Abstract
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 because of the heterogeneity and low immunogenicity of carbohydrates. Here, we report a hydrogen/deuterium exchange and MS approach to investigate the effect of N-glycosylation on the binding of antibodies against different hCG glycoforms. Hyperglycosylated hCG 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 hyperglycosylated hCG were compared using biolayer interferometry. The binding affinity of MCA1024 changed significantly in response to the alteration of hCG N-linked oligosaccharides. Hydrogen/deuterium exchange-MS reveals that the peptide β65-83 of the hCG β subunit is the epitope for MCA1024. Site-specific N-glycosylation analysis suggests that N-linked oligosaccharides at Asn-13 and Asn-30 on the β subunit affect the binding affinity of MCA1024. These results prove that some antibodies are sensitive to the structural change of N-linked oligosaccharides, whereas 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.
Highlights
The N-linked oligosaccharides of human chorionic gonadotropin become more complicated in cancer
Hydrogen/deuterium exchange-MS reveals that the peptide 65– 83 of the human chorionic gonadotropin (hCG)  subunit is the epitope for MCA1024
We demonstrated that the binding affinity of mAb MCA1024 to hCG was significantly affected by the N-glycosylation patterns, whereas mAb MCA329 exhibited no obvious changes in binding affinity to hCG with different N-linked oligosaccharides
Summary
The N-linked oligosaccharides of human chorionic gonadotropin become more complicated in cancer. Site-specific N-glycosylation analysis suggests that N-linked oligosaccharides at Asn-13 and Asn-30 on the  subunit affect the binding affinity of MCA1024. These results prove that some antibodies are sensitive to the structural change of N-linked oligosaccharides, whereas others are not affected by N-glycosylation. Extensive efforts have been made to develop mAbs that can distinguish the level of hCG-H from the total hCG in human serum or urine to improve the diagnostic specificity and accuracy for various cancers, including choriocarcinoma, placental site trophoblastic tumors, and testicular germ cell tumors [21]. The more branched and elongated N-linked oligosaccharides may change the spatial structure of hCG-H and affect its binding affinity to certain mAbs, especially ones with epitopes adjacent to the N-glycosylation sites. These findings have important implications for monitoring hyper-N-glycosylated hCG and diagnosing choriocarcinoma, germ cell tumors, and other diseases in which alterations to hCG N-glycosylation may be involved
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