Specificity of O-glycosylation by bovine colostrum UDP-GalNAc: polypeptide ?-N-acetylgalactosaminyltransferase using synthetic glycopeptide substrates

Abstract

The factors determining glycosylation of mucin type glycoproteins are not well understood. In the present work, we investigated the role of the peptide moiety and of the presence of O-glycan chains on O-glycosylation by UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyl-transferase (ppGalNAc-T). We used purified ppGalNAc-T from bovine colostrum and a series of synthetic glycopeptide and peptide substrates most of which contained sequences derived from the tandem repeat region of MUC2 mucin. The rate of incorporation of GalNAc into Thr was significantly greater than toward Ser residues. The presence of one or two GalNAc-Thr moieties in the substrate significantly reduced enzyme activity, and this effect was more pronounced when the disaccharide Gal beta 1-3GalNAc was present. Thus the sequential attachment of a second GalNAc residue in the vicinity of a pre-existing GalNAc-Thr or Gal beta 1-3GalNAc-Thr occurs at a slower rate than primary glycosylation of carbohydrate-free peptide. Analysis of products by HPLC showed that the enzyme was selective in glycosylating peptides or glycopeptides with the PTTTPIST sequence in that the preferred primary glycosylation site was the third Thr from the amino-terminal end; secondary glycosylation depended on the site of the primary glycosylation. Negatively but not positively charged amino acids on the carboxy-terminal side of the putative secondary glycosylation site resulted in high activity suggesting charge-charge interactions of substrates with the enzyme. These studies indicate that O-glycosylation by bovine colostrum ppGalNAc-T is a selective process dependent on both the amino acid sequence and prior glycosylation of peptide substrates.

Dynamic Epigenetic Regulation of InitialO-Glycosylation by UDP-N-Acetylgalactosamine:PeptideN-Acetylgalactosaminyltransferases

“…According to this concept, the site specificity of initial O-glycosylation would not merely be ruled by the peptide sequence around putative target sites. Evidence for negative effects on vicinal sites induced by mono-or disaccharide substituents has previously been reported for a series of glycopeptide substrates based on the MUC2 repeat peptide (12). Another in vitro glycosylation study indicated that positive regulatory effects on vicinal sites could also be responsible for the accelerated GalNAc transfer (7).…”

“…The samples were diluted with incubation buffer to give a total volume of 210 l prior to ultrafiltration through ultrafree MC membranes (Millipore, Eschborn, Germany) with a nominal cut-off of 10 kDa. Kinetic parameters were calculated by double reciprocal LineweaverBurk transformations at four substrate concentrations using assay conditions described by Brockhausen et al (12).…”

Determination of the Site-specific Oligosaccharide Distribution of the O-Glycans Attached to the Porcine Submaxillary Mucin Tandem Repeat

“…where the specificities of both the ppGalNAc and core 1 transferases were proposed to be affected by steric effects of neighboring glycosylated residues (22,23,32,33,58,59).…”

“…1 To date, 10 ppGalNAc transferase isoforms have been described (9 -16). Although each transferase has not been fully characterized, their peptide substrate specificities vary within the family (11,(17)(18)(19)(20)(21); many show sensitivity to prior glycosylation, and others require prior addition of GalNAc for activity (9,14,20,(22)(23)(24). The expression of ppGalNAc transferase isoforms with different peptide and/or glycopeptide specificities therefore represents the first step in the regulation of O-glycan structure by peptide sequence in vivo.…”

Initiation of Protein O Glycosylation by the Polypeptide GalNAcT-1 in Vascular Biology and Humoral Immunity

“…ppGal NAcT-1 exhibits a selective preference for some polypeptide sequences (7,16,60), further suggesting that a key function is likely provided by its activity among cells of intact organisms. The ppGalNAcT family of glycosyltransferases initiates coretype O-glycan formation, primarily consisting of core 1, core 2, core 3, and core 4 O-glycan subtypes that may be elaborated further by glycan linkages, contributing to selectin ligand-dependent control of leukocyte trafficking, regulation of CD8 ϩ T-cell apoptosis, and sensitivity to colitis and preventing the onset of Tn syndrome (11,27,42,59,65).…”