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Abstract
The objective of this study was to further characterize selenoprotein W using molecular biology techniques in order to obtain a greater understanding of the metabolic importance of this selenoprotein. The cDNAs for skeletal muscle selenoprotein W from human, rhesus monkey, sheep and mouse were cloned and compared to rat selenoprotein W cDNA, which was sequenced in previous studies. Selenocysteine insertion sequence (SECIS) elements were identified in the 3$\prime$ untranslated regions of selenoprotein W mRNAs. A feature of the rodent and sheep selenoprotein W mRNAs was that UGA was used for directing both selenocysteine incorporation and protein termination. Selenoprotein W coding sequences and their predicted amino acid sequences were respectively 80% and 83% identical among five species examined. Polyclonal antibodies raised against a rat selenoprotein W peptide failed to recognize selenoprotein W in primates. Antibodies raised against bacterially-expressed mutant human selenoprotein W (Secys $\rightarrow$ Cys) were successfully used in Western blots to detect selenoprotein W in primate tissues. Selenoprotein W was found in many tissues but was highest in the muscle and heart of rhesus monkeys fed a commercial chow. Selenoprotein W mRNA levels were estimated by Northern blots and shown to be highest in muscle and heart in both rhesus monkey and human tissues. Using MALDI mass spectrometry, selenoprotein W purified from monkey muscle revealed three forms with masses of 9330, 9371 and 9635 Daltons. The lowest mass form is in agreement with the theoretical mass deduced from the monkey selenoprotein W cDNA sequence. The highest mass form was shown to be the lowest mass form which contained glutathione bound through a disulfide bond. The binding site was tentatively identified as the 36th animo acid (cysteine). The other mass form, 9371 Da, is assumed to be the lowest mass form containing an unidentified small moiety (41 Da). Selenoprotein W gene expression was constitutive in cultured rat skeletal muscle cells. Reduction of the selenium concentration in the culture medium decreased the selenoprotein W mRNA levels. Nuclear run-on assays indicated that the transcription rate of the selenoprotein W gene is independent of selenium. Selenium supplementation increased the selenoprotein W mRNA half life. Regulation of selenoprotein W by selenium in rat skeletal muscle cells occurs at the post-transcriptional level.
