ISOFORMS OF SELENOPROTEIN-P IN RAT PLASMA - EVIDENCE FOR A FULL-LENGTH FORM AND ANOTHER FORM THAT TERMINATES AT THE 2ND UGA IN THE OPEN READING FRAME

Several forms of selenoprotein P that share the same N-terminal sequen ce have been identified in rat plasma, but only one selenoprotein P mR NA has been characterized. The open reading frame of the mRNA contains 10 UGAs that presumably code for selenocysteine residues. Using hepar in-Sepharose, we isolated two of the protein forms from immunoaffinity -purified selenoprotein P. One of the forms, Se-P-45B, migrates at 45 kDa on SDS-polyacrylamide gel electrophoresis, and the other, Se-P-57B , migrates at 57 kDa. These two forms were cleaved with cyanogen bromi de, and both yielded 40-kDa fragments that were consistent with those fragments being an inter-methionine peptide near the N terminus of the predicted polypeptide. A 20-kDa fragment present in the cleavage prod ucts of Se-P-57B was absent from the products of Se-P-45B. This result suggested that Se-P-45B lacks the C-terminal region of the predicted polypeptide. Carboxypeptidase P digestion of Se-P-45B indicated that i ts C-terminal amino acid is Ser(244), the amino acid immediately upstr eam from the predicted second selenocysteine. C-terminal analysis of S e-P-57B indicated that its final residue is Asn(366), the last amino a cid predicted by the cDNA sequence. Amino acid composition analyses of the two forms were consistent with both arising from the same mRNA Im munoaffinity-purified selenoprotein P was digested with proteases, and the resulting peptides were separated and sequenced. Only amino acid sequences predicted by the cDNA were found, and 80% of the predicted a mino acid sequence was confirmed. These results are compatible with Se -P-45B arising from termination of translation at the second in-frame UGA codon and all of the 10 in-frame UGA codons being read through to produce Se-P-57B. These findings demonstrate that selenoprotein P isof orms of differing peptide lengths are present in plasma. They raise th e possibility that the second UGA codon in selenoprotein P mRNA can ha ve alternative functions: coding for the incorporation of selenocystei ne or coding for termination of translation.