title: Determination of the site-specific O-glycosylation pattern of the porcine submaxillary mucin tandem repeat glycopeptide. Model proposed for the polypeptide:galnac transferase peptide binding site.
abstract: The heterogeneously glycosylated 81-residue tryptic tandem repeat glycopeptide from porcine submaxillary mucin (PSM) has been isolated and its glycosylation pattern determined by amino acid sequencing. Key to these studies is the ability to trim the structurally heterogeneous PSM oligosaccharide side chains to homogeneous GalNAc monosaccharide side chains by mild trifluoromethanesulfonic acid treatment. Trypsin treatment of trifluoromethanesulfonic acid-treated PSM releases the 81-residue tandem repeat as an ensemble of 81-residue glycopeptides with different glycosylation patterns. Automated amino acid sequencing using Edman degradative chemistry of the repeat was used to determine the extent of glycosylation of nearly every Ser and Thr residue. The Thr residues are all highly glycosylated within the range of 73-90%, giving an average Thr glycosylation of 83%. In contrast, the Ser residues display a wide range of glycosylations, ranging between 33 and 95%, giving an average Ser glycosylation of 74%. These data are consistent with the known elevated glycosylation of Thr peptides over Ser peptides for the porcine UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase. It is also observed that the extent of glycosylation of the repeat correlates poorly with published predictive methods. An examination of the sequences surrounding the glycosylation sites reveals that nearly all of the highly glycosylated sites have a penultimate Gly residue, whereas those that are less highly glycosylated have medium to large side chain penultimate residues. As observed by others, glycosylation also appears to be modulated by the presence of Pro residues. On the basis of these findings we suggest that the acceptor peptide binds the transferase in a beta-like conformation and that penultimate residue side chain steric interactions may play a role in determining extent that a given Ser or Thr is glycosylated. A model for the GalNAc transferase peptide binding site is proposed.
authors.affiliation: W. A. Bernbaum Center for Cystic Fibrosis Research, Case Western Reserve University, Cleveland, Ohio 44106-4948, USA. tag@glycocon.cwru.edu
authors.forename: T A
authors.initials: TA
authors.lastname: Gerken
authors.forename: C L
authors.initials: CL
authors.lastname: Owens
authors.forename: M
authors.initials: M
authors.lastname: Pasumarthy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006020:Glycopeptides / Q000737:chemistry* / Q000235:genetics; D006031:Glycosylation; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry*; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry* / Q000378:metabolism; D010449:Peptide Mapping; D012091:Repetitive Sequences, Nucleic Acid; D012694:Serine; D013363:Submandibular Gland / Q000737:chemistry*; D013552:Swine; D013912:Threonine
chemical_list: D006020:Glycopeptides; D009077:Mucins; D013912:Threonine; D012694:Serine; D017350:N-Acetylgalactosaminyltransferases