title: 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.
authors.affiliation: From the National Glycoengineering Research Center, Shandong University, Jinan 250100, China, the State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China.
authors.forename: Daoyuan
authors.initials: D
authors.lastname: Li
authors.affiliation: the Department of Obstetrics and Gynecology, The Second Hospital of Shandong University, Jinan 250033, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Zhang
authors.affiliation: From the National Glycoengineering Research Center, Shandong University, Jinan 250100, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Li
authors.affiliation: the School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, New York 11794, and.
authors.forename: Lequan
authors.initials: L
authors.lastname: Chi
authors.affiliation: the State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China.
authors.forename: Deyu
authors.initials: D
authors.lastname: Zhu
authors.affiliation: the Central Research Laboratory, The Second Hospital of Shandong University, Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Ministry of Public Health, Qilu Hospital, Shandong University, Jinan 250100 China wz.zhangqy@sdu.edu.cn.
authors.forename: Qunye
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: From the National Glycoengineering Research Center, Shandong University, Jinan 250100, China, the State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China, lianlichi@sdu.edu.cn.
authors.forename: Lianli
authors.initials: L
authors.lastname: Chi
mesh_terms: D000328:Adult; D020816:Amino Acid Motifs; D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry*; D006063:Chorionic Gonadotropin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D005260:Female; D006031:Glycosylation; D006801:Humans; D051379:Mice; D009844:Oligosaccharides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000175:diagnosis / Q000378:metabolism
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D006063:Chorionic Gonadotropin; D009844:Oligosaccharides
keywords: N-linked glycosylation; biomarker; cancer; epitope mapping; glycoprotein; human chorionic gonadotropin; hydrogen/deuterium exchange; mass spectrometry (MS)