Molecular cloning, expression, and characterization of UDP N-acetyl- α-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 4 from Cryptosporidium parvum

Publication date: Available online 23 March 2018 Source:Molecular and Biochemical Parasitology Author(s): Maria A. DeCicco RePass, Najma Bhat, Jamie Heimburg Molinaro, Stephen Bunnell, Richard D. Cummings, Honorine D. Ward Cryptosporidium spp. are the causative agents of diarrheal disease worldwide, but effective treatments are lacking. Cryptosporidium employs mucin-like glycoproteins with O-glycans to attach to and infect host intestinal epithelial cells. The Tn antigen (GalNAcα1-Ser/Thr) is an O-glycan essential for these processes, as Tn-specific lectins and a Tn-specific monoclonal antibody block attachment to and infection of host cells in vitro. The enzymes in Cryptosporidium catalyzing their synthesis, however, have not been studied. Previously, we identified four genes encoding putative UDP N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) in the genomes of three Cryptosporidium spp. Here we report the in silico analysis, cloning, expression, purification and characterization of one of the four enzymes Cryptosporidium parvum (Cp)-ppGalNAc-T4. This enzyme contains the characteristic domains and motifs conserved in ppGalNAc-Ts and is expressed at multiple time points during in vitro infection. Recombinant soluble Cp-ppGalNAc-T4 was enzymatically active against an unmodified EA2 peptide suggesting that it may function as an “initiating” ppGalNAc-T. Cp-ppGalNAc-T4 also exhibited a strong preference for UDP-GalNAc over ot...

Source: Molecular and Biochemical Parasitology - March 24, 2018 Category: Parasitology Source Type: research