Site-specific N-glycan Analysis of Antibody-binding Fc γ Receptors from Primary Human Monocytes

The composition of post-translational modifications, particularly carbohydrate chains attached to specific asparagine residue (N-glycans), that are attached to antibodies are well described in the human body and modulate the binding to Fc γ receptors. The N-glycan composition of these receptors likewise impacts antibody binding affinity, however receptor composition in the human body is unknown because of challenges associated with obtaining sufficient receptor from a single donor and limited analytical techniques. Here we characterize all N-glycosylation sites of CD16a and CD32a isolated from primary human monocytes that are isolated from a normally discarded filter following platelet and plasma donation. [Display omitted] Highlights •Monocytes are isolated from single donors after apheresis.•Monocytes process CD16a and CD32a N-glycosylation differently by site and donor.•CD16a with hybrid and oligomannose type N-glycans bind IgG1 Fc with stronger affinity than complex type. FcγRIIIa (CD16a) and FcγRIIa (CD32a) on monocytes are essential for proper effector functions including antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Indeed, therapeutic monoclonal antibodies (mAbs) that bind FcγRs with greater affinity exhibit greater efficacy. Furthermore, post-translational modification impacts antibody binding affinity, most notably the composition of the asparagine(N)-linked glycan at N162 of CD16a. CD16a is widely recognized as the key receptor for the monocyte response, however the post-translational modifications of CD16a from endogenous monocytes are not described. Here we isolated monocytes from individual donors and characterized the composition of CD16a and CD32a N-glycans from all modified sites. The composition of CD16a N-glycans varied by glycosylation site and donor. CD16a displayed primarily complex-type biantennary N-glycans at N162, however some individuals expressed CD16a V158 with ∼20% hybrid and oligomannose types which increased affinity for IgG1 Fc according to surface plasmon resonance binding analyses. The CD16a N45-glycans contain markedly less processing than other sites with >75% hybrid and oligomannose forms. N38 and N74 of CD16a both contain highly processed complex-type N-glycans with N-acetyllactosamine repeats and complex-type biantennary N-glycans dominate at N169. The composition of CD16a N-glycans isolated from monocytes included a higher proportion of oligomannose-type N-glycans at N45 and less sialylati