Fc gamma receptors (FcγRs) govern key functions of the human immune system. For example, triggering of FcγRIIIb on neutrophils by immunoglobulin G (IgG) induces phagocytosis and degranulation. Allelic polymorphisms of FcγRIIIb have been associated with autoimmune diseases. The influence of antibody glycosylation on FcγR binding is well established and led to therapeutic advances. Glycosylation of the FcγR itself also influences these interactions as it is essential for the 50-fold increased affinity of afucosylated IgG towards FcγRIIIa. Despite these crucial roles, FcγR glycosylation in humans is largely uncharacterized.
We are mapping the glycosylation of human FcγRs as a basis for studying its impact on IgG effector functions and its involvement in autoimmune diseases. Here, we present the glycosylation of FcγRIIIb obtained from the neutrophils of healthy donors. Our study represents one of the first site-specific glycosylation studies of a cellular FcγR and covers more glycosylation sites than recent reports .
Neutrophils were extracted from healthy donor plasma by density gradient centrifugation. FcγRIIIb was then purified by immunoprecipitation and subjected to in-gel glycoproteomics analysis. Proteolytic cleavage by chymotrypsin and endoproteinase GluC yielded separate glycopeptides for each theoretical glycosylation site. These were analysed by liquid chromatography – mass spectrometry (LC–MS) using an orbitrap Fusion Lumos MS. MS/MS spectra were obtained by high-energy collision induced dissociation at three energy levels and assigned by combining software tools and manual interpretation. All identified FcγR glycopeptide compositions were quantified on the MS level using LaCyTools.
The FcγRIIIb glycosylation appears to be quite different at the different glycosylation sites. For example, the presence of high mannose type (HM) glycans could be confirmed for FcγRIIIb and was attributed mainly to site N45. In contrast, N162 and N169 showed mainly di- and triantennary complex glycans. In the future, it will be interesting to also look at functional consequences of altered glycosylation at specific sites and its role in specific (patho)physiological conditions.
- Washburn N, et al. (17th Dec 2018) “Characterization of endogenous human FcγRIII by mass spectrometry reveals site, allele and sequence specific glycosylation” Mol Cell Proteomics papers in press