Several forms of selenoprotein P that share the same N-terminal sequence have been identified in rat plasma, but only one selenoprotein P mRNA has been characterized. The open reading frame of the mRNA contains 10 UGAs that presumably code for selenocysteine residues. Using heparin-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 bromide, 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 products 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 its C-terminal amino acid is Ser(244), the amino acid immediately upstream from the predicted second selenocysteine. C-terminal analysis of Se-P-57B indicated that its final residue is Asn(366), the last amino acid predicted by the cDNA sequence. Amino acid composition analyses of the two forms were consistent with both arising from the same mRNA Immunoaffinity-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 amino 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 isoforms of differing peptide lengths are present in plasma. They raise the possibility that the second UGA codon in selenoprotein P mRNA can have alternative functions: coding for the incorporation of selenocysteine or coding for termination of translation.
