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

Abstract

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.

N- and O-Glycosylation Pathways in the Microalgae Polyphyletic Group

“…For example, in Toxoplasma gondii, Cryptosporidium parvum, and Tetrahymena pyriformis, the luminal ALG responsible for the addition of the four last Man residues are absent. Thus, the synthesis of the oligosaccharide precursor stops prematurely leading to a structure harboring only five mannose residues (Yagodnik et al, 1987;Garénaux et al, 2008;Haserick et al, 2017). Moreover, in Cryptococcus neoformans, Trypanosoma brucei, and Trypanosoma cruzi, ALG6, ALG8 and ALG10 are missing, resulting in a non-glucosylated precursor Man 9 GlcNAc 2 -PP-Dol (De La Canal and Parodi, 1993).…”

Comparative Pathobiology of the Intestinal Protozoan Parasites Giardia lamblia, Entamoeba histolytica, and Cryptosporidium parvum

“…This result is of particular interest for the poultry industry where the closely related Eimeria constitute a major problem [132]. As a consequence, there is a constant and continuing effort to identify suitable candidates such as, for example, protein p23 [133], a rhomboid protein [134], or surface glycoproteins [135] as vaccine candidates, either as recombinant proteins, DNA vaccine or expressed in a suitable bacterial strain [136].…”

Glycopeptidomics Analysis of a Cell Line Model Revealing Pathogenesis and Potential Marker Molecules for the Early Diagnosis of Gastric MALT Lymphoma

“…Signal peptides and transmembrane helices were predicted using the online tool Phobius were mapped with all the peptides identified from all MS/MS experiments, were generated using the online software tool Protter v.1.0 [20]. The N-and O-glycosylation sites of specific proteins were predicted using the online software NetNGlyc 1.0 Server and NetOGlyc 4.0 Server [21], respectively.…”