Tatsuo Senshu

The major synovial targets of the rheumatoid arthritis-specific antifilaggrin autoantibodies are deiminated forms of the alpha- and beta-chains of fibrin.

IgG antifilaggrin autoantibodies (AFA) are the most specific serological markers of rheumatoid arthritis. In epithelial tissues, they recognize citrulline-bearing epitopes present on various molecular forms of (pro)filaggrin. Histological analysis of rheumatoid synovial membranes with an Ab to citrulline showed labeling of interstitial amorphous deposits and mononuclear cells of various types. Immunochemical analysis of exhaustive sequential extracts of the same tissues showed that they contain several deiminated (citrulline containing) proteins. Among them, two proteins, p64–78 and p55–61, present in urea-DTT and guanidine extracts, were shown by immunoblotting to be specifically targeted by AFA. By amino-terminal sequencing the proteins were identified as deiminated forms of the α- and β-chains of fibrin, respectively. Their identity was confirmed using several Abs specific for the Aα- and/or to the Bβ-chain of fibrin(ogen). Moreover, AFA-positive rheumatoid arthritis (RA) sera and purified AFA were highly reactive to the Aα- and Bβ-chains of human fibrinogen only after deimination of the molecules by a peptidylarginine deiminase. Autoantibodies affinity purified from a pool of RA sera onto deiminated fibrinogen were reactive toward all of the epithelial and synovial targets of AFA. This confirmed that the autoantibodies to the deiminated Aα-and Bβ-chains of fibrinogen, the autoantibodies to the synovial proteins p64–78 and p55–61, and, lastly, AFA, constitute largely overlapping autoantibody populations. These results show that deiminated forms of fibrin deposited in the rheumatoid synovial membranes are the major target of AFA. They suggest that autoimmunization against deiminated fibrin is a critical step in RA pathogenesis.

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.

Molecular Characterization of Peptidylarginine Deiminase in HL-60 Cells Induced by Retinoic Acid and 1α,25-Dihydroxyvitamin D3

Three types of peptidylarginine deiminase (PAD), which converts a protein arginine residue to a citrulline residue, are widely distributed in animal tissues. Little is known about PAD of hemopoietic cells. We found that PAD activity in human myeloid leukemia HL-60 cells was induced with the granulocyte-inducing agents retinoic acid and dimethyl sulfoxide and with the monocyte-inducing agent 1α,25-dihydroxyvitamin D3. We cloned and characterized a PAD cDNA from retinoic acid-induced cells. The cDNA was 2,238 base pairs long and encoded a 663-amino acid polypeptide. The HL-60 PAD had 50–55% amino acid sequence identities with the three known enzymes and 73% identity with the recently cloned keratinocyte PAD. The recombinant enzyme differs in kinetic properties from the known enzymes. Immunoblotting and Northern blotting with an antiserum against the enzyme and the cDNA, respectively, showed that a protein of approximately 67 kDa increased concomitantly with increase of mRNA of approximately 2.6 kilobases during granulocyte differentiation. During monocyte differentiation the same mRNA and protein increased as in granulocyte differentiation. Neither the enzyme activity nor the protein was found in macrophage-induced cells. These results suggested that expression of the PAD gene is tightly linked to myeloid differentiation.

Detection of citrulline residues in deiminated proteins on polyvinylidene difluoride membrane

We have developed a new detection method of deiminated proteins on polyvinylidene difluoride membranes. Citrulline residues in enzymatically deiminated histones were modified by incubating with diacetyl monoxime and antipyrine in a strong acid mixture. The products were injected to rabbits, and the antibodies obtained were affinity-purified using a modified citrulline column. Sample proteins blotted to the membrane were modified in a similar manner and incubated successively with the purified antibody and an alkaline phosphatase-conjugated second antibody. Detection was performed using a chemiluminescent substrate. The method enabled detection of 3-10 fmol of citrulline residues dot blotted as deiminated model proteins. It visualized numerous rat pituitary soluble proteins that had been enzymatically deiminated and Western blotted to the membrane. The data suggest usefulness of the method for detecting deiminated proteins regardless of the backbone protein molecules. Search for deiminated proteins on the Western blots of various rat tissue homogenates detected a single band on that of spinal cord, another band on that of uterus, and multiple bands on those of skin and hair root. The bands in the former two tissue homogenates comigrated with glial fibrillary acidic protein and vimentin, respectively.

