Site-specific N-glycosylation analysis of bovine immunoglobulin M and its secretory form

Bovine immunoglobulin M (IgM) from serum and colostrum has got considerable attention due to its immunological property against certain human pathogens. N-glycosylation being a key structural modification of IgM wherein glycan diversity exhibits significant role in maintaining its structural integrity and also bio functionalities; glycan heterogeneity in bovine serum and colostrum IgM complexes have not been explored till date. In this study, we have evaluated site-specific glycan heterogeneity of both commercially available bovine serum IgM as well as secretory IgM (sIgM) complex that was purified from bovine colostrum through the generation of N-glycopeptides followed by UHPLC-MS/MS analysis. N-glycosylation sites- N91ST, N260VS, and N307IS in μ heavy chain constant region (IGHM) displayed bi- and tri-antennary complex glycans with varying degree of core fucosylation and sialyation. While N329DT contained mainly oligomannose as well as hybrid glycans, only oligomannose types were present at N490VS. Both N307IS and N490VS indicated differential glycosylation. Glycans of joining chain (JC) were of bi-antennary complex glycans in both serum and colostrum IgM complexes. Glycans processing at individual sites of IGHM and JC were found in part similar to human counterparts yet bovine-specific glycan heterogeneity was observed. Secretory component (SC) of colostrum sIgM revealed mainly LacdiNAc containing bi-antennary complex glycans that differed substantially from human SC containing only LacNAc complex types with both core and terminal fucsoylation. Interestingly, CD5L was found associated with both serum and colostrum IgM complexes. Thus, the insights obtained in the study would form the basis for extended applications of bovine IgM complexes.