Cloning, expression, and characterization of stratum corneum chymotryptic enzyme. A skin-specific human serine proteinase.
The cDNA encoding human stratum corneum chymotryptic enzyme (SCCE); an epidermal serine-proteinase which was recently purified from human stratum corneum, was isolated from a keratinocyte derived library. The obtained nucleotide sequence contained an open reading frame sufficient to encode a preproprotein consisting of 253 amino acid residues. Expression of two mRNA species hybridizing with SCCE cDNA, 1.2 and 2.0 kilobases, respectively, was detected to human skin. These two forms differ with respect to the length of the 3'-untranslated sequence. Analysis of mRNA derived from various human tissues showed that abundant expression of the SCCE gene was restricted to human skin. The cloned cDNA was introduced to a bovine papilloma virus-based expression system and recombinant protein was purified and characterized. The results show that recombinant SCCE is produced with a 22-amino acid residue signal peptide and a propeptide of 7 amino acid residues. Tryptic digestion removed this propeptide and yielded a proteolytically active protein with the same NH2-terminal amino acid sequence as native SCCE. The deduced amino acid sequence contains the conserved active site regions of serine proteinases. The calculated molecular mass of unglycosylated active SCCE was 24.4 kDa. The sequence indicates one tentative N-glycosylation site located near the C terminus. Recombinant SCCE was found to be heterogenous regarding glycosylation in a manner similar to that of the native enzyme.
Symbicom AB, Umeå, Sweden.
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L
Hansson
M
M
Strömqvist
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A
Bäckman
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P
Wallbrandt
A
A
Carlstein
T
T
Egelrud
D000005:Abdomen; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004730:Endothelium, Vascular; D004817:Epidermis / Q000201:enzymology*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007610:Kallikreins; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014471:Umbilical Veins
D018076:DNA, Complementary; D011498:Protein Precursors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; C082472:KLK7 protein, human; D007610:Kallikreins; D012697:Serine Endopeptidases