Prediction of a coding sequence for a novel type II keratin from N-terminal sequences of mouse epidermal proteins site-specifically deiminated in embryonic development.

BACKGROUND: Epidermal keratinization involves various post-translational modifications including the deimination of arginine residues. Major deiminated proteins are derived from keratin K1. Two preferred deimination sites were identified in the V subdomain of mouse K1. An antibody against one of the deiminated peptide sequences (ACP) recognized deiminated mouse and human K1, and stained the cornified layers of human and infant mouse epidermis. ACP also stained the outermost layer of mouse embryonic epidermis. Western blotting revealed minor proteins showing strong ACP-positive signals in the mouse embryonic epidermal extract in which deiminated K1 derivatives were hardly detected. OBJECTIVE: To characterize ACP-positive proteins expressed in mouse embryonic epidermis. METHODS: ACP-positive proteins were isolated by preparative gel electrophoresis for N-terminal sequencing followed by blast searches for matching sequences in the protein and nucleotide database. RESULTS: We obtained N-terminal sequences of two ACP-positive proteins. A cDNA clone in the est_mouse database has an open reading frame for 202 amino acid residues containing both sequenced peptides. The deduced sequence shows typical features of the N-terminal portion of type II keratins. A virtually identical sequence to this reading frame is present in a genomic contig of chromosome 15 on which keratin type II genes are clustered. Sequential searches for overlapping cDNA clones in the est_mouse database along with similar searches in the genomic contig formulated a hypothetical cDNA sequence encoding a putative protein of 572 amino acid residues tentatively called K1-emb. CONCLUSION: We predicted a sequence of novel type II keratin site-specifically deiminated in embryonic mouse epidermis.

Changing pattern of deiminated proteins in developing human epidermis.

Peptidylarginine deiminases are widely distributed, calcium-ion-dependent enzymes that convert arginine residues of proteins into citrulline residues. This reaction, deimination, is thought to be an important event during the final stage of epidermal differentiation, possibly associated with integration and disintegration of keratin filaments. To elucidate the possible roles of protein deimination during human epidermal development we investigated localization of deiminated proteins using anti-citrulline peptide antibody, which preferentially recognizes citrulline residues in the V subdomains of keratin 1, and anti-chemically modified citrulline antibody, which enables detection of citrulline residues independent of amino acid sequences. Anti-chemically modified citrulline antibody, but not anti-citrulline peptide antibody stained the periderm in two-layered epidermis of 49 d and 57 d estimated gestational age. In the stratified epidermis of 88 d, 96 d, and 108 d estimated gestational age fetal skin, anti-citrulline peptide antibody and anti-chemically modified citrulline antibody staining was seen in the periderm and intermediate cell layers. After periderm cells regressed and keratinization began in the interfollicular epidermis, anti-citrulline peptide antibody and anti-chemically modified citrulline antibody were restricted to the cornified cell layers of the interfollicular epidermis, similar to the distribution patterns of that in adult epidermis. Postembedding immunoelectron microscopy showed anti-citrulline peptide antibody immunogold labeling over the cytoplasmic intermediate filament network in the periderm and the intermediate cell layers. These results demonstrate an orderly formation of deiminated proteins in different layers of embryonic epidermis and suggest important roles for peptidylarginine deiminases in human epidermal morphogenesis.

Sequential reorganization of cornified cell keratin filaments involving filaggrin-mediated compaction and keratin 1 deimination.

The final step of keratinocyte differentiation, transition from the granular cells to the cornified cells, involves various post-translational modifications that include deimination of arginine residues. Major deiminated epidermal proteins are derived from K1. Two preferred deimination sites were identified in mouse K1, one in the V1 and the other in the V2 subdomains. An antibody against the deiminated peptide sequence in the V2 subdomain recognized not only deiminated mouse K1 but also deiminated human K1. In this study we analyzed distribution of deiminated K1 in normal human skin and in bullous congenital ichthyosiform erythroderma at light and electron microscopic levels. In normal skin the first few (1-3) cornified cell layers were positive for filaggrin and negative for the antibody against deiminated mouse K1 peptide, whereas the more superficial cells were negative for filaggrin and strongly positive for the antibody against deiminated mouse K1 peptide, indicating slightly delayed onset of K1 deimination at the initial stage of cornification. The clumped keratin in bullous congenital ichthyosiform erythroderma that was not properly compacted with filaggrin was poorly positive to the antibody against deiminated mouse K1 peptide. In addition, K1 derivatives in bullous congenital ichthyosiform erythroderma reacted poorly with the antibody against deiminated mouse K1 peptide compared with the normal control in immunoblot analyses. Our results suggest sequential reorganization of cornified cell keratin filaments involving filaggrin-mediated compaction and K1 deimination. Abnormal keratin aggregation in bullous congenital ichthyosiform erythroderma is likely to disturb the normal deimination of K1.

Deimination of arginine residues in nucleophosmin/B23 and histones in HL-60 granulocytes.

