The role of constant and variable region glycosylation in assembly and secretion of antibody.

摘要

Human immunoglobulins are glycoproteins, with N-linked glycans attached to constant and sometimes variable (V) regions of all isotypes, and O-linked glycans attached to the hinge regions of IgA1 and IgD. These two isotypes were used to analyze the effects of glycosylation on antibody folding and secretion. IgD lacking all three N-glycans is not secreted. To analyze the contribution of each individual glycan, point mutations in the delta gene that eliminated each carbohydrate singly and in combination were made. Results showed that only the hinge-proximal carbohydrate was critical for assembly and secretion. In its absence, heavy chains were withheld inside the endoplasmic reticulum (ER), as determined by immunofluorescent staining and confocal microscopy. Using a drug that inhibits elongation of O-linked glycan chains, we found that both IgD and IgA1 containing truncated hinge glycans were assembled and secreted.; We studied V region glycosylation, focusing on a glycan in complementarity-determining region 2 (CDR2) of an anti-dextran IgG antibody. The glycan is noteworthy because it is high mannose, despite being located on an exposed protein loop. We made alterations in the overall structure of the antibody in order to determine whether processing of this carbohydrate would change. It remained high mannose in all cases. However, when CDR2 was grafted onto another V region, the new, hybrid antibody was not secreted. It appeared to misfold in the presence of the carbohydrate and was retained in the ER.; Finally, we analyzed the role of N- and O-linked oligosaccharides in providing protection from or susceptibility to bacterial enzymes known to cleave IgA1 in the hinge. By inhibiting N-glycosylation or complete O-glycosylation, we found that for two enzymes, there was no difference between cleavage rates of any glycoform of IgA1 and for another, the presence or absence of N-glycans changed the rate of hydrolysis, but O-glycans made no difference.; Carbohydrates on glycoproteins often play a critical role in expression, structure and function. Studies into the exact nature of these roles and the factors controlling carbohydrate structure are critical towards understanding how to better design recombinant proteins for diagnostic, analytical and therapeutic use.