Glycoproten biosynthesis in Trypanosoma brucei. The glycosylation of Glycoproteins located in and attached to the plasma membrane.
1. Glycosyltransferase activity incorporating N-[14C]acetylglucosamine ([14C]GlcNAc) from uridine diphosphate N-[14C]acetylglucosamine (UDP-[14C]GlcNAc) into endogenous proitein acceptors was localized primarily in the plasma membrane of Trypanosoma brucei. 2. The acceptor site for the nucleotide sugar was further localized exclusively to the cytoplasmic face of the plasma membrane. 3. The glycosyltransferase produced elongation of the growing oligosaccharide chains while they were attached to their peptide acceptors. 4. This glycosyltransferase activity was incapable of initiating sugar attachment directly to amino acid residues within peptide acceptors. 5. The dolichyl-phosphate-sugar pathway for glycoprotein biosynthesis was either absent of only present at a very low level in T. brucei when compared to rat liver. 6. All oligosaccharide chains accepting GlcNAc were of the same or very similar lengths. 7. Both O-glycosidic (26%) and N-glycosidic (74%) linkages (exclusive of hydroxylysine attachment) were found. 8. Glycosyltransferase activity required either Mn2+ or Mg2+, had a pH optimum of 6.5 and was temperature-dependent. 9. The kinetics of incorporation were complex, probably a result of multiple acceptors or glycosyltransferases whose activities were characterized by a Km of 30 microM for UDP-GlcNAc with a V of 40 pmol x mg protein -1 x min-1 for the highest affinity system and a Km of approximately 2 mM for UDP-GlcNAc with a V of approximately 400 pmol x mg protein-1 x min-1 for the lowest affinity system. 10. Glycosyltransferases using UDP-GlcNAc, uridine diphosphate glucose, uridine diphosphate galactose and guanidine diphosphate mannose as glycosyl donors were observed. Each peptide acceptor was specific for a singloe labelled sugar in the absence of other unlabelled nucleotide sugars. 11. The final extent of incorporation of GlcNAc was due primarily to exhaustion of peptide acceptor. 12. An inhibitor of UDP-[14C]GlcNAc incorporation into plasma membranes was found in the cytoplasmic fraction.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D004288:Dolichol Phosphates / Q000378:metabolism; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008565:Membrane Proteins / Q000096:biosynthesis*; D009844:Oligosaccharides / Q000032:analysis; D013347:Subcellular Fractions / Q000201:enzymology; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D004288:Dolichol Phosphates; D006023:Glycoproteins; D008565:Membrane Proteins; D009844:Oligosaccharides; D008345:Manganese; D005964:Glucosyltransferases; C026900:UDPacetylglucosamine-protein acetylglucosaminyltransferase; D008274:Magnesium