Functional control of polypeptide GalNAc-transferase 3 through an acetylation site in the C-terminal lectin domain (original) (raw)

2017, Biological Chemistry

Glycan biosynthesis occurs mainly in Golgi. Molecular organization and functional regulation of this process are not well understood. We evaluated the extrinsic effect of lectin domains (␤-trefoil fold) of polypeptide GalNAc-transferases (ppGalNAc-Ts) on catalytic activity of glycosyltransferases during O-GalNAc glycan biosynthesis. The presence of lectin domain T3lec or T4lec during ppGalNAc-T2 and ppGalNAc-T3 catalytic reaction had a clear inhibitory effect on GalNAc-T activity. Interaction of T3lec or T4lec with ppGalNAc-T2 catalytic domain was not mediated by carbohydrate. T3lec, but not T2lec and T4lec, had a clear activating effect on Drosophila melanogaster core 1 galactosyltransferase enzyme activity and a predominant inhibitory effect on in vivo human core 1 glycan biosynthesis. The regulatory role of the ␤-trefoil fold of ppGalNAc-Ts in enzymatic activity of glycosyltransferases involved in the O-glycan biosynthesis pathway, described here for the first time, helps clarify the mechanism of biosynthesis of complex biopolymers (such as glycans) that is not template-driven. Glycobiology is the study of biosynthesis, structure, function, and evolution of naturally occurring glycans and the proteins that recognize them. Glycan biosynthesis consists of the polymerization of sugars in an "assembly line" in which enzymes (glycosyltransferases (glycosyl-Ts) 2) catalyze the synthesis of glycosidic linkages by saccharide transfer from sugar donor to an acceptor. In contrast to synthesis of DNA, RNA, or protein molecules, glycosylation is not template-driven; rather the monosaccharide sequence is determined by the action of specific glycosyl-Ts (1). Glycan biosynthesis occurs mainly in Golgi. Molecular organization and functional regulation of this process are not well understood. A self-assembled enzyme complex has been proposed as a mechanism responsible for correct positioning of glycosyl-Ts in Golgi (2, 3). According to this model, glycosyl-T domains facilitate protein/protein interactions related to formation of a "low affinity/high specificity" complex that controls enzyme ratios and optimizes flow in the sugar assembly line. In O-GalNAc glycan (mucin) biosynthesis, the first step is the transfer of N-acetylgalactosamine (GalNAc) from the sugar donor, UDP-GalNAc, to selected Ser/Thr residues of the acceptor protein to yield GalNAc␣1-O-Ser/Thr (Tn antigen) (4). This key initial step is catalyzed by a multigene family of enzyme polypeptide GalNAc-transferases (ppGalNAc-Ts). Various ppGalNAc-T isoforms are differentially expressed in mammalian cells and tissues during development and differentiation. The second monosaccharide linked to GalNAc␣1-O-Ser/Thr may be galactose (Gal) or N-acetylglucosamine (GlcNAc) to yield core 1 (Gal␤3GalNAc␣1-O-Ser/Thr) or core 3 (GlcNAc␤3GalNAc␣1-O-Ser/Thr) glycan, respectively. Core 1 glycan biosynthesis involves core 1 ␤3Gal-T (C1GalT), a ubiquitous enzyme found in most mammalian cells. Core 3 ␤3GlcNAc-T catalyzes biosynthesis of core 3 glycan, the predominant glycan in colonic and salivary mucins (5). Core 2 and core 4 glycans are the products of ␤6-GlcNAc-T action on GalNAc␣ of core 1 and core 3, respectively. Addition of repeating Gal␤3/4GlcNAc units gives rise to the backbone region of glycans. The peripheral regions of saccharide chains display high structural diversity. Fucose and N-acetylneuraminic acid are frequent capping residues in these regions. In studies of structure-function relationships, ppGalNAc-Ts were shown to be type II transmembrane proteins containing a short N-terminal cytoplasmic tail, a small transmembrane anchor, and a Golgi lumenal region. The lumenal region consists of a stem section, catalytic domain, and C terminus. This C-terminal domain has sequence and predicted structural homology to a lectin. Lectins are glycan-binding proteins that play important roles in mammalian cellular homeostasis (6). Some lectins have bifunctional properties; they have both a glycanbinding site and a pocket involved in protein/protein interac-* This work was supported by funding from Argentinean Institutions Consejo