Molecular cloning, expression, and characterization of UDP N-acetyl-α-Dgalactosamine: Polypeptide N-acetylgalactosaminyltransferase 4 from Cryptosporidium parvum

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 (GaINAcal-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-a-o-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) in the genomes of three Cryptosporidium spp. Here we report the in silico analysis, cloning, ex pression, 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 a 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-GaINAc over other nucleotide sugar donors and was active agains unmodified and 0-glycosylated versions of the C. parvum gp40-derived peptide, with a preference for the former suggesting it may play a role in modifying this glycoprotein in vivo. Given the importance of mucin-type 01 glycosylation in Cryptosporidium spp., the enzymes that catalyze their synthesis may serve as potential therapeutic targets.