The use of UDP-Glc:glycoprotein glucosyltransferase for radiolabeling protein-linked high mannose-type oligosaccharides.
A simple method for specifically radiolabeling high mannose-type oligosaccharides linked to protein backbones has been developed. The method is based on the fact that incubation of rat liver UDP-Glc:glycoprotein glucosyltransferase, glucose-labelled UDP-Glc and a denatured high mannose-type glycoprotein target leads to the glucosylation of the oligosaccharide. In the case described here it allowed to follow easily the purification, by HPLC and affinity chromatography, of labelled glycopeptides obtained by controlled proteolysis of cruzipain, a cysteine proteinase isolated from the human pathogen Trypanosoma cruzi. It was thus determined that the N-glycosylation site in Asn33 of cruzipain is occupied by high mannose-type oligosaccharides.
Metzner
S I
SI
Instituto de Investigaciones Bioquímicas Fundación Campomar, Buenos Aires, Argentina.
Sousa
M C
MC
Hellman
U
U
Cazzulo
J J
JJ
Parodi
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000737:chemistry; D005964:Glucosyltransferases / Q000378:metabolism*; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D008358:Mannose / Q000737:chemistry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D015800:Protozoan Proteins; D051381:Rats; D014349:Trypanosoma cruzi / Q000201:enzymology
D002250:Carbon Radioisotopes; D006020:Glycopeptides; D006023:Glycoproteins; D009844:Oligosaccharides; D015800:Protozoan Proteins; D005964:Glucosyltransferases; D003546:Cysteine Endopeptidases; C063214:cruzipain; D008358:Mannose