Human Peptidylarginine Deiminase Type III: Molecular Cloning and Nucleotide Sequence of the cDNA, Properties of the Recombinant Enzyme, and Immunohistochemical Localization in Human Skin

Abstract

Peptidylarginine deiminase catalyzes the post-translational modification of proteins through the conversion of arginine to citrulline in the presence of calcium ions. In rodents, peptidylarginine deiminase has been classified into four isoforms, types I, II, III, and IV, which are distinct in their molecular weights, substrate specificities, and tissue localization. Of these isoforms, only type III was detected in epidermis and hair follicles. Although the role of this enzyme in these tissues is not yet clear, indirect data have shown that several structural proteins such as filaggrin, trichohyalin, and keratin are substrates for peptidylarginine deiminase. In this study, we cloned the full-length cDNA of human peptidylarginine deiminase type III (3142 bp) from cultured human keratinocytes by reverse transcription-polymerase chain reaction and by rapid amplification of cDNA ends methods. This cDNA contained a 1995 bp open reading frame encoding 664 amino acids (Mr = 74 770). To explore the physicochemical and enzymatic properties of human peptidylarginine deiminase type III, we constructed a plasmid for producing a recombinant human peptidylarginine deiminase type III in bacteria. The enzymatic characteristics of the recombinant enzyme were very similar to those of the rodent peptidylarginine deiminase type III. The recombinant enzyme showed the catalytic activities toward structural proteins of epidermis and hair follicle, filaggrin and trichohyalin, in which the deiminations maxima of about 60% and 13% arginine residues were observed in filaggrin and trichohyalin, respectively. An immunohistochemical study of human scalp skin with a monospecific anti-peptidyl-arginine deiminase type III antibody revealed that the type III enzyme was localized to the inner root sheath and outer root sheath of hair follicles. Peptidylarginine deiminase type III in the inner root sheath was notable between supramatrix and keratogenous zone and was scarcely detected in cornified hair zone. The enzyme was also expressed in the cuticle layer of hair. On the other hand, expression of the enzyme in the epidermis was very low. These data imply that human peptidylarginine deiminase type III is the predominant isoform in hair follicles and may function as a modulator of hair structural proteins, including trichohyalin during hair and hair follicle formation.

Specific Citrullination Causes Assembly of a Globular S100A3 Homotetramer

“…PAD2 was observed as granular patches from the early differentiation stages of all hair epithelial matrix daughter cells; however, as each cellular differentiation advanced, PAD2 appeared in the cytoplasm and partially colocalized with S100A3 in the cuticular and cortical cells. Anti-PAD3 antibody intensely labeled the differentiating cells within the inner root sheath and cuticular layers and medulla, but a PAD3 signal was absent from the matrix in the lower follicle bulb and hair cortex, as previously reported (35). Cytoplasmic distribution of PAD3 was seen at an earlier cuticular differentiation stage than for PAD2 and almost completely co-localized with S100A3.…”

“…In Vitro Modification of S100A3-Active recombinant human PAD1, PAD2, and PAD3 enzymes were prepared according to the previously reported procedures (18,35). Conventionally, 1 g of recombinant S100A3 was reacted with 25 milliunits of PAD enzymes in 20 l of 100 mM Tris-HCl buffer (pH 7.5) containing 10 mM CaCl 2 and 5 mM DTT at 37°C.…”

“…Conventionally, 1 g of recombinant S100A3 was reacted with 25 milliunits of PAD enzymes in 20 l of 100 mM Tris-HCl buffer (pH 7.5) containing 10 mM CaCl 2 and 5 mM DTT at 37°C. One unit was defined as the amount of PAD able to citrullineate 1 mol of benzoyl-L-arginine ethyl ester in the same reaction buffer for 1 h at 55°C (35).…”

“…In hair follicles, PAD3 is located in the cuticular cells expressing S100A3 (24 -26) as well as in the inner root sheaths and the medulla, where it co-localizes with trichohyalin (35,40). We determined how PAD isozymes co-localize with S100A3 in order to elucidate the role of PAD isozymes in S100A3 citrullination.…”

Identification of 45 novel SNPs in the 83-kb region containing peptidylarginine deiminase types 1 and 3 loci on chromosomal band 1p36.13

“…To date, four human PADI genes, type 1 to type 4, have been isolated and characterized by cDNA sequences, expression patterns, and biochemical analyses (Nakashima et al 1999;Kanno et al 2000;Ishigami et al 2002;Guerrin et al 2003). Three of four genes, PADI1, PADI3, and PADI4, lie within an approximately 160-kb region on chromosome 1p36.13.…”