Site-Specific Classification of N -Linked Oligosaccharides of the Extracellular Regions of Fcγ Receptor IIIb Expressed in Baby Hamster Kidney Cells

Human Fcγ receptor III (FcγRIII) consists of two isoforms that are encoded by two individual genes: transmembrane FcγRIIIa and glycosylphosphatidylinositol-linked FcγRIIIb. Both isoforms can exist as a soluble form (sFcγRIII), which is composed of their extracellular region produced by proteolytic cleavage. FcγRIII-mediated immunological functions such as antibody-dependent cell-mediated cytotoxicity and phagocytosis critically depend on the N-glycosylation of FcγRIII molecules. In our previous study, high-performance liquid chromatography-based profiling indicated that N-linked oligosaccharides released from the NA2 allele of human sFcγRIIIb expressed in baby hamster kidney cells are composed of high-mannose-type oligosaccharides and core-fucosylated complex-type oligosaccharides. Here we successfully classified the N-glycans of this glycoprotein into these two types at each of the six N-glycosylation sites by liquid chromatography (LC)-electrospray tandem mass spectrometry analysis combined with endoglycosidase treatments. Our results indicated that four sites of sFcγRIIIb, Asn38, Asn74, Asn162, and Asn169, expressed only complex-type oligosaccharides, while the remaining two sites, Asn45 and Asn64 (both are not conserved in the NA1 allele), were occupied by not only complextype oligosaccharides but also high-mannose-type oligosaccharides, which are thought to be involved in the interaction of FcγRIIIb with complement receptor type 3. Together with the previously reported site-specific N-glycosylation profiling of recombinant sFcγRIIIa, this study underlines that both sFcγRIIIa and sFcγRIIIb produced in different production vehicles express core-fucosylated complex-type oligosaccharides as the major glycoforms at Asn74 and Asn162. These finding provide insights into the design and development of therapeutic antibodies because the Asn162 N-glycan significantly contributes to immunoglobulin G binding.