title: Cell type-specific and site directed N-glycosylation pattern of FcγRIIIa.
abstract: Human leukocyte receptor IIIa (hFcγRIIIa) plays a prominent role in the elimination of tumor cells by antibody-based cancer therapies. In previous studies, a major impact of the presence of carbohydrates at Asn-162 on the binding between the receptor and the Fc part of wild type fucosylated or glycoengineered nonfucosylated antibodies has been shown. In this study, we performed a site directed carbohydrate analysis at hFcγRIIIa derived from human embryonic kidney (HEK) and Chinese hamster ovary (CHO) cells, respectively. Using mass spectrometry (MS) and a multienzyme protein digest, we analyzed the proteolysis-generated glycopeptides in detail. We could show that hFcγRIIIa expressed by HEK cells was mostly bearing multifucosylated biantennary Asn162-glycans with a major fraction terminating with GalNAc residues replacing the more common Gal. We could demonstrate that the glycan antennae with terminal GalNAc could be sialylated as indicated by a novel reporter ion HexNAcHexNAcNeuAc(+) (m/z 698.28) using a source induced dissociation (SID) scan in the MS cycle. In contrast to the hFcγRIIIa Asn-162 glycosylation pattern from HEK cells, the CHO cells derived receptor contains bi- and triantennary galactosylated and highly sialylated carbohydrates. Our data suggest that the type of expression host system was a dominating factor for formation of distinct glycopatterns of hFcγRIIIa, while the protein sequence and the site of glycosylation remained unchanged for both types of cells. Using surface plasmon resonance (SPR) interaction analysis, we show that the cell type and site specific glycosylation pattern of hFcγRIIIa influences its binding behavior to immunoglobulin molecules.
authors.affiliation: Proteinanalytics, Biologics Research (pRED), Roche Diagnostics GmbH, Penzberg, Germany. anne.zeck@nmi.de
authors.forename: Anne
authors.initials: A
authors.lastname: Zeck
authors.forename: Gottfried
authors.initials: G
authors.lastname: Pohlentz
authors.forename: Tilman
authors.initials: T
authors.lastname: Schlothauer
authors.forename: Jasna
authors.initials: J
authors.lastname: Peter-Katalinić
authors.forename: Jörg Thomas
authors.initials: JT
authors.lastname: Regula
mesh_terms: D000116:Acetylgalactosamine / Q000276:immunology / Q000378:metabolism; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D001665:Binding Sites / Q000276:immunology*; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D003412:Cricetulus; D005643:Fucose / Q000276:immunology / Q000378:metabolism; D006020:Glycopeptides / Q000737:chemistry / Q000276:immunology; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism*; D007962:Leukocytes / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding / Q000276:immunology*; D017452:Receptors, IgG / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D020349:Surface Plasmon Resonance; D053719:Tandem Mass Spectrometry; D014162:Transfection
chemical_list: C496124:FCGR3A protein, human; D006020:Glycopeptides; D007074:Immunoglobulin G; D011134:Polysaccharides; D017452:Receptors, IgG; D011994:Recombinant Proteins; D005643:Fucose; D001216:Asparagine; D000116:Acetylgalactosamine