Combined biochemical and immunochemical comparison of peptidylarginine deiminases present in various tissues

We have performed a combined biochemical and immunochemical study on the identity of peptidylarginine deiminases (EC 3.5.3.15) present in various mammalian tissues. First, we purified peptidylarginine deiminase from rat skeletal muscle. It gave a single band of molecular weight 83,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Next we immunized rabbits with the purified enzyme. The resulting antibodies reacted specifically with the antigen in Western blot assay. Most of the enzyme activities present in rat skeletal muscle, brain, spinal cord, submaxillary gland and spleen could be characterized as the same muscle-type enzyme by immunoprecipitation and Western blot assay. The antibodies did not react with enzyme samples obtained from rat hair follicles and bovine epidermis. The lack of immunoreactivity of the epidermal enzyme could not be accounted for by the species difference, since the antibodies reacted with a 83 kDa polypeptide of bovine brain, which was thought to represent a bovine counterpart of the muscle-type enzyme. The epidermal enzyme could be distinguished from the other enzyme samples by its high activity towards benzoylarginine. These data suggest the existence of at least three types of peptidylarginine deiminase in mammalian tissues, i.e., a muscle type, a hair follicle type, and an epidermal type.

Immunocytochemical localization of peptidylarginine deiminase in human eosinophils and neutrophils

Peptidylarginine deiminase, registered as PAD V in the DDBJ/GenBank/EMBL data banks, is expressed in HL‐60 cells differentiated into granulocytes or monocytes. We analyzed PAD activities in density‐fractionated human peripheral blood cell fractions. PAD activity with similar substrate specificity to that of PAD V was found in the eosinophil and neutrophil fractions, which showed single bands comigrating with authentic PAD V on immunoblotting with an anti‐PAD V antibody. Both the biochemical and immunoblotting analyses showed marked enrichment of PAD V in the eosinophil fraction. Its immunoreactivity appeared to localize in eosinophilic granules at high density and in myeloperoxidase‐negative cytoplasmic granules of neutrophils at low density, as determined by confocal laser‐scanning microscopy. Possible roles of PAD V in myeloid differentiation and granulocyte function are discussed. In addition, we present evidence for the presence of PAD(s) that are antigenically different from PAD V in monocytes and lymphocytes.

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.

DEIMINATION OF 70-KD NUCLEAR PROTEIN DURING EPIDERMAL APOPTOTIC EVENTS IN VITRO

Peptidylarginine deiminase (PAD) is the enzyme responsible for converting protein-bound arginine residues to citrulline. It has recently been shown that a number of epidermal proteins, including filaggrin, trichohyalin, and keratins, are deiminated by the action of PAD, suggesting a possible role for protein deimination during the final stages of epidermal differentiation. We report here a novel PAD substrate found during the course of identifying deiminated proteins in cultured rat epidermal keratinocytes. We found that a 70-kD protein localized to the periphery of the nucleus was preferentially deiminated after ionomycin treatment in the presence of 2 mM calcium and was associated with apoptotic events in these cells. Furthermore, we discovered that the deimination of nuclear protein could be induced by transfection of a PAD cDNA into rat epidermal keratinocytes. These data suggest that PAD may act on the 70-kD nuclear protein to induce disassembly of the nuclear lamina and promote apoptosis during terminal epidermal differentiation.

Localization of peptidylarginine deiminase type II in a stage-specific immature oligodendrocyte from rat cerebral hemisphere.

Myelin basic protein (MBP) is composed of multiple charged isomers as the products of various posttranslational modifications. The least cationic component contains six citrulline residues converted from arginine residues by peptidylarginine deiminase (PAD). The modified MBP differs markedly from unmodified MBP in the ability to aggregate acidic lipid vesicles. However, the localization of PAD in brain has remained rather elusive. We performed Western blotting and immunocytochemical analyses of PAD type II and found that it was present in stage-specific immature oligodendrocytes but not in either type-1 astrocytes or neurons. We also confirmed that only the oligodendrocyte homogenate contained the PAD activity utilizing a sensitive method to detect citrulline-containing proteins. These data suggest that PAD type II localized in oligodendrocytes is responsible for deiminating MBP.

Studies on specificity of peptidylarginine deiminase reactions using an immunochemical probe that recognizes an enzymatically deiminated partial sequence of mouse keratin K1.

Citrulline residues are detected in keratins and filaggrin in the cornified layers of mammalian epidermis. Such citrulline residues are formed by the enzymatic deimination of arginine residues by peptidylarginine deiminases (EC 3.5.3.15). Major deiminated keratins are derived from keratin K1. Two arginine residues identified as preferred deimination sites in mouse K1 are located in its V subdomains. To develop an immunochemical probe which recognizes the deiminated peptide sequence specifically, we enzymatically deiminated an undecapeptide corresponding to the deiminated peptide sequence identified in the V2 subdomain for immunizing rabbits. An IgG fraction obtained from the antiserum was affinity-purified using an immobilized peptide column. The affinity-purified IgG showed high specificity towards partially degraded keratin K1 obtained from the cornified layer of 3-day-old mouse epidermis. It also yielded intense signals of unidentified minor components localized in the cornified layers of late embryonic and early postnatal mouse epidermis. Comparative studies using different types of the enzymes suggested that peptidylarginine deiminase type I acted on the arginine residue in the V2 subdomain of keratin K1 more readily than peptidylarginine deiminase type II. The data are discussed in conjunction with possible factors influencing the specificity of the enzyme reaction.