cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type VI

Peptidylarginine deiminase (PAD) catalyzes the post-translational modification of protein through the conversion of arginine to citrulline in the presence of calcium ions. Human, similar to rodents, has four isoforms of PAD (type I, II, III and IV/V), each of which is distinct in substrate specificity and tissue specific expression. In our large-scale sequencing project, we identified a new human PAD cDNA from a human fetal brain cDNA library. The putative protein encoded by this cDNA is designated hPADVI. Expression analysis of hPADVI showed that it is mainly expressed in adult human ovary and peripheral blood leukocytes. We conclude that hPADVI may be orthologous to mouse ePAD, basing on sequence comparison, chromosome localization and exon-intron structure analysis. PAD-mediated deimination of epithelial cell keratin resulting in cytoskeletal remodeling suggests a possible role for hPADVI in cytoskeletal reorganization in the egg and in early embryo development. This study describes a new important member of the human PAD family.     

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.     

Functional role of dimerization of human peptidylarginine deiminase 4 (PAD4)

Peptidylarginine deiminase 4 (PAD4) is a homodimeric enzyme that catalyzes Ca²⁺-dependent protein citrullination, which results in the conversion of arginine to citrulline. This paper demonstrates the functional role of dimerization in the regulation of PAD4 activity. To address this question, we created a series of dimer interface mutants of PAD4. The residues Arg8, Tyr237, Asp273, Glu281, Tyr435, Arg544 and Asp547, which are located at the dimer interface, were mutated to disturb the dimer organization of PAD4. Sedimentation velocity experiments were performed to investigate the changes in the quaternary structures and the dissociation constants (K _d) between wild-type and mutant PAD4 monomers and dimers. The kinetic data indicated that disrupting the dimer interface of the enzyme decreases its enzymatic activity and calcium-binding cooperativity. The K _d values of some PAD4 mutants were much higher than that of the wild-type (WT) protein (0.45 µM) and were concomitant with lower k _cat values than that of WT (13.4 s⁻¹). The K _d values of the monomeric PAD4 mutants ranged from 16.8 to 45.6 µM, and the k _cat values of the monomeric mutants ranged from 3.3 to 7.3 s⁻¹. The k _cat values of these interface mutants decreased as the K _d values increased, which suggests that the dissociation of dimers to monomers considerably influences the activity of the enzyme. Although dissociation of the enzyme reduces the activity of the enzyme, monomeric PAD4 is still active but does not display cooperative calcium binding. The ionic interaction between Arg8 and Asp547 and the Tyr435-mediated hydrophobic interaction are determinants of PAD4 dimer formation.