytes. Three experimental approaches were adopted to elucidate the functional role of N-linked carbohydrate side chains of immunoglobulin molecules in the efficient membrane deposition of these molecules. In one case it was found that non-N-glycosylated IgM could be demonstrated at the cell membrane of tunicamycin-treated cells: this data was shown to be incorrect by more rigorous experimental analyses. It is concluded that N-glycosylation of immunoglobulins is necessary for efficient membrane deposition of IgM and IgA. In contrast, HLA-A,-B and -C alloantigens and also HLA-DR alloantigens were detected at the membrane of tunicamycin-treated cells and, in the case of HLA-A2 and HLA-B7, the non-glycosylated molecules were alloantigenic as judged by their serological reactivity with alloantisera. Biosynthesis of membrane and secretory forms ofy -heavy chain polypeptides was studied in the B-lymphoblastoid cell line Bec-11. Normal Bec-11 cells synthesised three intracellular forms of Y-Chain of AMWs 64K, 55K and 52K and two of these chains are secreted into the culture supernatant (52K and 55K). The 64K species was presumed to be the membrane form of y-heavy chain and this hypothesis was supported by the finding that IgG isolated from aliquots of radioiodinated Bec-11 membrane proteins contained y-heavy chains of AMW 64K. (Abstract shortened by ProQuest.)