Eukaryotic expression and characterization of two α1,2-fucosyltransferase genes in the histo-blood group antigens synthesis pathway of the Pacific oyster (Crassostrea gigas).

Abstract

Norovirus (NoV), an important cause of human viral gastroenteritis worldwide, recognizes human histo-blood group antigens (HBGAs) as receptors. Oysters are a vector of foodborne transmission of NoV, and HBGAs have been found in oyster tissues. In this study, CgFUT1 and CgFUT2, the key genes involved in the synthesis of HBGAs in Crassostrea gigas, were successfully expressed in Pichia pastoris, and 32.6 kDa target proteins were obtained after purification, concentration, and dialysis treatments. Western blot analysis using FUT1 and FUT2 antibodies showed that CgFUT1 and CgFUT2 have antigenic similarity as human FUT1 and FUT2. Enzyme catalysis assays using Galβ1-3GlcNAc and Galβ1-4GlcNAc as substrates showed that these substrates react with GDP-Fuc to generate Fucα1-2GalβGlcNAc under the action of CgFUT1 and CgFUT2. High-resolution mass spectrometry analysis revealed that CgFUT1 and CgFUT2 have the same substrate specificity, both reacting with Galβ1-3GlcNAc and Galβ1-4GlcNAc. The results of this study demonstrate the probable role of CgFUT1 and CgFUT2 in regulating substrates for H antigen synthesis in oysters and provide a reference for future studies into the functions of these genes. The study also lays a foundation for further exploration of the molecular mechanisms underlying NoV accumulation in oysters.