期刊
JOURNAL OF PROTEOME RESEARCH
卷 10, 期 7, 页码 3031-3039出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr1012653
关键词
Fc gamma RIIIa Asn-162; N-linked Fc oligosaccharides; HEK; CHO; LC-ESI; tandem mass spectrometry; GalNAc; PC-293 determinant; recombinant glycoprotein; surface plasmon resonance
Human leukocyte receptor IIIa (hFc gamma RIIIa) 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 hFc gamma RIIIa 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 hFc gamma RIIIa 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 hFc gamma RIIIa 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 hFc gamma RIIIa, 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 hFc gamma RIIIa influences its binding behavior to immunoglobulin molecules.
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