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Recognition of N-glycoforms in human chorionic gonadotropin by monoclonal antibodies and their interaction motifs.

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.
AuthorsDaoyuan Li, Ping Zhang, Fei Li, Lequan Chi, Deyu Zhu, Qunye Zhang, Lianli Chi
JournalThe Journal of biological chemistry (J Biol Chem) Vol. 290 Issue 37 Pg. 22715-23 (Sep 11 2015) ISSN: 1083-351X [Electronic] United States
PMID26240146 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Copyright© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Chemical References
  • Antibodies, Monoclonal, Murine-Derived
  • Chorionic Gonadotropin
  • Oligosaccharides
Topics
  • Adult
  • Amino Acid Motifs
  • Animals
  • Antibodies, Monoclonal, Murine-Derived (chemistry)
  • Chorionic Gonadotropin (chemistry, genetics, metabolism)
  • Deuterium Exchange Measurement
  • Female
  • Glycosylation
  • Humans
  • Mice
  • Oligosaccharides (chemistry, genetics, metabolism)
  • Ovarian Neoplasms (diagnosis, metabolism)

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