The most used cancer serum biomarker is the CA125 immunoassay for ovarian cancer that detects the mucin MUC16. However, the monoclonal antibodies (OC125 and M11) used in the serum assay have epitopes that remain elusive. In this study, we demonstrate for the first time that neither N- nor O-glycosylation appear to substantially influence binding of OC125 and M11 mAbs and narrowed down the epitopes to a segment containing parts of two consecutive SEA domains with a linker. These findings suggest that binding epitopes of mAbs OC125 and M11 are dependent on conformation but not on glycosylation. This information is relevant both for improving existing serum assays and for development of vaccines.
Authors and Affiliations:
Lara Marcos-Silva 1,2,3, Yoshiki Narimatsu1,4, Adnan Halim 1, Diana Campos 1,2,3, Zhang Yang 1,4, Mads A. Tarp 1, Pedro J. B. Pereira 5, Ulla Mandel 1, Eric P. Bennett 1, Sergey Y. Vakhrushev 1, Steven B. Levery 1, Leonor David 2,3, and Henrik Clausen 1,4
1Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark
2IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal
3Faculty of Medicine of the University of Porto, Al. Prof. Hernâni Monteiro, 4200-319 Porto, Portugal
4The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Alle 6, 2970 Hørsholm, Denmark
5IBMC, Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal
Abstract:
The most used cancer serum biomarker is the CA125 immunoassay for ovarian cancer that detects the mucin glycoprotein MUC16. Several monoclonal antibodies (mAbs) including OC125 and M11 are used in CA125 assays. However, despite considerable efforts, our knowledge of the molecular characteristics of the recognized epitopes and the role played by glycosylation has remained elusive. Here a comprehensive set of recombinant MUC16 tandem repeats (TRs) expressed in glycoengineered mammalian cells and E. coli, together with overlapping peptides, was used to probe antigen-binding epitopes. We present a complete analysis of N- and O-glycosylation sites of a MUC16 TR expressed in CHO cells and demonstrate that neither N- nor O-glycosylation appear to substantially influence binding of OC125 and M11 mAbs. A series of successive N- and C-terminal truncations of a MUC16 TR construct expressed in E. coli narrowed down the epitopes for OC125 and M11 to a segment containing parts of two consecutive SEA domains with a linker. Thus, a complete SEA domain is not required. These findings suggest that binding epitopes of mAbs OC125 and M11 are dependent on conformation but not on glycosylation. The availability of recombinant TR constructs with and without aberrant glycosylation now opens the way for vaccine studies.
Journal:
Journal of Proteome Research
Link: http://pubs.acs.org/doi/abs/10.1021/pr500215g
