The Catalytic and Lectin Domains of UDP-GalNAc:Polypeptide α-N-Acetylgalactosaminyltransferase Function in Concert to Direct Glycosylation Site Selection

Abstract

UDP-GalNAc:polypeptide ␣-N-Acetylgalactosaminyltransferases (ppGalNAcTs), a family (EC 2.4.1.41) of enzymes that initiate mucin-type O-glycosylation, are structurally composed of a catalytic domain and a lectin domain. Previous studies have suggested that the lectin domain modulates the glycosylation of glycopeptide substrates and may underlie the strict glycopeptide specificity of some isoforms (ppGalNAcT-7 and -10). Using a set of synthetic peptides and glycopeptides based upon the sequence of the mucin, MUC5AC, we have examined the activity and glycosylation site preference of lectin domain deletion and exchange constructs of the peptide/glycopeptide transferase ppGalNAcT-2 (hT2) and the glycopeptide transferase ppGalNAcT-10 (hT10). We demonstrate that the lectin domain of hT2 directs glycosylation site selection for glycopeptide substrates. Pre-steady-state kinetic measurements show that this effect is attributable to two mechanisms, either lectin domain-aided substrate binding or lectin domain-aided product release following glycosylation. We find that glycosylation of peptide substrates by hT10 requires binding of existing GalNAcs on the substrate to either its catalytic or lectin domain, thereby resulting in its apparent strict glycopeptide specificity. These results highlight the existence of two modes of site selection used by these ppGalNAcTs: local sequence recognition by the catalytic domain and the concerted recognition of distal sites of prior glycosylation together with local sequence binding mediated, respectively, by the lectin and catalytic domains. The latter mode may facilitate the glycosylation of serine or threonine residues, which occur in sequence contexts that would not be efficiently glycosylated by the catalytic domain alone. Local sequence recognition by the catalytic domain differs between hT2 and hT10 in that hT10 requires a pre-existing GalNAc residue while hT2 does not.The first committed step in mucin-type O-glycan biosynthesis is the transfer of GalNAc from UDP-GalNAc to Ser/Thr residues of proteins catalyzed by a large family of evolutionarily conserved enzymes, the ppGalNAcTs 2 (1). The collective substrate specificity of the individual family members defines the mucin-type glycome, i.e. the Ser/Thr residues on a protein that acquire mucin-type O-glycans. Despite observations of biases in amino acid composition flanking known sites of O-glycosylation (2-5), no rigid consensus sequence for the prediction of sites has emerged. Consequently, current predictive methods are limited in their accuracy (6 -8). In vitro studies using peptide acceptors have demonstrated that ppGalNAcT isoforms have distinct but overlapping substrate specificities (2, 9 -12). Although a majority of these isoforms can glycosylate both naked peptides and glycopeptides (9 -11), a subset, notably isoforms ppGalNAcT-7 (13) and ppGalNAcT-10 (14), has an apparent strict requirement for prior glycosylation of their substrates to add additional GalNAc.ppGalNAcTs are type II transmembrane proteins with a short cyt...

Glycosylation of α-Dystroglycan

“…How the extant GalNAc on glycopeptide substrates is recognized by the ppGalNAc-T enzymes is not completely understood. However, previous studies suggest that both the catalytic and lectin domains can influence the sites of subsequent GalNAc addition (23). In the case of ppGalNAc-T2, the lectin domain appears to be responsible for recognizing extant GalNAcs, whereas the catalytic domain takes on this function in ppGalNAc-T10 (23).…”

“…In the in vivo environment there are likely additional factors and components that impact this process, including potential interactions involving the lectin domains of the ppGalNAc-Ts, which may preferentially direct activity to sites based on pre-existing glycosylation on ␣-DG (23). This may explain why we did not observe GalNAc added to Ser 485 in our experiments, although it was observed in one of the glycoforms of the 479 -489 tryptic fragment from rabbit muscle ␣-DG (10).…”

Glycosyltransferase Function in Core 2-Type Protein O Glycosylation

“…Core-type protein O glycosylation is initiated in the secretory pathway by the covalent addition of a N-acetylgalactosamine (GalNAc) to the hydroxyl group of serine or threonine residues by one of multiple polypeptide GalNAc transferases (ppGalNAcTs) (20,44,57,58). After linkage of the GalNAc monosaccharide to serine or threonine, other glycosyltransferases sequentially and sometimes competitively elaborate the repertoire of O-glycan structures to include different core subtypes (31,42,48,49).…”

UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase- 6 (pp-GalNAc-T6): Role in Cancer and Prospects as a Drug Target

“…The lectin domain recognises glycosylated polypeptide substrates [26]. The domain contains the three repeats of a CCQxW motif, in subdomains α, β and γ.…”