Structural Analysis of Peptide Substrates for Mucin-Type O-Glycosylation

Abstract

The structures of three nine-residue peptide substrates that show differential kinetics of O-linked glycosylation catalyzed by distinct recombinant uridine diphosphate-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases) were investigated by NMR spectroscopy. A combined use of NMR data, molecular modeling techniques, and kinetic data may explain some structural features required for O-glycosylation of these substrates by two GalNAc transferases, GalNAc-T1 and GalNAc-T3. In the proposed model, the formation of an extended backbone structure at the threonine residue to be glycosylated is likely to enhance the O-glycosylation process. The segment of extended structure includes the reactive residue in a beta-like or an inverse gamma-turn conformation and flanking residues in a beta-strand conformation. The hydroxyl group of the threonine to be glycosylated is exposed to solvent, and both the amide proton and carbonyl oxygen of the peptide backbone are exposed to solvent. The exchange rate of the amide proton for the reactive threonine correlated well with substrate efficiency, leading us to hypothesize that this proton may serve as a donor for hydrogen bonding with the active site of the enzyme. The oxygens of the residue to be glycosylated and several flanking residues may also be involved in a set of hydrogen bonds with the GalNAc-T1 and -T3 transferases.

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

“…The lack of a strong correlation for Thr may be the result of the relatively high and more uniform glycosylation of Thr residues by GalNAc relative to Ser. This is consistent with most in vitro studies showing that Thr residues are typically an order of magnitude more readily glycosylated compared with Ser (11,(17)(18)(19)(20)(21). Note that in Fig.…”

“…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 O-Glycan Synthesis in IgA1 Hinge Region Is Determined by a Single Enzyme, UDP-N-Acetyl-α-d-galactosamine:PolypeptideN-Acetylgalactosaminyltransferase 2

“…It has also been reported that the initial O-glycosylation of a peptide substrate influences the subsequent glycosylation, probably due to conformational change of the peptide and the accessibility of pp-GalNAc-Ts for particular acceptor sites (57). There are two consecutive Ser and Thr residues, such as Ser-3/Thr-4 and Ser-11/Thr-12, in HRP.…”

O-linked glycosylation of von Willebrand factor modulates the interaction with platelet receptor glycoprotein Ib under static and shear stress conditions

“…Blocking glycosylation at T1255A might influence subsequent glycosylation events by inducing conformational changes in the peptide backbone and altering the accessibility of particular glycosylation sites for N-acetylgalactosaminyltransferases. [47][48][49] However, to date it has not been possible to ascertain whether mutation of a given OLG site of VWF affects glycosylation of nearby residues.…”

“…Blocking glycosylation at T1255A might influence subsequent glycosylation events by inducing conformational changes in the peptide backbone and altering the accessibility of particular glycosylation sites for N-acetylgalactosaminyltransferases. [47][48][49] However, to date it has not been possible to ascertain whether mutation of a given OLG site of VWF affects glycosylation of nearby residues.Together, the presented data support the hypothesis that OLG(s) present in the hinge, linker region connecting the D3-A1 domains, modulates VWF-platelet interaction both in the presence of ristocetin and shear stress. Importantly, however, the effect of the OLGs seems to be mediated by altering accessibility of the A1 domain in these conditions rather than by an effect on VWF A1-GPIb␣ bond.…”