cDNA cloning and expression of UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase T1 from Toxoplasma gondii.
We report the cloning, expression, and characterization of the first UDP-GalNAc:polypetide N-acetylgalactosaminyltransferase (ppGalNAc-T) from the human disease-causing parasite, Toxoplasma gondii. This enzyme is also the first characterized ppGalNAc-T of protozoan origin. This type of enzyme catalyzes the initial step of mucin-type O-glycosylation, that is, the transfer of GalNAc in O-glycosidic linkage to serine and threonine residues in polypeptides. We used polymerase chain reaction amplification with degenerate primers and hybridization screening of a T. gondii cDNA library to identify this enzyme. The resulting 84-kDa type II membrane protein contains a 49-amino acid N-terminal cytoplasmic domain, a 22-amino acid hydrophobic transmembrane domain, and a 680-amino acid C-terminal lumenal domain. Conceptual translation of the cDNA sequence reveals a relatively long (i.e. 135 amino acids) stem region and the presence of several important sequence motifs. The latter include a glycosyltransferase 1 (GT1) motif containing a DXH sequence, a Gal/GalNAc-T motif, and a region homologous to ricin lectin. Northern blot analysis identified a single 5.5-kb ppGalNAc-T transcript. Comparison of the cDNA and genomic DNA sequences reveals that this transferase is encoded by 10 exons in a 10 kb region. When the recombinant construct was expressed in stably transfected Drosophila melanogaster S2 cells, the purified protein exhibited transferase activity in vitro. The identification of this enzyme in T. gondii demonstrates that this human parasite has its own enzymatic machinery for the O-glycosylation of toxoplasmal proteins.
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D015723:Gene Library; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D014122:Toxoplasma / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D017350:N-Acetylgalactosaminyltransferases; D011994:Recombinant Proteins; D000097763:Polypeptide N-acetylgalactosaminyltransferase