No abstract

The binding of Bandeiraea simplicifolia lectin-I isolectin B 4 on the endogenous glycoproteins of different insect cell lines led us to characterize for the first time a UDP-Gal:Gal␤1-3GalNAc ␣1,4-galactosyltransferase in a Mamestra brassicae cell line (Mb). The study of the acceptor specificity indicated that the Mb ␣-galactosyltransferase prefers Gal␤1-3-R as acceptor, and among such glycans, the relative substrate activity V max /K m was equal to 20 l⅐mg ؊1 ⅐h ؊1 for Gal␤l-3Glc-NAc␤1-O-octyl and to 330 l⅐mg ؊1 ⅐h ؊1 for Gal␤1-3Gal-NAc␣-1-O-benzyl, showing clearly that Gal␤1-3GalNAc disaccharide was the more suitable acceptor substrate for Mb ␣-galactosyltransferase activity. Nuclear magnetic resonance and mass spectrometry data allowed us to establish that the Mb ␣-galactosyltransferase synthesizes one unique product, Gal␣1-4Gal␤1-3GalNAc␣1-Obenzyl. The Gal␤1-3GalNAc disaccharide is usually present on O-glycosylation sites of numerous asialoglycoproteins and at the nonreducing end of some glycolipids. We observed that Mb ␣1,4-galactosyltransferase catalyzed the transfer of galactose onto both natural acceptors. Finally, we demonstrated that the trisaccharide Gal␣1-4Gal␤1-3GalNAc␣1-O-benzyl was able to inhibit anti-P K monoclonal antibody-mediated hemagglutination of human blood group P K 1 and P K 2 erythrocytes.In recent years, the development of therapeutic glycoprotein production using the baculovirus expression system in insect cells (1-4) has stimulated concerns to determine the posttranslational modifications capacity, in particular the glycosylation potential of insect cells. In fact, it is now well established that protein-bound carbohydrate side chains play important roles on the physicochemical properties and functions of glycoproteins such as antigenicity, immunogenicity, and metabolic clearance (5-8).The N-glycosylation pattern of recombinant glycoproteins expressed by lepidopteran cells using the baculovirus vector is now well documented and revealed that the N-linked oligosaccharides found are essentially of high mannose type (Man 9 -5 GlcNAc 2 ) and short truncated structures (Man 3-2 (Fuc 0 -1 )GlcNAc 2 ) 1 frequently substituted by a Fuc residue ␣1,6-linked to the asparagine-bound GlcNAc residue (9 -24). Only a few studies have shown complex N-linked glycans in insect cells. Recent data suggest that the insect cell lines Estigmena acrea and Trichoplusia ni clone Tn-5-B14 (Tn) can add some terminal galactose residues to the recombinant human interferon ␥ and to the heavy chain of a heterologous murin IgG (25, 26), respectively. Only one group has reported that recombinant human plasminogen carries complex type N-glycans with terminal sialic acid residues when expressed in Spodoptera frugiperda clone Sf-21 (Sf-21) or Mamestra brassicae clone SPCMb-92-C6 (Mb) (27-29).As mainly observed on recombinant glycoproteins expressed by the baculovirus/insect cell system, endogenous N-glycan structures found in insect cell glycoproteins have no complex type glycans (16, 30 -32), but a small amount of olig...