The beginnings of mucin biosynthesis: The crystal structure of UDP-GalNAc:polypeptide α- N -acetylgalactosaminyltransferase-T1

Abstract

UDP-GalNAc:polypeptide ␣-N-acetylgalactosaminyltransferases (ppGaNTases) initiate the formation of mucin-type, O-linked glycans by catalyzing the transfer of ␣-N-acetylgalactosamine from UDP-GalNAc to Ser or Thr residues of core proteins to form the Tn antigen (GalNAc-␣-1-O-Ser͞Thr). ppGaNTases are unique among glycosyltransferases in containing a C-terminal lectin domain. We present the x-ray crystal structure of a ppGaNTase, murine ppGaNTase-T1, and show that it folds to form distinct catalytic and lectin domains. The association of the two domains forms a large cleft in the surface of the enzyme that contains a Mn 2؉ ion complexed by invariant D209 and H211 of the ''DXH'' motif and by invariant H344. Each of the three potential lectin domain carbohydrate-binding sites (␣, ␤, and ␥) is located on the active-site face of the enzyme, suggesting a mechanism by which the transferase may accommodate multiple conformations of glycosylated acceptor substrates. A model of a mucin 1 glycopeptide substrate bound to the enzyme shows that the spatial separation between the lectin ␣ site and a modeled active site UDP-GalNAc is consistent with the in vitro pattern of glycosylation observed for this peptide catalyzed by ppGaNTase-T1. The structure also provides a template for the larger ppGaNTase family, and homology models of several ppGaNTase isoforms predict dramatically different surface chemistries consistent with isoform-selective acceptor substrate recognition.glycosyltransferase ͉ mucin

The Lectin Domain of the Polypeptide GalNAc Transferase Family of Glycosyltransferases (ppGalNAc Ts) Acts as a Switch Directing Glycopeptide Substrate Glycosylation in an N- or C-terminal Direction, Further Controlling Mucin Type O-Glycosylation

“…Such domain mobility is supported by the superimposition of the x-ray crystal structures of ppGalNAc T1, T2, and T10, as shown in Fig. 6B (42,60,61). For those transferases with similar N-and C-terminal glycopeptide enhancements (Fig.…”

The LHβ Gene of Several Mammals Embeds a Carboxyl-terminal Peptide-like Sequence Revealing a Critical Role for Mucin Oligosaccharides in the Evolution of Lutropin to Chorionic Gonadotropin in the Animal Phyla

“…glycans may result from amino acids surrounding the potential acceptors in the boCTP, causing misalignment of the primary sequence that prevent the GalNac transferase from recognizing the Ser/Thr targets. Alternatively, regions of the cryptic CTP may inhibit the accessibility of the GalNac transferase(s) to potential acceptor sites, as discussed previously (57,58). The high Pro content of the boCTP (about 25% of the residues) is indicative for a little secondary structure, which is a favorable feature for O-glycosylation.…”

Genetic and Molecular Basis of Kingella kingae Encapsulation

“…Given the novelty of the CsaA glycosyltransferase, we used BLAST and PHYRE2 to compare its amino acid sequence and predicted protein structure to those of other known glycosyltransferases. At the N terminus of CsaA, we found a GT-A-type glycosyltransferase superfamily domain that has homology to the N-terminal domain of ␣-N-acetylgalactosaminyltransferase-T1, which catalyzes the transfer of ␣-N-acetylgalactosamine from UDP-GalNAc to Ser or Thr residues of core proteins to form the Tn antigen (GalNAc-␣-1-O-Ser/Thr) in mice, suggesting that CsaA is a GalNAc transferase (22). In support of this hypothesis, CsaA has Asp residues at positions 99 and 101, similarly to the DXD motif in GT-A structural superfamily glycosyltransferases (23).…”