Abstract
Cryptosporidium parvum causes severe diarrhea in infants in developing countries and in immunosuppressed persons, including those with AIDS. We are interested in the Asn-linked glycans (N-glycans) of C. parvum, because (1) the N-glycan precursor is predicted to contain five mannose and two glucose residues on a single long arm versus nine mannose and three glucose residues on the three-armed structure common in host N-glycans, (2) C. parvum is a rare eukaryote that lacks the machinery for N-glycan-dependent quality control of protein folding in the lumen of the Endoplasmic Reticulum (ER), and (3) ER and Golgi mannosidases, as well as glycosyltransferases that build complex N-glycans, are absent from the predicted proteome. The C. parvum N-glycans reported here, which were determined using a combination of collision-induced dissociation and electronic excitation dissociation, contain a single, unprocessed mannose arm ± terminal glucose on the trimannosyl chitobiose core. Upon nanoUPLC-MS/MS separation and analysis of the C. parvum tryptic peptides, the total ion and extracted oxonium ion chromatograms delineated 32 peptides with occupied N-glycan sites; these were derived from 16 glycoproteins. Although the number of potential N-glycan sites with Thr (NxT) is only about twice that with Ser (NxS), almost 90% of the occupied N-glycan sites contain NxT. The two most abundant C. parvum proteins modified with N-glycans were an immunodominant antigen on the surface of sporozoites (gp900) and the possible oocyst wall protein 1 (POWP1). Seven other glycoproteins with N-glycans were unique to C. parvum; five shared common ancestry with other apicomplexans; two glycoproteins shared common ancestry with many organisms. In summary, C. parvum N-glycans are remarkable for the absence of ER and Golgi modification and for the strong bias toward occupancy of N-glycan motifs containing Thr.
Highlights
From the ‡Center for Biomedical Mass Spectrometry, Department of Biochemistry, Cell Biology and Genomics, Boston University School of Medicine, Boston, Massachusetts 02118 and §Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts 02118
The C. parvum N-glycans are much simpler than those of calf glycoproteins, suggesting oocysts, which are washed with phosphate buffered saline (PBS) and purified on a CsCl gradient, are clean of host tissues [56]
If branching were present in this tetrasaccharide moiety, there would be a gap in the linear series, and double glycosidic bond fragments might be observed along the chain
Summary
HCD MS2 spectra containing oxonium ions were manually interpreted to determine the peptide sequence and the linear arrangement of the glycan. All spectra that were assigned by the software as possible N-glycosylated peptides were manually reviewed for quality and proper assignment to compile the final lists of glyopeptides and proteins that are available in supplemental table Excel S3.
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