Identification of two distinct galactosyltransferase activities acting on the variant surface glycoprotein of Trypanosoma brucei.
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.
Pingel
S
S
Physiologisch-Chemisches Institut, Universität Tübingen, Federal Republic of Germany.
Duszenko
M
M
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000378:metabolism; D005696:Galactosidases; D005700:Galactosyltransferases / Q000378:metabolism*; D006017:Glycolipids / Q000378:metabolism; D017261:Glycosylphosphatidylinositols; D007700:Kinetics; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000378:metabolism*; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase / Q000378:metabolism
D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D009844:Oligosaccharides; D010716:Phosphatidylinositols; D014643:Variant Surface Glycoproteins, Trypanosoma; D005700:Galactosyltransferases; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase; D005696:Galactosidases; D005690:Galactose