ACS DIVISION OF BIOLOGICAL CHEMISTRY

April 21st, 2014 by Nonaka, M., Fukuda, M. N., Gao, C., Li, Z., Zhang, H., Greene, M., Peehl, D. M., Feizi, T., Fukuda, M.

This study reports determination of the carbohydrate epitope of monoclonal antibody F77 previously raised against human prostate cancer PC-3 cells (Zhang, G., Zhang, H., Wang, Q., Lal, P., Carroll, A.M., de la Llera-Moya, M., Xu, X., and Greene, M.I. (2010) Proc Natl Acad Sci U S A 107, 732-737). We performed a series of co-transfections using mammalian expression vectors encoding specific glycosyltransferases. We thereby identified branching enzymes and FUT1 (required for Fucα1→2Gal linkage) as being essential for F77 antigen formation. When immortalized normal prostate 267B1 cells were transfected with FUT1 alone, cells showed weak expression of F77 antigen. By contrast, cells co-transfected with FUT1 plus either GCNT1, GCNT2 or GCNT3 (an enzyme required to form GlcNAcβ1→6Gal/GalNAc) showed robust F77 antigen expression, suggesting that F77 specifically binds to Fucα1→2Galβ1→4GlcNAcβ1→6Gal/GalNAc. RT-PCR for FUT1, GCNT1, GCNT2, and GCNT3 showed that F77-positive cell lines indeed express transcripts encoding FUT1 plus one GCNT. F77-positive prostate cancer cells transfected with siRNAs targeting FUT1, GCNT2 and GCNT3 showed significantly reduced F77 antigen, confirming the requirement of these enzymes for epitope synthesis. We also found that hypoxia induces F77 epitope expression in immortalized prostate RWPE1 cells, which express F77 antigen moderately under normoxia but at an elevated level under hypoxia. Quantitative RT-PCR demonstrated upregulation of FUT1, GCNT2 and GCNT3 transcripts in RWPE1 cells under hypoxia, suggesting that hypoxia upregulates glycosyltransferase expression required for F77 antigen synthesis. These results define the F77 epitope and provide a potential mechanism for F77 antigen synthesis in malignant prostate cancer.