title: Site directed processing: role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type O-glycan core 2.
abstract: BACKGROUND
  The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 beta3-Gal-transferase (C1GalT) and core 2 beta6-GlcNAc-transferase (C2GnT). Beta4-Gal-transferase (beta4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates.
  
  
  METHODS
  We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6.
  
  
  RESULTS
  Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the +3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galbeta1-3GalNAcalpha-TAGV was identified as an efficient C2GnT substrate.
  
  
  CONCLUSIONS
  The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion.
  
  
  CONCLUSIONS
  Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease.
authors.affiliation: Department of Medicine, Division of Rheumatology, Queen's University, Kingston, Ontario, Canada K7L 3N6. brockhau@queensu.ca
authors.forename: Inka
authors.initials: I
authors.lastname: Brockhausen
authors.forename: Thomas
authors.initials: T
authors.lastname: Dowler
authors.forename: Hans
authors.initials: H
authors.lastname: Paulsen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D055262:Mucin-2 / Q000737:chemistry / Q000378:metabolism*; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D005700:Galactosyltransferases; D006020:Glycopeptides; D055262:Mucin-2; D017350:N-Acetylgalactosaminyltransferases; D017351:N-Acetylglucosaminyltransferases; D010446:Peptide Fragments; D011134:Polysaccharides; D011994:Recombinant Proteins; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C505293:C1GALT1 protein, human; C419407:beta-1,4-galactosyltransferase II; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase