Antibodies and their receptors are critical glycoproteins that bridge the innate and adaptive immune systems to provide protection during infection. The isotype/subclass of the antibody, and post-translational modifications like glycosylation on the CH2 domain, are the two known modifications of the Fc domain that program antibody effector function. All of the isotypes/subclasses of antibodies have distinct functions, respond to a wide range of infections and are expressed in different tissues. It has been established that addition and removal of certain glycan structures can influence the potency of antibodies, highlighting the importance of understanding antibody glycosylation. Although IgGs have a single N-glycosylation site and are very well studied, other antibody isotypes, like IgA and IgM, that are the first responders in certain diseases, bear multiple potential N-linked glycosylation sites ranging from 2-5 sites/monomer of antibody, and very little is known about their actual glycosylation. Here we employ an LC-MS/MS method to isolate, enhance and separate very small amounts of the purified glycoproteins, to identify the glycan reportoire and site occupancy of bulk serum antibodies. High throughput analysis using our in-house automated glycopeptide analysis software helped elucidate the diversity of site-specific N-linked glycan signatures after protease digestion of purified isotypes, and showed that these range from high mannose to complex bi- and triantennary glycans on the different antibody isotypes. In addition, within the same serum sample, we were also able to assess their site occupancy and determine the various glycoforms on a single site. Using this method, we have established the N-linked glycan repotoire for bulk IgG1-4, IgA1 and IgA2, and IgM in healthy patients. Characterization of bulk antibodies gives us a platform to study the impact of glycosylation on antigen- and pathogen-specific glycan profiles and glycan-mediated effector functions. This ultimately aids in defining disease-specific glycan signatures for different isotypes that help tune antibodes to induce protection.

Glycosylation impacts antibody Fc receptor function and is tuned by the immune system

256th National Meeting (2018)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.