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.

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.