Glycoprotein Biosynthesis in Trypanosoma brucei. The Glycosylation of Glycoproteins Located in and Attached to the Plasma Membrane

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Abstract
Glycosyltransferase activity incorporating N-[14C]acetylglucosamine ([14C]GlcNAc) from UDP N-[14C]acetylglucosamine (UDP-[14C]GlcNAc) into endogenous protein acceptors was localized primarily in the plasma membrane of T. brucei. The acceptor site for the nucleotide sugar was further localized exclusively to the cytoplasmic face of the plasma membrane. The glycosyltransferase produced elongation of the growing oligosaccharide chains while they were attached to their peptide acceptors. This glycosyltransferase activity was incapable of initiating sugar attachment directly to amino acid residues within peptide acceptors. The dolichyl-phosphate-sugar pathway for glycoprotein biosynthesis was either absent or only present at a very low level in T. brucei when compared to rat liver. All oligosaccharide chains accepting GlcNAc were of the same or very similar lengths. Both O-glycosidic (26%) and N-glycosidic (74%) linkages (exclusive of hydroxylysine attachments) were found. Glycosyltransferase activity required either Mn2+ or Mg2+, had a pH optimum of 6.5 and was temperature-dependent. The kinetics of incorporation were complex, probably a result of multiple acceptors or glycosyltransferases whose activities were characterized by a Km of 30 .mu.M for UDP-GlcNAc with a V of 40 pmol .times. mg protein-1 .times. min-1 for the highest affinity system and a Km of .apprxeq. 2 mM for UDP-GlcNAc with a V of .apprx. 400 pmol .times. mg protein-1 .times. min-1 for the lowest affinity system. Glycosyltransferases using UDP-GlcNAc, UDP-glucose, UDP-galactose and GDP-mannose as glycosyl donors were observed. Each peptide acceptor was specific for a single labeled sugar in the absence of other unlabeled nucleotide sugars. The final extent of incorporation of GlcNAc was due primarily to exhaustion of peptide acceptor. An inhibitor of UDP-[14C]GlcNAc incorporation into plasma membranes was found in the cytoplasmic fraction.