Establishment of a novel 70K Mac-2 binding protein antibody through screening of fucosylation-related antibodies

Abstract

Mac-2 binding protein (Mac-2bp) is a serum glycoprotein that contains seven N-glycans, and Mac-2bp serum levels are increased in patients with several types of cancer and liver disease. Mac-2bp glycosylation isomer has been applied as a clinical biomarker of several diseases, including liver fibrosis. In the present study, we identified fucosylated Mac-2bp in the conditioned medium of cancer cells resistant to anticancer therapies using glycoproteomic analyses. Fucosylation is one of the most important types of glycosylation involved in carcinogenesis and cancer stemness. To establish a next-generation glycan antibody for fucosylated Mac-2bp, we used fucosylation-deficient HEK293T cells to prepare reference Mac-2bp antigens and performed antibody screening. Unexpectedly, the 19-8H mAb obtained with our screen recognized 70K Mac-2bp, which is C-terminus-truncated product, rather than specifically recognizing fucosylated Mac-2bp. We performed immunocytochemistry using our novel 19-8H mAb, which resulted in strong cell surface staining of anticancer drug-resistant cancer cells. Therefore, our novel 19-8H mAb represents a valuable tool for cancer biology research that can help elucidate the biological function of 70K Mac-2bp.

Keywords: 70K Mac-2bp. Abbreviations: AAL, Aleuria aurantia lectin; Mac-2bp, Mac-2 binding protein; Mac-2bp OE, Mac-2bp over expression; 19-8H mAb, 19-8H monoclonal antibody; Mac-2bp pAb, Mac-2bp polyclonal antibody; CA19-9, carbohydrate antigen 19-9; GMDS, GDP-mannose 4,6-dehydratase; ELISA, enzyme-linked immunosorbent assay; Mac-2bp OE cells, Mac-2bp over-expressing HEK293T; Mac-2bp OE and GMDS KO cells, Mac-2bp overexpressing HEK293T with GMDS knockout cells; Mac-2 binding protein; fucosylation; glycan antibody; pancreatic cancer.