Identification of two distinct galactosyltransferase activities acting on the variant surface glycoprotein of Trypanosoma brucei

• Published in: Biochemical journal Vol. 283 ( Pt 2); no. 2; pp. 479 - 485
• Main Authors: Pingel, S, Duszenko, M
• Format: Journal Article
• Language: English
• Published: England 15.04.1992
• Subjects: Animals; beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase - metabolism; Electrophoresis, Polyacrylamide Gel; Galactose - metabolism; Galactosidases; Galactosyltransferases - metabolism; Glycolipids - metabolism; Glycosylphosphatidylinositols; Kinetics; Oligosaccharides - isolation & purification; Oligosaccharides - metabolism; Phosphatidylinositols - metabolism; Substrate Specificity; Trypanosoma brucei brucei - enzymology; Variant Surface Glycoproteins, Trypanosoma - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2830479

Variant surface glycoproteins (VSGs) of Trypanosoma brucei contain two distinct glycosylation sites: (1) N-linked glycans within the protein portion of the molecules, and (2) the glycosyl-phosphatidylinositol (GPI) membrane anchor. Since galactose residues show uncommon alpha-glycosidic linkages in the GPI membrane anchor, we were prompted to investigate galactosylation of the GPI anchor. On comparing a trypanosome clone galactosylated exclusively in N-glycans (clone MITat 1.5) with clones galactosylated predominantly in the glypiated membrane anchor (clones MITat 1.4, MITat 1.6 and AnTat 1.8), clone MITat 1.5 showed a 10-fold increased enzyme activity when using a protocol including Triton X-100 to assay UDPgalactose:N-acetylglucosaminyl glycopeptide beta 1,4-galactosyltransferase (EC 2.4.1.38). Only the VSG of clone MITat 1.5 could be radiochemically labelled with UDP[14C]galactose, and galactosylation of N-glycans was confirmed by digestion with peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase F). However, in a modified enzyme assay without detergent, galactosyltransferase activity was increased considerably (15-fold) in clone MITat 1.4. VSG galactosylation of clones MITat 1.4, MITat 1.6 and AnTat 1.8 was readily detected by fluorography of the respective SDS/polyacrylamide gels, suggesting that galactosyltransferase activity modifies the VSG membrane anchor in these clones. In this case, [14C]galactose labelling of immunoprecipitated VSG (clone MITat 1.4) was resistant to the release of N-glycans by PNGase F treatment, and thus revealed galactosylation in vitro of a VSG membrane anchor. Exoglycosidase digestions of VSG MITat 1.4 confirmed the presence of alpha-linked galactose residues. We suggest that these specific alpha-galactosyltransferases are inhibited by the action of detergent, but can be activated in a detergent-free buffer system.