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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.

Studies of heparan sulfate biosynthesis on beta-D-xylosides have led to the hypothesis that heparan sulfate alpha-N-acetylglucosaminyltransferase I (alpha-GlcNAc-TI) recognizes structures at the reducing end of the proteoglycan linkage tetrasaccharide. We report here the in vivo and in vitro testing of this hypothesis using four synthetic substrates, benzyl- and 2-naphthalenemethanyl-beta-D-xylosides, and two proteoglycan linkage tetrasaccharides containing benzyl alcohol or naphthalmethanol aglycones, viz., GlcAbeta(1 --> 3)Gal beta(1 --> 3)Gal beta(1 --> 4)Xyl beta-O-Bn (BNT) and GlcAbeta(1 --> 3)Gal beta(1 --> 3)Gal beta(1 --> 4)Xyl beta-O-NM (NMT). The aryl tetrasaccharides were chemically synthesized and the 1H and 13C resonances were assigned by two-dimensional NMR spectroscopy. The inter-residue spatial constraints, determined by the 2D NOESY data, revealed essentially identical conformations for the interglycosidic linkages and Xyl-O-CH2Ar linkages in both compounds. Interestingly, the aromatic rings in both tetrasaccharides undergo rapid internal rotation across the CH2-Ar bond. These tetrasaccharides were used to assay heparan sulfate alpha-GlcNAc-TI from homogenates of wild-type CHO cells. alpha-GlcNAc-TI was also purified approximately 900-fold from rat liver and assayed with BNT and NMT. At nearly all concentrations tested, alpha-GlcNAc-TI activity from both CHO cell homogenates and rat liver was greater with the NMT. When fed to CHO cells, benzyl-beta-D-xyloside primed heparan sulfate poorly relative to 2-naphthalenemethanyl-beta-D-xyloside. Thus, the in vitro enzyme activity is consistent with the in vivo priming data that suggests that alpha-GlcNAc-TI can directly recognize structure at the reducing end of the linkage tetrasaccharide. These studies provide an in vivo basis for the possible role of core protein sequences in the biosynthesis of specific glycosaminoglycans.