Mass spectrometric identification of N- and O-glycosylation sites of full-length rat selenoprotein P and determination of selenide-sulfide and disulfide linkages in the shortest isoform.
Rat selenoprotein P is an extracellular glycoprotein of 366 amino acid residues that is rich in cysteine and selenocysteine. Plasma contains four isoforms that differ principally by length at the C-terminal end. Mass spectrometry was used to identify sites of glycosylation on the full-length protein. Of the potential N-glycosylation sites, three located at residues 64, 155, and 169 were occupied, while the two at residues 351 and 356 were not occupied. Threonine 346 was variably O-glycosylated. Thus, full-length selenoprotein P is both N- and O-glycosylated. The shortest isoform of selenoprotein P, which terminates at residue 244, was analyzed for selenide-sulfide and disulfide linkages. In this isoform, a single selenocysteine and seven cysteines are present. Mass spectrometric analysis indicated that a selenide-sulfide bond exists between Sec40 and Cys43. Two disulfides were also detected as Cys149-Cys167 and Cys153-Cys156. The finding of a selenide-sulfide bond in the shortest isoform is compatible with a redox function of this pair that might be analogous to the selenol-thiol pair near the C terminus of animal thioredoxin reductase. The disulfide formed by Cys153-Cys156 also has some characteristics of a redox active pair.
Ma
Shuguang
S
Mass Spectrometry Research Center, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Hill
Kristina E
KE
Burk
Raymond F
RF
Caprioli
Richard M
RM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002240:Carbohydrate Sequence; D004220:Disulfides / Q000737:chemistry*; D006031:Glycosylation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D020033:Protein Isoforms; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012643:Selenium / Q000737:chemistry*; D017279:Selenocysteine / Q000737:chemistry; D051149:Selenoprotein P; D051140:Selenoproteins; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013440:Sulfides / Q000737:chemistry*
D004220:Disulfides; D010446:Peptide Fragments; D020033:Protein Isoforms; D011506:Proteins; D051149:Selenoprotein P; D051140:Selenoproteins; D013440:Sulfides; D017279:Selenocysteine; D012643:Selenium