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 other
nucleotide sugar donors and was active against unmodified and O-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 O-glycosylation in Cryptosporidium spp., the
enzymes that catalyze their synthesis may serve as potential therapeutic targets.