Three isozymes of peptidylarginine deiminase in the chicken: molecular cloning, characterization, and tissue distribution

Abstract

Peptidylarginine deiminase (PAD; EC 3.5.3.15) is a post-translational modification enzyme that catalyzes the conversion of protein-bound arginine to citrulline (deimination) in a calcium ion dependent manner. Although PADI genes are widely conserved among vertebrates, their function in the chicken is poorly understood. Here, we cloned and sequenced three chicken PADI cDNAs and analyzed the expression of their proteins in various tissues. Immunoblotting analysis showed that chicken PAD1 and PAD3 were present in cells of several central neuron system tissues including the retina; the chicken PAD2 protein was not detected in any tissue. We expressed recombinant chicken PADs in insect cells and characterized their enzymatic properties. The chicken PAD1 and PAD3 recombinant proteins required calcium ions as an essential cofactor for their catalytic activity. The two recombinant proteins showed similar substrate specificities toward synthetic arginine derivatives. By contrast to them, chicken PAD2 did not show any activity. We found that one of the conserved active centers in mammalian PADs had been altered in chicken PAD2; we prepared a reverse mutant but we did not detect an activity. We conclude that chicken PAD1 and PAD3 might play specific roles in the nervous system, but that chicken PAD2 might not be functional under normal physiological conditions.

Keywords: Ac-l-Arg; Ac-l-Arg-O-Me; BAEE, benzoyl-l-arginine-O-ethylester; Bz-l-Arg; Bz-l-Arg-O-Et; Bz-l-Arg-O-NH(2); CNS; Calcium-ion-dependent catalytic activity; Central nervous system; Chicken; PAD; Peptidylarginine deiminase; RACE; RCC2; RT-PCR; Retina; SDHB; Tos-l-Arg-O-Me; Tris; acetyl-l-arginine; acetyl-l-arginine-O-methylester; anti-chicken GAPDH; anti-chicken glyceraldehyde-3-phosphate dehydrogenase; benzoyl-l-arginine; benzoyl-l-arginine-O-amide; cPAD; central nervous system; chicken PAD; l-Arg-O-Me; l-arginine-O-methylester; peptidylarginine deiminase; rapid amplification of cDNA ends; regulator of chromosome condensation 2; reverse-transcription polymerase chain reaction; succinate dehydrogenase; tosyl-l-arginine-O-methylester; tris(hydroxymethyl) aminomethane.