Human peptidylarginine deiminase type II: molecular cloning, gene organization, and expression in human skin.
Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that convert protein arginine to citrulline residues in a calcium ion-dependent manner. Rodents have four isoforms of PAD (types I, II, III, and IV), each of which is distinct in substrate and tissue specificity. In fact, the only tissue in which all four PAD mRNAs have been detected is the epidermis. In this study, we found PAD activity in HSC-1 human cutaneous squamous carcinoma cells in vitro, and this activity increased during cultivation. Using a homology-based strategy, we cloned a full-length cDNA encoding human PAD type II. The cDNA was 2348 bp long and encoded a 665-amino-acid sequence with a predicted molecular mass of 75 kDa. The predicted protein shared 93% identity with the rat and mouse PAD type II sequence. Alignment of the amino acid sequences from both species revealed notable conservation in the C-terminal region, suggesting the presence of a functional region such as an enzyme catalytic site and/or a calcium-binding domain. Gene organization analysis established that human PAD type II on chromosome 1p35.2-p35.21 spanned more than 50 kb and contained 16 exons and 15 introns. A recombinant PAD protein subsequently produced in Escherichia coli proved to be enzymatically active, with substrate specificities similar to those of the rat PAD type II. In an immunohistochemical study of human skin, the type II enzyme was expressed by all the living epidermal layers, suggesting that PAD type II is functionally important during terminal differentiation of epidermal keratinocytes.
Ishigami
Akihito
A
Department of Molecular Pathology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo, Japan. ishigami@tmig.or.jp
Ohsawa
Takako
T
Asaga
Hiroaki
H
Akiyama
Kyoichi
K
Kuramoto
Masashi
M
Maruyama
Naoki
N
D000595:Amino Acid Sequence; D002294:Carcinoma, Squamous Cell / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D005091:Exons; D005260:Female; D040461:Gene Components; D015870:Gene Expression; D006801:Humans; D006867:Hydrolases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D007438:Introns; D015603:Keratinocytes / Q000201:enzymology; D008875:Middle Aged; D008969:Molecular Sequence Data; D000080064:Protein-Arginine Deiminase Type 2; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012867:Skin / Q000201:enzymology*; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D006867:Hydrolases; C461539:Padi2 protein, rat; D000080064:Protein-Arginine Deiminase Type 2