Characterization of a UDP-Gal:Galβ1–3GalNAc α1,4-Galactosyltransferase Activity in a Mamestra brassicaeCell Line

Abstract The binding of Bandeiraea simplicifolia lectin-I isolectin B4 on the endogenous glycoproteins of different insect cell lines led us to characterize for the first time a UDP-Gal:Galβ1–3GalNAc α1,4-galactosyltransferase in a Mamestra brassicae cell line (Mb). The study of the acceptor specificity indicated that the Mb α-galactosyltransferase prefers Galβ1–3-R as acceptor, and among such glycans, the relative substrate activityV max/K m was equal to 20 μl·mg−1·h−1 for Galβl-3GlcNAcβ1-O-octyl and to 330 μl·mg−1·h−1 for Galβ1–3GalNAcα-1-O-benzyl, showing clearly that Galβ1–3GalNAc disaccharide was the more suitable acceptor substrate for Mb α-galactosyltransferase activity. Nuclear magnetic resonance and mass spectrometry data allowed us to establish that the Mb α-galactosyltransferase synthesizes one unique product, Galα1–4Galβ1–3GalNAcα1-O-benzyl. The Galβ1–3GalNAc disaccharide is usually present onO-glycosylation sites of numerous asialoglycoproteins and at the nonreducing end of some glycolipids. We observed that Mb α1,4-galactosyltransferase catalyzed the transfer of galactose onto both natural acceptors. Finally, we demonstrated that the trisaccharide Galα1–4Galβ1–3GalNAcα1-O-benzyl was able to inhibit anti-PK monoclonal antibody-mediated hemagglutination of human blood group PK 1 and PK 2 erythrocytes.