Dynamic Association between the Catalytic and Lectin Domains of Human UDP-GalNAc:Polypeptide α-N-Acetylgalactosaminyltransferase-2

Abstract

The family of UDP-GalNAc:polypeptide ␣-N-acetylgalactosaminyltransferases (ppGalNAcTs) is unique among glycosyltransferases, containing both catalytic and lectin domains that we have previously shown to be closely associated. Here we describe the x-ray crystal structures of human ppGalNAcT-2 (hT2) bound to the product UDP at 2.75 Å resolution and to UDP and an acceptor peptide substrate EA2 (PTTDSTTPAPTTK) at 1.64 Å resolution. The conformations of both UDP and residues Arg 362 -Ser 372 vary greatly between the two structures. In the hT2-UDP-EA2 complex, residues Arg 362 -Ser 373 comprise a loop that forms a lid over UDP, sealing it in the active site, whereas in the hT2-UDP complex this loop is folded back, exposing UDP to bulk solvent. EA2 binds in a shallow groove with threonine 7 positioned consistent with in vitro data showing it to be the preferred site of glycosylation. The relative orientations of the hT2 catalytic and lectin domains differ dramatically from that of murine ppGalNAcT-1 and also vary considerably between the two hT2 complexes. Indeed, in the hT2-UDP-EA2 complex essentially no contact is made between the catalytic and lectin domains except for the peptide bridge between them. Thus, the hT2 structures reveal an unexpected flexibility between the catalytic and lectin domains and suggest a new mechanism used by hT2 to capture glycosylated substrates. Kinetic analysis of hT2 lacking the lectin domain confirmed the importance of this domain in acting on glycopeptide but not peptide substrates. The structure of the hT2-UDP-EA2 complex also resolves long standing questions regarding ppGalNAcT acceptor substrate specificity.The first committed step of carbohydrate addition to mucin-type glycoproteins is catalyzed by a family of UDP-GalNAc:polypeptide ␣-Nacetylgalactosaminyltransferases (ppGalNAcTs), 2 yielding the Tn antigen (GalNac-␣-1-O-Ser/Thr). This family is large (with Ϸ24 mammalian isoforms) and phylogenetically conserved with Drosophila expressing 14 isoforms, at least one of which is essential for development (1, 2), and Caenorhabditis elegans expressing 9 isoforms (3). Subsequent elongation of the Tn structure yields an array of eight distinct "core" glycans that can be further modified by many of the glycosyltransferases resident in the Golgi. The embryonic lethality resulting from the knock-out of one of these core glycosyltransferases (the core 1 ␤1,3-galactosyltransferase) in mice underscores the biological importance of mucin-type glycans (4). The repertoire of O-glycans has been implicated in diverse biological processes including host defense (5), lymphocyte homing (6), and tumor metastasis (7), and the first example of a human disease (familial tumoral calcinosis) caused by the loss of function of a ppGalNAcT-T (ppGalNAcT-3) was recently reported (8). However, there appears to be functional redundancy among ppGalNAcT members because mice in which isoforms 4, 5, or 13 are ablated do not present with any obvious phenotype (9 -11), whereas mice in which ppGalNAcT-1 has been ablated a...

Mechanism of a cytosolic O -glycosyltransferase essential for the synthesis of a bacterial adhesion protein

“…In mucin-synthesizing ppGalNAcT enzymes, an active domain with a GT-A fold is able to modify unmodified substrates without the help of any other protein/ domain. However, the additional attachment of sugars to modified segments requires a sugar-binding lectin domain (42)(43)(44). Thus, different O-glycosylation enzymes have found distinct ways to cope with the problem of a progressively changing substrate structure.…”

Golgi Glycosylation

“…Some GTs contain two functional domains. For example, several polypeptide GalNAcTs have a lectin domain that binds to GalNAc and a catalytic domain that transfers GalNAc (Hassan et al 2000;Fritz et al 2006); a family of N-deacetylase/N-sulfotransferases are important in heparan sulfate biosynthesis (Aikawa et al 2001); and the putative glycosyltransferase Large has two glycosyltransferase domains (Longman et al 2003;Aguilan et al 2009). GTs are often glycosylated by other GTs or, in some cases, by autocatalytic transferase activity.…”

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

“…1 for the crystal structure of ppGalNAc T2 with bound EA2 peptide substrate (42)). Presently, the roles of the catalytic and lectin domains in peptide and glycopeptide recognition and specificity remain unclear.…”