| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
Peptidylarginine deiminase (PAD) was a post-translational modification enzyme that converts the guanidino group of arginine in proteins to the ureido group of citrulline in a calcium-dependent manner. To study the regulation of mouse PAD type II and human PAD type III expression, we have cloned the mouse PAD type II and human PAD type III gene from the mouse and human genomic libraries and PCR. The overall structure of both of mouse PAD type II human PAD type III genes consist of 16 exons interrupted by 15 introns, spanning over 37-38 kb. The promoter activity of the mouse PAD type II and human PAD type III were evaluated by transfecting rodent cells and normal human epidermis keratinocyte cells (NHEK), immortalized epidermis cell line (HaCaT), normal human fibroblast cells (TIG-114), HeLa, and COS-7 cells with a chimeric fusion construct containing the 5' flanking sequence and luciferase reporter gene. The promoter activity of mouse contruct was observed at the 5' flanking region of s
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everal cells. The promoter activity of human construct was 48-fold higher in NHEK cells and 57-fold higher in HaCaT cells than the promoter-less vector. Deletion of sequences at the 5' end of the pGB-2336/+40 construct from nucleotide -2336 to -1898 (pGB-1898/+40), from -2336 to -1498 (pGB-1498/+40), from -2336 to -1071 (pGB-1071/+40), from -2336 to -671 (pGB-671/+40), and from -2336 to -276 (pGB-276/+40) increased luciferase activity. Further deletion of the 5' sequence from nucleotide -2336 to -94 (pGB-94/+40) decreased activity by about 70%. These results suggested that the sequence from nucleotide -277 to -95 is involved in positive regulation and necessary for the essential high activity and that the sequence between nucleotide -1898 and -1071 contains an suppressor element(s). Promoter activity was significantly high in the epidermal keratinocyte cells compared with in the stratified squamous epithelial and non-epithelial cell lines, suggesting a basis for the epidermal keratinocyte-specific expression of the human PAD type III. Less
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