Recognition of N-glycoforms in human chorionic gonadotropin by monoclonal antibodies and their interaction motifs [Glycobiology and Extracellular Matrices]

August 3rd, 2015 by Li, D., Zhang, P., Li, F., Chi, L., Zhu, D., Zhang, Q.,

The glycosylation of human chorionic gonadotropin (hCG) plays an important role in reproductive tumors. Detecting hCG N-glycosylation alteration may significantly improve the diagnostic accuracy and sensitivity of related cancers. However, developing an immunoassay directly against the N-linked oligosaccharides is unlikely due to the heterogeneity and low immunogenicity of carbohydrates. Here, we report a hydrogen/deuterium exchange (HDX) and MS approach to investigate the effect of N-glycosylation on the binding of antibodies against different hCG glycoforms. Hyperglycosylated hCG (HCG-H) was purified from the urine of invasive mole patients, and the structure of its N-linked oligosaccharides was confirmed to be more branched by MS. The binding kinetics of the anti-hCG antibodies MCA329 and MCA1024 against hCG and hCG-H were compared using biolayer interferometry (BLI). The binding affinity of MCA1024 changed significantly in response to the alteration of hCG N-linked oligosaccharides. HDX-MS reveals that the peptide β65-83 of the hCG beta subunit is the epitope for MCA1024. Site-specific N-glycosylation analysis suggests that N-linked oligosaccharides at Asn-13 and Asn-30 on the beta subunit affect the binding affinity of MCA1024. These results prove that some antibodies are sensitive to the structural change of N-linked oligosaccharides, while others are not affected by N-glycosylation. It is promising to improve glycoprotein biomarker-based cancer diagnostics by developing combined immunoassays that can determine the level of protein and measure the degree of N-glycosylation simultaneously.
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