The influence of flanking sequence on the O-glycosylation of threonine in vitro.
To investigate the influence of flanking amino acid sequence on the O-glycosylation of a single threonine residue in vitro, we have examined a series of 52 related peptides. The substrates were based upon a sequence from human von Willebrand factor which is known to be glycosylated in vivo (-6PHMAQVTVGPGL+5). Each residue of the parent peptide was substituted, in turn, with isoleucine, alanine, proline, glutamic acid, or arginine. Peptides were glycosylated using a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase purified 15,000-fold from bovine colostrum by chromatography on DEAE-Sephacel, SP-Sephadex, Sephacryl S-300, Affi-Gel Blue, and 5-mercuri-UDP-GalNAc thiopropyl-Sepharose. Single amino acid changes in the sequences flanking the threonine could profoundly alter the glycosylation of the substrate peptides. Substitution of any amino acid tested at positions +3, -3, and -2 markedly decreased O-glycosylation, as did the presence of a charged residue at position -1. The substitution of amino acids at the other positions of the peptide substrate had little effect on the incorporation of GalNAc. Statistical analysis of sequences flanking known glycosylated threonine and serine residues suggests that they should be glycosylated with equal efficiency in the same sequence context (O'Connell et al., 1991). However, the bovine colostrum transferase failed to glycosylate a peptide derived from human erythropoietin which contains a serine that is glycosylated in vivo (-5PPDAASAAPLR+5). When a threonine was substituted for the serine in this peptide (-5PPDAATAAPLR+5), the substrate proved to be an excellent acceptor of GalNAc. These observations indicate that although flanking amino acid sequence is important for the O-glycosylation of specific hydroxyamino acids, discrete threonine- and serine-specific transferases may exist.
O'Connell
B C
BC
Department of Dental Research, School of Medicine and Dentistry, University of Rochester, New York 14642.
Hagen
F K
FK
Tabak
L A
LA
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003126:Colostrum / Q000201:enzymology; D005260:Female; D006031:Glycosylation; D007700:Kinetics; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism*; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D017350:N-Acetylgalactosaminyltransferases; D009842:Oligopeptides; D013912:Threonine; D000097763:Polypeptide N-acetylgalactosaminyltransferase