Dataset Information

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 ?65-83 of the hCG ? subunit is the epitope for MCA1024. 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. 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.

SUBMITTER: Li D

PROVIDER: S-EPMC4566243 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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

Li Daoyuan D Zhang Ping P Li Fei F Chi Lequan L Zhu Deyu D Zhang Qunye Q Chi Lianli L

The Journal of biological chemistry 20150803 37

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-glycosylatio ...[more]

PMID: 26240146

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Project description:With the rapid growth of complex heterogeneous biological molecules, effective techniques that are capable of rapid characterization of biologics are essential to ensure the desired product characteristics. To address this need, we have developed a method for analysis of intact glycoproteins based on high-resolution capillary electrophoretic separation coupled to an LTQ-FT mass spectrometer. We evaluated the performance of this method on the alpha subunit of mouse cell line-derived recombinant human chorionic gonadotrophin (r-alpha hCG), a protein that is glycosylated at two sites and is part of the clinically relevant gonadotrophin family. Analysis of r-alpha hCG, using capillary electrophoresis (CE) with a separation time under 20 min, resulted in the identification of over 60 different glycoforms with up to nine sialic acids. High-resolution CE-Fourier transform mass spectrometry (FT-MS) allowed separation and analysis of not only intact glycoforms with different numbers of sialic acids but also intact glycoforms that differed by the number and extent of neutral monosaccharides. The high mass resolution of the FT-MS enabled a limited mass range to be targeted for the examination of the protein glycoforms, simplifying the analysis without sacrificing accuracy. In addition, the limited mass range resulted in a fast scan speed that enhanced the reproducibility of the relative quantitation of individual glycoforms. The intact glycoprotein analysis was complemented with the analysis of the tryptic glycopeptides and glycans of r-alpha hCG to enable the assignment of glycan structures to individual sites, resulting in a detailed characterization of the protein. Samples of r-alpha hCG obtained from a CHO cell line were also analyzed and briefly shown to be significantly different from the murine cell line product. Taken together, the results suggest that the CE coupled to high-resolution FT-MS can be one of the effective tools for in-process monitoring as well as for final product characterization.

Project description:Immunopathological outcomes in Systemic Lupus Erythematosus (SLE; or lupus) are believed to be autoantibody-mediated. Conditions which promote a Th2 skew (such as pregnancy) should encourage antibody production, worsening antibody-mediated diseases while ameliorating Th1/Th17-mediated diseases. Although an increased propensity toward autoreactivity can be observed in pregnant lupus patients and in pregnant lupus-prone mice, whether a unique human pregnancy-specific factor can contribute to such effects is unknown. This study assessed whether human chorionic gonadotropin (hCG, a pregnancy-specific hormone of diverse function) at physiological concentrations could mediate stimulatory influences on immune parameters in non-pregnant, lupus-prone mice, in light of the hormone's ameliorating effects on Th1-mediated autoimmunity in murine models. Results demonstrate that administration of hCG heightened global autoreactivity in such mice; antibodies to dsDNA, RNP68, Protein S, Protein C, ?2-glycoprotein 1, and several phospholipids were enhanced, and hormone administration had adverse effects on animal survival. Specifically in splenic cell cultures containing cells derived from lupus-prone mice, hCG demonstrated synergistic effects with TLR ligands (up-modulation of costimulatory markers on B cells) as well as with TCR stimuli (enhanced proliferative responses, enhanced levels of cytokines, and the phosphorylation of p38). In both instances, enhanced synthesis of lupus-associated cytokines was observed, in addition to the heightened generation of autoantibodies reactive toward apoptotic blebs. These results suggest that selective transducive, proliferative, and differentiative effects of hCG on adaptive immune cells may drive autoreactive responses in a lupus environment, and may also potentially provide insights into the association between the presence of higher hCG levels (or the administration of hCG) with the presence (or appearance) of humoral autoimmunity.

Dataset Information

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

Publications

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

Similar Datasets

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