Characterization of a UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase activity in a Mamestra brassicae cell line.
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:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase in a Mamestra brassicae cell line (Mb). The study of the acceptor specificity indicated that the Mb alpha-galactosyltransferase prefers Galbeta1-3-R as acceptor, and among such glycans, the relative substrate activity Vmax/Km was equal to 20 microliters.mg-1.h-1 for Galbetal-3GlcNAcbeta1-O-octyl and to 330 microliters.mg-1.h-1 for Galbeta1-3GalNAcalpha-1-O-benzyl, showing clearly that Galbeta1-3GalNAc disaccharide was the more suitable acceptor substrate for Mb alpha-galactosyltransferase activity. Nuclear magnetic resonance and mass spectrometry data allowed us to establish that the Mb alpha-galactosyltransferase synthesizes one unique product, Galalpha1-4Galbeta1-3GalNAcalpha1-O-benzyl. The Galbeta1-3GalNAc disaccharide is usually present on O-glycosylation sites of numerous asialoglycoproteins and at the nonreducing end of some glycolipids. We observed that Mb alpha1,4-galactosyltransferase catalyzed the transfer of galactose onto both natural acceptors. Finally, we demonstrated that the trisaccharide Galalpha1-4Galbeta1-3GalNAcalpha1-O-benzyl was able to inhibit anti-PK monoclonal antibody-mediated hemagglutination of human blood group PK1 and PK2 erythrocytes.
Lopez
M
M
Laboratoire de Chimie Biologique, Unité Mixte de Recherche du CNRS 111, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq, France.
Gazon
M
M
Juliant
S
S
Plancke
Y
Y
Leroy
Y
Y
Strecker
G
G
Cartron
J P
JP
Bailly
P
P
Cerutti
M
M
Verbert
A
A
Delannoy
P
P
D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism; D002460:Cell Line; D005690:Galactose / Q000378:metabolism; D005700:Galactosyltransferases / Q000737:chemistry / Q000378:metabolism*; D006017:Glycolipids / Q000378:metabolism; D006801:Humans; D037102:Lectins / Q000378:metabolism; D009036:Moths / Q000166:cytology / Q000201:enzymology*; D037121:Plant Lectins; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera / Q000166:cytology; D013379:Substrate Specificity
D001212:Asialoglycoproteins; D006017:Glycolipids; C045475:Griffonia simplicifolia lectins; D037102:Lectins; D037121:Plant Lectins; C071307:GalNAc beta1-4 GlcNAc-R beta1-3-galactosyltransferase; D005700:Galactosyltransferases; D005690:Galactose