Cryptosporidium parvum vaccine candidates are incompletely modified with O-linked-N-acetylgalactosamine or contain N-terminal N-myristate and S-palmitate

Cryptosporidium parvum (studied here) and Cryptosporidium hominis are important causes of diarrhea in infants and immunosuppressed persons. C . parvum vaccine candidates, which are on the surface of sporozoites, include glycoproteins with Ser- and Thr-rich domains (Gp15, Gp40, and Gp900) and a low complexity, acidic protein (Cp23). Here we used mass spectrometry to determine that O -linked GalNAc is present in dense arrays on a glycopeptide with consecutive Ser derived from Gp40 and on glycopeptides with consecutive Thr derived from Gp20, a novel C . parvum glycoprotein with a formula weight of ~20 kDa. In contrast, the occupied Ser or Thr residues in glycopeptides from Gp15 and Gp900 are isolated from one another. Gly at the N-terminus of Cp23 is N -myristoylated, while Cys, the second amino acid, is S -palmitoylated. In summary, C . parvum O -GalNAc transferases, which are homologs of host enzymes, densely modify arrays of Ser or Thr, as well as isolated Ser and Thr residues on C . parvum vaccine candidates. The N-terminus of an immunodominant antigen has lipid modifications similar to those of host cells and other apicomplexan parasites. Mass spectrometric demonstration here of glycopeptides with O -glycans complements previous identification C . parvum O -GalNAc transferases, lectin binding to vaccine candidates, and human and mouse antibodies binding to glycopeptides. The significance of these post-translational modifications is discussed with regards to the function of these proteins and the design of serological tests and vaccines.