Molecular cloning of cDNAs of mouse peptidylarginine deiminase type I, type III and type IV, and the expression pattern of type I in mouse

Abstract

Peptidylarginine deiminases (PADs), a group of post-translational enzymes, catalyze the conversion of proteinbound arginine residues to citrulline residues in a calcium ion-dependent manner and are widely distributed in various organs of vertebrates. Although the existence of four isoforms of PAD (types I, II, III, and IV) is reported in rodents, the relative functions of the isoforms with respect to their colocation in the tissues have yet to be explored. In this study, we cloned the full-length cDNA encoding mouse PAD type I by screening a uterine cDNA library and using the RACE method. This cDNA consists of an open reading frame of 1989 bases encoding 662 amino acids (73 823 Da), a 5 H -untranslated region of 127 bases and a 3 H -untranslated region of 1639 bases. Comparative reverse transcription-PCR and Northern-blot analyses detected PAD type I mRNA only in the epidermis and uterus. Administration of estrogen to adult ovariectomized mice increased the content of PAD type I mRNA in the uterus, providing evidence that its expression is under the control of the sex steroid hormone. We also cloned the full-length cDNAs of mouse PAD type III and type IV by the reverse transcription-PCR and RACE methods. The primary structure of PAD type III contains 664 amino acids (75 098 Da) deduced from the coding region of 1995 bases, and the primary structure of PAD type IV consists of 666 amino acids (74 475 Da) deduced from the coding region of 2001 bases. Comparison of the deduced amino acid sequences of all four isoforms of PAD showed about 50% identity with each other, the 3 H regions being highly homologous compared with the 5 H regions.

Peptidylarginine deiminase 2 (PAD2) overexpression in transgenic mice leads to myelin loss in the central nervous system

“…Mobilization of PAD4 into the nucleus may be an additional mechanism that contributes to oligodendrocyte apoptosis. generous gift from Dr A. P. Nicholas, University of Alabama at Birmingham, AL), which recognizes peptidylcitrulline; (3) modified citrulline detection kit (Upstate Biotech, Lake Placid, NY) containing rabbit anti-citrulline (modified) and goat anti-rabbit IgG HRP, which was also used for the detection of citrulline-containing proteins; (4) rabbit anti-pan PAD (a gift from Dr H. Takahara, Ibaraki University, Ibaraki, Japan), which recognizes all isoforms of PAD (Takahara et al, 1989;Terakawa et al, 1991;Nishijyo et al, 1997;Rus'd et al, 1999), and rabbit anti-PAD2 polyclonal antibody (Abcam, Cambridge, MA), which recognizes only PAD2; (5) rabbit anti-histone H3 polyclonal antibody Ab1791 (Abcam); and (6) rabbit anti-GFAP IgG (DAKO, Glostrup, Denmark). The secondary antibodies used were goat anti-mouse and goat anti-rabbit IgG HRP conjugate (Bio-Rad Laboratories, Hercules, CA).…”

The peptidylarginine deiminases expressed in human epidermis differ in their substrate specificities and subcellular locations

“…Recently, in our laboratory, the family of human PADs was fully characterized at the genomic level as five clustered paralogous genes (PADI1, 2, 3, 4 and 6) on chromosome 1p35-36 [5]. Human PADs are highly conserved, with percentages of identity around 80 % for cDNA and 50 % for amino acid sequences [5][6][7][8][9][10]. PADs have been detected in numerous organs of humans and rodents by activity measurements and RT-PCR analysis [5,[10][11][12][13].…”

Methods for the Detection of Peptidylarginine Deiminase (PAD) Activity and Protein Citrullination

“…In humans, five different PAD isotypes exist, PAD1-4 and PAD6, which have ϳ50% sequence similarity (2). PAD enzymes are distributed over a wide range of cells and tissues and each isotype has a tissue-specific expression pattern (3)(4)(5)(6). They have been reported to be involved in hair growth, myelin formation, the regulation of gene expression and many other processes (reviewed in (7)).…”