Glycosylation of proteins is an essential process in all eukaryotes and a great diversity in types of protein glycosylation exists in animals, plants and microorganisms. Mucin-type O-glycosylation, consisting of glycans attached via O-linked N-acetylgalactosamine (GalNAc) to serine and threonine residues, is one of the most abundant forms of protein glycosylation in animals. Although most protein glycosylation is controlled by one or two genes encoding the enzymes responsible for the initiation of glycosylation, i.e. the step where the first glycan is attached to the relevant amino acid residue in the protein, mucin-type O-glycosylation is controlled by a large family of up to 20 homologous genes encoding UDP-GalNAc:polypeptide GalNAc-transferases (GalNAc-Ts) (EC 2.4.1.41). Therefore, mucin-type O-glycosylation has the greatest potential for differential regulation in cells and tissues. The GalNAc-T family is the largest glycosyltransferase enzyme family covering a single known glycosidic linkage and it is highly conserved throughout animal evolution, although absent in bacteria, yeast and plants. Emerging studies have shown that the large number of genes (GALNTs) in the GalNAc-T family do not provide full functional redundancy and single GalNAc-T genes have been shown to be important in both animals and human. Here, we present an overview of the GalNAc-T gene family in animals and propose a classification of the genes into subfamilies, which appear to be conserved in evolution structurally as well as functionally.

Mucin-type linkages (GalNAcalpha1-O-Ser/Thr) are initiated by a family of glycosyltransferases known as the UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases, EC 2.4.1.41). These enzymes transfer GalNAc from the sugar donor UDP-GalNAc to serine and threonine residues, forming an alpha anomeric linkage. Despite the seeming simplicity of ppGaNTase catalytic function, it is estimated on the basis of in silico analysis that there are 24 unique ppGaNTase human genes. ppGaNTase isoforms display tissue-specific expression in adult mammals as well as unique spatial and temporal patterns of expression during murine development. In vitro assays suggest that a subset of the ppGaNTases have overlapping substrate specificities, but at least two ppGaNTases (ppGaNTase-T7 and -T9 [now designated -T10]) appear to require the prior addition of GalNAc to a synthetic peptide before they can catalyze sugar transfer to this substrate. Site-specific O-glycosylation by several ppGaNTases is influenced by the position and structure of previously added O-glycans. Collectively, these observations argue in favor of a hierarchical addition of core GalNAc residues to the apomucin. Various forms of O-glycan pathobiology may be reexamined in light of the existence of an extensive ppGaNTase family of enzymes. Recent work has demonstrated that at least one ppGaNTase isoform is required for normal development in Drosophila melanogaster. Structural insights will no doubt lead to the development of isoform-specific inhibitors. Such tools will prove valuable to furthering our understanding of the functional roles played by O-glycans.

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...

Inhibitors of glycosylation provide a tool for studying the biology of glycoconjugates. Another class of inhibitors consists of glycosides that resemble biosynthetic intermediates involved in glycoconjugate assembly. These compounds act as substrates and produce free oligosaccharides, diverting the assembly of chains from glycoconjugates to the added acceptors. The first type of inhibitor in this class was described >20 years ago by Okayama et aL (7).They showed that f3-D-xylosides stimulate the synthesis of free glycosaminoglycan (GAG) chains and competitively inhibit GAG formation on proteoglycan core proteins (7). The free GAG chains can have desirable biological properties as well. For example, heparan sulfate chains produced on Xyl,3-0-2-naphthol (naphthol-o3-D-xyloside)t will bind to basic fibroblastThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. growth factor, facilitating its interaction with high-affinity receptors (8,9). Recent studies have shown that f3-D-xylosides will prime ganglioside GM3-like compounds and partially I inhibit glycolipid biosynthesis (10). In a similar way, GalNAca-O-benzyl stimulates mucin oligosaccharide synthesis and inhibits 0-linked glycoprotein synthesis (11, 12). Altering glycoprotein synthesis in HL-60 cells in this way inhibits the I expression of sialyl Lewis X [sLex; NeuAca2-*3Gal1,1--> 4(Fuca1->3)GlcNAc-] ligands and adhesion to activated endothelial cells (13).Acceptors consisting of two or more sugars would make this strategy more useful and selective since many glycosyltransferases prefer disaccharides or larger oligosaccharides as substrates (4-6). However, poor transfer of disaccharides across cell membranes severely limits this approach. In this report, we show that decreasing the number of free hydroxyl groups to <5 solves the uptake problem for disaccharides linked to 2-naphthol. Acetylation of the sugars also allows disaccharides to enter the Golgi and prime oligosaccharide chains. MATERIALS AND METHODSSynthesis of Glycosides. The syntheses of Xylp3-0-2-naphthol and L-Araa-0-2-naphthol have been described (8). Gal/3-O-9-phenanthrol, Gal,Bl->3Galf3-O-9-phenanthrol, Gal3l --3Gal3-0-2-naphthol, and Galf3 -4Xyl,B-0-2-naphthol were prepared by reacting the bromo sugar with the sodium salt of the alcohol (A.K.S. and J.D.E., unpublished results). XylJ31-*6Galf3-0-2-naphthol was obtained by reacting acetobromoxylose with Gal,3-0-2-naphthol (Sigma) in the presence of silver carbonate (8). The disaccharide intermediate Xyl(Ac)3f31->6Galf3-O-naphthol was partially methylated by reaction with trimethyloxonium tetrafluoroborate in the presence of 2,6-di(tert-butyl)trimethyl pyridine and the acetyl groups were subsequently removed with sodium methoxide (A.K.S. and J.D.E., unpublished results). Gal,1-*4GlcNAc,3-O-naphthalenemethanol was made by coupling acetylated Galf3-S-C2H5 and 3,6-di-O-benzoylGlcNAcf3-O-n...