Molecular analysis of a UDP-GlcNAc:polypeptide α-N-acetylglucosaminyltransferase implicated in the initiation of mucin-type O-glycosylation in Trypanosoma cruzi

Abstract

Trypanosoma cruzi, the causative agent of Chagas disease, is surrounded by a mucin coat that plays important functions in parasite survival/invasion and is extensively O-glycosylated by Golgi and cell surface glycosyltransferases. The addition of the first sugar, alpha-N-acetylglucosamine (GlcNAc) linked to Threonine (Thr), is catalyzed by a polypeptide alpha-GlcNAc-transferase (pp-alphaGlcNAcT) which is unstable to purification. Here, a comparison of the genomes of T. cruzi and Dictyostelium discoideum, an amoebazoan which also forms this linkage, identified two T. cruzi genes (TcOGNT1 and TcOGNT2) that might encode this activity. Though neither was able to complement the Dictyostelium gene, expression in the trypanosomatid Leishmania tarentolae resulted in elevated levels of UDP-[(3)H]GlcNAc:Thr-peptide GlcNAc-transferase activity and UDP-[(3)H]GlcNAc breakdown activity. The ectodomain of TcOGNT2 was expressed and the secreted protein was found to retain both activities after extensive purification away from other proteins and the endogenous activity. Product analysis showed that (3)H was transferred as GlcNAc to a hydroxyamino acid, and breakdown was due to hydrolysis. Both activities were specific for UDP-GlcNAc relative to UDP-GalNAc and were abolished by active site point mutations that inactivate a related Dictyostelium enzyme and distantly related animal pp-alphaGalNAcTs. The peptide preference and the alkaline pH optimum were indistinguishable from those of the native activity in T. cruzi microsomes. The results suggest that mucin-type O-glycosylation in T. cruzi is initiated by conserved members of CAZy family GT60, which is homologous to the GT27 family of animal pp-alphaGalNAcTs that initiate mucin-type O-glycosylation in animals.

Golgi UDP-GlcNAc:Polypeptide O -α- N -Acetyl- d -Glucosaminyltransferase 2 (TcOGNT2) Regulates Trypomastigote Production and Function in Trypanosoma cruzi

“…TcOGNT2 evolved from an ancient lineage of polypeptide ␣HexNAc transferases that also provided cytoplasmic hydroxyproline pp␣GlcNAcTs in other protists and the well-known pp␣GalNAcT family in animals (41) but was specific for Thr as the amino acid acceptor and UDP-GlcNAc as the donor. As expected for this lineage, both activities depended on a DxD-like DxH sequence motif and a divalent cation (21). In comparison to the single Golgi pp␣GlcNAcT (DdGnt2) from Dictyostelium (42,43), however, the T. cruzi enzyme was not expressed well and exhibited low in vitro specific activity and high hydrolysis activity.…”

“…The prediction that the initial step involves only two enzyme isoforms, in contrast to the myriad subsequent processing glycosyltransferases (GTs), implies a potential gatekeeper role which can now be analyzed owing to identification of the genes (21). Here we show that in comparison to proliferating insect-form epimastigotes, differentiated infective MCTs and TCTs downregulate their Golgi-associated pp␣GlcNAcT activities, which is explained at least in part by reduced levels of the TcOGNT2 protein.…”

“…In comparison to the single Golgi pp␣GlcNAcT (DdGnt2) from Dictyostelium (42,43), however, the T. cruzi enzyme was not expressed well and exhibited low in vitro specific activity and high hydrolysis activity. These differences, observed using several peptide substrates, imply that other factors may be important for TcOGNT2 function in the cell, such as its transmembrane anchor, posttranslational modifications, or availability of a limiting factor, such as TcOGNT1 or TcOGNTL (21).…”

“…Major glycoproteins of insect-derived stages are the 35-to 50-kDa mucin-like glycoproteins, NETNES, mucin-associated surface proteins (MASPs), active and inactive trans-sialidases, and peptidases, including gp63-like metalloprotease and isoforms of the cysteine proteinase cruzipain (10)(11)(12)(13)(14)(15)(16). The mucinlike glycoproteins are extensively modified by O-glycans attached via a GlcNAc␣1-Thr linkage (17)(18)(19), whose formation is under the control of UDP-GlcNAc:polypeptide N-acetyl-␣-glucosaminyltransferases (pp␣GlcNAcTs) (20,21). The N-acetyl-␣-D-glucosamine (␣GlcNAc) residue is extended, depending on the parasite strain, at its O-6 and/or O-4 position by ␤-galactopyranose and/or ␤-galactofuranose residues, respectively, which are themselves subject to extension by other ␤Gal residues before capping by terminal ␣Gal or ␣2,3-sialic acid (22).…”

“…TcOGNT2 was heterologously expressed in Leishmania tarentolae as an N-terminally truncated secreted protein, purified to near homogeneity, and found to possess peptide-dependent pp␣GlcNAcT activity and peptide-independent UDP-GlcNAc hydrolytic activity (21). TcOGNT2 evolved from an ancient lineage of polypeptide ␣HexNAc transferases that also provided cytoplasmic hydroxyproline pp␣GlcNAcTs in other protists and the well-known pp␣GalNAcT family in animals (41) but was specific for Thr as the amino acid acceptor and UDP-GlcNAc as the donor.…”