Asparagine-Linked Glycans ofCryptosporidium parvumContain a Single Long Arm, Are Barely Processed in the Endoplasmic Reticulum (ER) or Golgi, and Show a Strong Bias for Sites with Threonine.
Cryptosporidium parvumcauses 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) ofC. parvum, because (1) theN-glycan precursor is predicted to contain five mannose and two glucose residues on a single long armversusnine mannose and three glucose residues on the three-armed structure common in hostN-glycans, (2)C. parvumis a rare eukaryote that lacks the machinery forN-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 complexN-glycans, are absent from the predicted proteome. TheC. 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 theC. parvumtryptic peptides, the total ion and extracted oxonium ion chromatograms delineated 32 peptides with occupiedN-glycan sites; these were derived from 16 glycoproteins. Although the number of potentialN-glycan sites with Thr (NxT) is only about twice that with Ser (NxS), almost 90% of the occupiedN-glycan sites contain NxT. The two most abundantC. parvumproteins modified withN-glycans were an immunodominant antigen on the surface of sporozoites (gp900) and the possible oocyst wall protein 1 (POWP1). Seven other glycoproteins withN-glycans were unique toC. 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 ofN-glycan motifs containing Thr.
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.
John R
JR
Haserick
From the ‡Center for Biomedical Mass Spectrometry, Department of Biochemistry, Cell Biology and Genomics, Boston University School of Medicine, Boston, Massachusetts 02118 and.
Deborah R
DR
Leon
§Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts 02118.
John
J
Samuelson
From the ‡Center for Biomedical Mass Spectrometry, Department of Biochemistry, Cell Biology and Genomics, Boston University School of Medicine, Boston, Massachusetts 02118 and cecmsms@bu.edu.
Catherine E
0000-0003-1594-5122
CE
Costello
D001216:Asparagine / Q000378:metabolism; D001665:Binding Sites; D016785:Cryptosporidium parvum / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D015394:Molecular Structure; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D040901:Proteomics / Q000379:methods; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D006023:Glycoproteins; D011134:Polysaccharides; D015800:Protozoan Proteins; D012694:Serine; D001216:Asparagine