Peptidylarginine deiminases (PADs) convert arginine residues in proteins into citrulline residues Ca(2+)-dependently. PAD V was recently found in granulocyte-differentiated HL-60 cells. To find a target of PAD V, we incubated HL-60 granulocytes with the calcium ionophore A23187 and studied deiminated proteins by immunocytochemistry and immunoblotting using a monospecific antibody to modified citrulline residues. Immunocytochemical signals were found in the nucleus upon incubation with A23187. Immunoblotting indicated that 40-, 18-, 17-, and 14-kDa proteins were preferentially deiminated. The 40-kDa protein, which was focused to pI 5.0 on two-dimensional gel electrophoresis, was identified as nucleophosmin/B23 by mass spectrometry. The 18-, 17-, and 14-kDa proteins extracted with 0.4 N H(2)SO(4) comigrated with histones H3, H2A, and H4, respectively, on two-dimensional gel electrophoresis specialized for histones. The citrulline content of histones amounted to about 10% of the histone molecules. We discuss the implications of deimination of these proteins for their nuclear functions.

Rheumatoid arthritis specific anti-Sa antibodies target citrullinated vimentin.

Antibodies directed to the Sa antigen are highly specific for rheumatoid arthritis (RA) and can be detected in approximately 40% of RA sera. The antigen, a doublet of protein bands of about 50 kDa, is present in placenta and in RA synovial tissue. Although it has been stated that the Sa antigen is citrullinated vimentin, experimental proof for this claim has never been published. In this study, we investigated the precise nature of the antigen. Peptide sequences that were obtained from highly purified Sa antigen were unique to vimentin. Recombinant vimentin, however, was not recognized by anti-Sa reference sera. In vivo, vimentin is subjected to various post-translational modifications, including citrullination. Since antibodies to citrullinated proteins are known to be highly specific for RA, we investigated whether Sa is citrullinated and found that Sa indeed is citrullinated vimentin. Anti-Sa antibodies thus belong to the family of anticitrullinated protein/peptide antibodies. The presence of the Sa antigen in RA synovial tissue, and the recent observation that vimentin is citrullinated in dying human macrophages, make citrullinated vimentin an interesting candidate autoantigen in RA and may provide new insights into the potential role of citrullinated synovial antigens and the antibodies directed to them in the pathophysiology of RA.

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

Peptidylarginine deiminases (PADs) catalyse a post-translational modification of proteins through the conversion of arginine residues into citrullines. The existence of four isoforms of PAD (types I, II, III and IV) encoded by four different genes, which are distinct in their substrate specificities and tissue-specific expression, was reported in rodents. In the present study, starting from epidermis polyadenylated RNA, we cloned by reverse transcriptase-PCR a full-length cDNA encoding human PAD type I. The cDNA was 2711 bp in length and encoded a 663-amino-acid sequence. The predicted protein shares 75% identity with the rat PAD type I sequence, but displays only 50-57% identity with the three other known human isoforms. We have described the organization of the human PAD type I gene on chromosome 1p36. A recombinant PAD type I was produced in Escherichia coli and shown to be enzymically active. Human PAD type I mRNAs were detected by reverse transcriptase-PCR not only in the epidermis, but also in various organs, including prostate, testis, placenta, spleen and thymus. In human epidermis extracts analysed by Western blotting, PAD type I was detected as a 70 kDa polypeptide, in agreement with its predicted molecular mass. As shown by immunohistochemistry, the enzyme was expressed in all the living layers of human epidermis, with the labelling being increased in the granular layer. This is the first description of the human PAD type I gene and the first demonstration of its expression in epidermis.

Citrullination of synovial proteins in murine models of rheumatoid arthritis.

OBJECTIVE: Antibodies directed to citrulline-containing proteins are highly specific for rheumatoid arthritis (RA) and can be detected in up to 80% of patients with RA. Citrulline is a nonstandard amino acid that can be incorporated into proteins only by posttranslational modification of arginine by peptidylarginine deiminase (PAD) enzymes. The objective of this study was to investigate the presence of anticitrulline antibodies, PAD enzymes, and citrullinated antigens in mouse models of both acute and chronic destructive arthritis: streptococcal cell wall (SCW)-induced arthritis and collagen-induced arthritis (CIA), respectively. METHODS: Synovial tissue biopsy specimens were obtained from naive mice, mice with CIA, and mice with SCW-induced arthritis. The expression of messenger RNA (mRNA) for PAD enzymes was analyzed by reverse transcriptase-polymerase chain reaction; the presence of PAD proteins and their products (citrullinated proteins) was analyzed by Western blotting and by immunolocalization. The presence of anticitrullinated protein antibodies was investigated by an anti-cyclic citrullinated peptide (anti-CCP) enzyme-linked immunosorbent assay (ELISA) and an ELISA using in vitro citrullinated fibrinogen. RESULTS: In both mouse models, PAD type 2 (PAD2) mRNA was present in the synovium but was not translated into PAD2 protein. In contrast, PAD4 mRNA, although absent from healthy synovium, was readily transcribed and translated by polymorphonuclear neutrophils infiltrating the synovial tissue during inflammation. As a consequence, several synovial proteins were subjected to citrullination. One of these proteins was identified as fibrin, which has been reported to be citrullinated also in synovium of patients with RA. Although generation of citrullinated antigens during synovial inflammation in the mice was eminent, no anti-CCP antibodies could be detected. CONCLUSION: Citrullination of synovial antigens is an active process during joint inflammation in both mice and humans, but the induction of autoantibodies directed to these proteins is a more specific phenomenon, detectable only in human RA patients.