Sulfhydryl oxidase (SOx) from mouse epidermis: molecular cloning, nucleotide sequence, and expression of recombinant protein in the cultured cells.
Skin sulfhydryl oxidase (SOx) is an enzyme that catalyzes disulfide (S-S) cross-linking through the oxidation of sulfhydryl compounds in the skin. In this study, using the enzyme purified from rat seminal vesicle, we obtained peptide sequences for SOx by mass spectrometry. We then searched for SOx nucleotides corresponding highly to the rat peptide sequences by assembling murine-expressed sequence tags (ESTs) from the GeneBank database. The assembled mouse SOx cDNA has an open reading frame of 1704-bp nucleotides, translating into a size of 568 amino acids. The calculated molecular mass of the mouse SOx protein is 65 kDa. This mouse sequence can be amplified from total RNAs of various mouse tissue samples by reverse transcription polymerase chain reaction, especially highly amplified from those of the seminal vesicles and epidermis. The cDNA fragment was subsequently cloned into the mammalian expression vector (pTARGET-MSSOx), allowing us to express mouse recombinant SOx protein in cultured cells. When pTARGET-MSSOx was transfected, Western blot analysis using anti-SOx antiserum could detect a 65 kDa-band of recombinant SOx in both samples from the whole cell extract and the medium after the harvest of the HEK cells. In immunohistochemical analysis, the Pt-K2 cells, following the introduction of pTARGET-MSSOx, seemed to generate a SOx protein reactive to anti-SOx antiserum in the cells. Moreover, the indirect staining of the S-S bonds using N-(7-dimethylamino-4-methyl coumarinyl) maleimide (DACM), following the addition of N-ethylmaleimide and dithiothreitol, showed that the formation of S-S bridges almost matched the localization of SOx expression in the Pt-K2 cells after the transfection. In essence, we cloned skin SOx cDNA and characterized it as one of the S-S cross-linking enzymes. The SOx clone from mouse epidermis seems to be useful for investigating the potential function of the enzyme in the epidermis, especially for understanding the physiological role of SOx in the differentiation of keratinocytes.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004817:Epidermis / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012669:Seminal Vesicles / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D050862:Oxidoreductases Acting on Sulfur Group Donors
D018076:DNA, Complementary; D010088:Oxidoreductases; D012313:RNA; D011994:Recombinant Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors