Functional Characterization of Drosophila melanogaster Peptide O-Xylosyltransferase, the Key Enzyme for Proteoglycan Chain Initiation and Member of the Core 2/I N-Acetylglucosaminyltransferase Family

Abstract

Chondroitin and heparan sulfates are essential players in animal development and are synthesized by a series of glycosyltransferases, the first of which is UDP-␣-D-xylose:proteoglycan core protein ␤-D-xylosyltransferase (EC 2.4.2.26). In the present study, a Drosophila melanogaster gene (CG17771), previously designated as a homologue of core 2 and I ␤1,6-N-acetylglucosaminyltransferases, was shown to encode an active peptide O-xylosyltransferase. A novel coupled assay using matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated transfer of xylose to the peptide DDDSIEGSGGR. Analysis of sequences of various peptide O-xylosyltransferase and ␤1,6-N-acetylglucosaminyltransferase sequences indicates that they are members of a large multifunctional protein family with a range of roles in ␤-glycosylation of either peptide or glycan substrates. Because in contrast to mammals, there is only one fly peptide O-xylosyltransferase gene, it is anticipated that, given the key roles of proteoglycans, the hereby designated oxt gene is essential for viability.

Plasmodium falciparum ookinetes require mosquito midgut chondroitin sulfate proteoglycans for cell invasion

“…3a, in bold). Unlike in mammals, for Drosophila (and putatively for other dipterans) the activity of a CS GalNAc-transferase I (GalNAcT-I) as opposed to an N-acetylglucosaminyl transferase I (GlcNAcT-I) determine the initiation and progression of the chain toward either a CSGAG or HSGAG, respectively (16)(17)(18) (Fig. 3a).…”

“…As with human and Drosophila OXTs, AgOXT1 is a type II transmembrane protein, with a WSC motif glycanbinding domain (GBD) and glycosyltransferase family 14 domains ( Fig. 3 b and c), but lacks the characteristic Asp-Xaa-Asp (DXD) motif present in other family members and is involved in coordination of glycan substrates (16). The functional relevance of the lack of a DXD motif in AgOXT1 is not readily apparent, although it has been shown that nematode OXT, which also lacks this motif, is enzymatically active (15).…”

“…CSGAG dissacharide units are attached to a tetrasaccharide core composed of a GlcA, two galactose (Gal) units and a xylose (Xyl) residue (GlcA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-Ser) that is glycosidically linked to the hydroxyl group of serines on proteoglycan polypeptides (15). CSGAGs vary in the degree and pattern of sulfation through the differential action of carbon 2-O, 4-O, and 6-O sulfotransferases acting on GalNAc and the combination of A-E units (16). In mammals, the addition of Xyl by a peptide-O-xylosyltransferase (OXT) to the Ser residue on a proteoglycan core initiates the sequential formation of the tetrasaccharide core (Fig.…”

“…3a). Anopheles homologs of Drosophila melanogaster GAG biosynthetic enzymes, including OXT, as well as the genes encoding several proteoglycans have not been characterized (16,19). An An.…”

Proteoglycan UDP-Galactose:β-Xylose β1,4-Galactosyltransferase I Is Essential for Viability inDrosophila melanogaster

“…Biochemical analysis of GAGs has demonstrated that both Caenorhabditis elegans and Drosophila melanogaster have HS and CS (11,12). Recently, Drosophila peptide O-XylT has been reported to transfer xylose (Xyl) to the syndecan peptide (13). But it is still unknown which Drosophila ␤4GalT works as proteoglycan ␤4GalTI.…”