Asparagine-Linked Glycans of Cryptosporidium parvum Contain a Single Long Arm, Are Barely Processed in the Endoplasmic Reticulum (ER) or Golgi, and Show a Strong Bias for Sites with Threonine

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.

Fig. 1.

MALDI-TOF MS: released, deutero-reduced, permethylated N-glycans [M + Na]¹⁺. Total N-glycans released from C. parvum glycoproteins with PNGase-F, reduced with sodium borodeuteride, and permethylated. Only two glycoforms are observed: Hex₅HexNAc₂ and Hex₆HexNAc₂.

Fig. 2.

Topology of the most abundant glycoform Hex₆HexNAc₂ determined by EED FT-ICR MS/MS. 14-eV EED FT-ICR MS/MS: Hex₆HexNAc₂ [M + Na]¹⁺ m/z 1800.9192. Glycosidic fragments provide topological information. The spectrum is labeled only with glycosidic fragments that indicate the topology of the glycoform, revealing a single long arm and an unmodified core. All assignments can be viewed in supplemental table Excel S1.

Fig. 3.

Glycosidic linkage determination of the most abundant glycoform Hex₆HexNAc₂ determined by EED FT-ICR MS/MS. 14-eV EED FT-ICR MS/MS: Hex₆HexNAc₂ [M+Na]¹⁺ m/z 1800.9192. Cross-ring fragments provide linkage information. The spectrum is labeled with only the informative cross ring fragments that provide linkage information. All assignments can be viewed in supplemental table Excel S1.

Fig. 4.

De novo identification of glycopeptides from a whole C. parvum oocyst lysate. A, Base peak and extracted oxonium ion chromatograms from a reversed phase C18 separation. The top trace shows the base peak chromatogram from the MS. The middle and bottom chromatograms are extracted oxonium ion chromatograms (XIC) from the 35-V HCD MS/MS spectra, corresponding to m/z 204.08 (HexNAc) and m/z 366.13 (HexNAc-Hex), respectively. The carat located at 15 min. indicates the time point for recording of the MS/MS spectrum shown in Fig. 4B and 4C. B, 35-V HCD MS/MS spectrum of an N-glycosylated peptide: NSTTEVR modified with Hex₆HexNAc₂, [M + 2H]²⁺ m/z 1092.9426. Prominent glycosidic bond fragmentation is observed, delineating the sequence of the glycan. C, Peptide sequence of the aglycon. Lower intensity y-ion peptide backbone fragments are observed in the same spectrum. Peptide fragment ion assignments are shown on this magnified view; these extend from y1 (Arg) at m/z 175.1189 to the complete aglycon, m/z 806.3992, defining the complete peptide sequence as NSTTEVR.

Fig. 5.

Occupied N-glycosylation sites of POWP1 (cgd2_490). The most densely N-glycosylated protein is represented as a cartoon schematic. The occupied peptides are shown; the bold and italicized asparagine residues indicate the site of attachment of the glycan.

Fig. 6.

Total and occupied N-glycosylation sequons in the observed N-glycosylated proteins. A, Web-Logos, generated using WebLogo 3.5.0, were compiled for all the peptides containing the canonical N-glycosylation sequons from proteins observed to be glycosylated (Total). B, Peptides observed to be occupied are represented by the (Occupied) logo.