Identification of the major sites of enzymic glycosylation of myelin basic protein.
In the presence of porcine submaxillary N-acetylgalactosaminyltransferase and uridine diphospho-N-acetyl-D-galactosamine, approx. 1.2-1.5 mol of N-acetylgalactosamine were transfered per mol of myelin basic protein. Tritium-labelled N-acetylgalactosamine-labelled basic protein was digested with trypsin and the peptides were separated by HPLC and the radioactivity measured. Most of the radioactivity was associated with three peptide peaks (I, II and III) containing 17, 69 and 6% of the total radioactivity, respectively. The remaining radioactivity was distributed amongst several peptides, each containing less than 2.5% of the total radioactivity. Glycosylation of the basic proteins isolated from human, bovine and guinea pig myelins showed that they were all equally good acceptors. In spite of differences in the peptide profiles of the basic proteins from different species, the distribution of radioactivity between the three peptide peaks was similar for all the species studied. The transfer of N-acetylgalactosamine to peptide II was much faster than to peptides I and III. The apparent Km values of the three peptides were within a narrow range of 0.52-0.63 mM, whereas the Vmax values were considerably different. The glycosylated peptide peaks (I, II and III) were separated by electrophoresis, the radioactivity measured, and amino acid compositions determined after hydrolysis. The major radioactive peptides of the human basic protein were identified with tryptic peptides containing the following sequences: (formula; see text)
D000116:Acetylgalactosamine / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D005688:Galactosamine / Q000031:analogs & derivatives*; D005700:Galactosyltransferases / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis*; D006168:Guinea Pigs; D006801:Humans; D004676:Myelin Basic Protein / Q000378:metabolism*; D017350:N-Acetylgalactosaminyltransferases; D013045:Species Specificity; D013552:Swine
D006023:Glycoproteins; D004676:Myelin Basic Protein; D005688:Galactosamine; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C003742:UDPgalactosamine-galactose acetylgalactosaminyltransferase; D000116:Acetylgalactosamine