Characterization of a UDP-Gal : Gal beta 1-3GalNAc alpha 1,4-galactosyltransferase activity in a Mamestra brassicae cell line

The binding of Bandeiraea simplicifolia lectin-I isolectin B-4 on the endog enous glycoproteins of different insect cell lines led us to characterize f or the first time a UDP-Gal:Gal beta 1-3GalNAc alpha 1,4-galactosyltransfer ase in a Mamestra brassicae cell line (Mb). The study of the acceptor speci ficity indicated that the Mb alpha-galactosyltransferase prefers Gal beta 1 -3-R as acceptor, and among such glycans, the relative substrate activity V -max/K-m was equal to 20 mu l.mg(-)1.h(-1) for Gal beta 1-3GlcNAc beta 1-O- octyl and to 330 mu l.mg(-1).h(-1) for Gal beta 1-3GalNAc alpha-1-O-benzyl, showing clearly that Gal beta 1-3GalNAc disaccharide was the more suitable acceptor substrate for Rib alpha-galactosyltransferase activity. Nuclear m agnetic resonance and mass spectrometry data allowed us to establish that t he Mb alpha-galactosyltransferase synthesizes one unique product, Gal alpha 1-4Gal beta 1-3GalNAc alpha 1-O-benzyl, The Gal beta 1-3GalNAc disaccharid e is usually present on O-glycosylation sites of numerous asialoglycoprotei ns and at the nonreducing end of some glycolipids. We observed that Mb alph a 1,4-galactosyltransferase catalyzed the transfer of galactose onto both n atural accepters. Finally, we demonstrated that the trisaccharide Gal alpha 1-4Gal beta 1-3GalNAc alpha 1-O-benzyl was able to inhibit anti-PK monoclo nal antibody-mediated hemagglutination of human blood group P-1(K) and P-2( K) erythrocytes.