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

John R Haserick,Joshua A Klein,John Samuelson,Catherine E Costello,Silvia N Moreno

doi:10.1371/journal.pone.0182395

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.

Highlights

C. parvum infects humans and cows, while C. hominis only infects humans [1,2,3]
Peptides obtained from trypsin digestion of total proteins of C. parvum oocysts were separated using a Ultra Performance Liquid Chromatography (UPLC) reversed phase C18 column that was online with a mass spectrometer
Since there was no enrichment for glycoproteins in the protein preparations, we identified the most abundant glycopeptides without selection bias

Summary

Introduction

C. parvum infects humans and cows, while C. hominis only infects humans [1,2,3]. C. parvum was first identified as an opportunistic pathogen and cause of severe diarrhea in AIDS patients [4, 5]. The presence of O-glycans (most likely O-GalNAc) on C. parvum glycoproteins has not previously been detected by mass spectrometry, but it has been suggested on the basis of the following five observations: (1) The C. parvum genome predicts four O-GalNAc transferases (O-GalNAcTs), and parasite lysates add O-GalNAc to synthetic peptides [32]. Mass spectrometric studies of C. parvum sporozoites and oocysts have identified numerous peptides from Gp40, Gp15, Gp900, and Cp23, but none of these studies localized post-translational modifications (PTMs), which may include O-linked glycans, Asn-linked glycans (N-glycans), and fatty acyl chains [15, 22, 32, 33, 46,47,48,49,50,51]. Mass spectrometry of hydrophobic peptides detected the addition of myristoyl and palmitoyl groups to the first and second residues, respectively, at the N-terminus of Cp23 [56, 57]

Materials and methods

Results

Discussion