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.

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 alpha- and beta-chains of fibrin, respectively. Their identity was confirmed using several Abs specific for the A alpha- and/or to the B beta-chain of fibrin(ogen). Moreover, AFA-positive rheumatoid arthritis (RA) sera and purified AFA were highly reactive to the A alpha- and B beta-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 A alpha-and B beta-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.

A case of erythrokeratoderma variabilis without mutations in connexin 31.

Erythrokeratoderma (EK) variabilis is a heterogeneous group of diseases characterized by migratory erythematous patches and hyperkeratotic plaques. Mutations in connexin 31 have recently been found to underlie several cases of EK variabilis. We describe a Japanese girl with extensive lesions that appeared to be a form of EK variabilis, clinically resembling genodermatose en cocardes (Degos). Our patient had characteristic migratory rosette or target-like erythematous keratotic plaques with peripheral scaling in addition to relatively fixed keratotic plaques. Sequencing of the connexin 31 gene did not detect mutations. Skin biopsy showed parakeratotic hyperkeratosis with hypergranulosis. Immunohistochemically, suprabasal keratins, involucrin and profilaggrin were unequivocally expressed, while loricrin expression was greatly diminished and deiminated K1 was undetectable. Our results confirm aetiological heterogeneity in EK. The histological features suggest disruption of keratinocyte terminal differentiation at a very late stage.

Decreased deiminated keratin K1 in psoriatic hyperproliferative epidermis.

Citrulline-containing proteins, mainly originating from keratin K1 and formed by enzymatic deimination of arginine residues, have been identified in the cornified layers of human epidermis. We analyzed the localization and nature of the deiminated proteins in psoriatic epidermis. Immunostaining based on chemical modification of citrulline residues showed that the normal and psoriatic uninvolved epidermis contained deiminated proteins diffusely in the cornified cell layer, whereas the involved epidermis had no detectable or markedly reduced levels of deiminated proteins. Immunolabeling with polyclonal antibodies against a synthetic citrulline-containing peptide corresponding to a deiminated sequence of mouse K1 also suggested markedly decreased deiminated K1 in psoriatic involved lesions. Keratin analyses indicated that deiminated K1 present in normal and psoriatic uninvolved epidermis was not detected in the psoriatic involved epidermis. Double staining with a monoclonal antibody, 34betaB4, and the polyclonal antibodies demonstrated that epidermis with low suprabasal keratin expression was negative for deiminated K1. In contrast, intralesional acrosyringia showing decreased suprabasal keratin immunoreactivity like that of the surrounding psoriatic epidermis showed strong deiminated K1 staining. This suggests that abnormal keratin deimination is restricted to the psoriatic hyperproliferative epidermis, without affecting sweat ductal epithelia.

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.

Identification of citrulline residues in the V subdomains of keratin K1 derived from the cornified layer of newborn mouse epidermis.

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 deiminase (EC 3.5.3.15). Major deiminated keratins are thought to be partially degraded/disulfide-cross-linked keratin K1 based on the immunoblotting profiles. In order to obtain more definitive evidence of the deimination of keratin K1 and also to investigate its functional significance, we attempted to identify its preferred acting sites of peptidylarginine deiminase. A partially degraded keratin K1 fraction obtained from the cornified layer of newborn mouse epidermis was subjected to limited proteolytic cleavages, and the resulting deiminated peptides were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or reverse-phase high-performance liquid chromatography for N-terminal sequencing and/or amino acid analysis. At least two sites were identified, one in the V1 and the other in the V2 subdomains of keratin K1. An undecapeptide sequence covering the latter shows about 70% homology with an undecapeptide sequence in the V2 subdomain of human K1, a presumptive site of deimination. We speculated that the deimination of arginine residues in these subdomains might modulate their interactions with epidermal proteins other than keratins and filaggrin during the terminal stage of 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.

Molecular characterization of peptidylarginine deiminase in HL-60 cells induced by retinoic acid and 1alpha,25-dihydroxyvitamin D(3).

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 1alpha,25-dihydroxyvitamin D(3). 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.

Dynamic aspects of protein deimination in developing mouse epidermis.

The cornified layer of mammalian epidermis contains deiminated keratins and filaggrin whose arginine residues are partly converted to citrulline residues by peptidylarginine deiminase (EC 3.5.3.15). We have attempted to study dynamic aspects of protein deimination using late embryonic to early postnatal mouse skin. The epidermis was separated from the dermis by brief immersion of skin into a weakly alkaline ammonium chloride solution. The total homogenate of the epidermis was subjected to western blotting analyses for quantitative densitometry of major keratins, deiminated proteins and immunoreactive filaggrin. We found marked increases in both deiminated keratins and deiminated filaggrin from the 18th day of gestation to 2 h after birth followed by rapid decreases to minimum levels at 6 h and subsequent gradual increases surpassing the earlier levels by 72 h after birth. Such variations were associated with consistent changes of the intensity of deiminated proteins stained immunocytochemically. These results suggest that the protein deimination might play a role in dealing with the drastic environmental change after birth. Furthermore, we found compartmentalization of both total and deiminated filaggrins into soluble and particulate fractions. The soluble compartment contained relatively more deiminated filaggrin than the particulate fraction.

Collateral occurrence of deimination of keratins with differentiation of an immortalized newborn rat keratinocyte cell line cultured at air-liquid interface.

We devised a simple method to maintain an immortalized newborn rat keratinocyte cell line at the air-liquid interface using a tissue culture insert fitted with a microporous membrane. The cells formed stratified layers of flattened and anucleated cells resembling stratum corneum of the epidermis. Deiminated proteins, which are localized in the cornified layer of epidermis as the reaction products of peptidylarginine deiminase were detected immunohistochemically in the differentiated cells. Western blot analyses revealed that major deiminated proteins were type I keratins K10 and K14. Deiminated products of type II keratin K5 were found as minor components. Our observations show that deimination of keratins might be correlated with terminal differentiation of the immortalized keratinocyte cell line.

The epitopes targeted by the rheumatoid arthritis-associated antifilaggrin autoantibodies are posttranslationally generated on various sites of (pro)filaggrin by deimination of arginine residues.

Antifilaggrin autoantibodies (AFA) are a population of IgG autoantibodies associated to rheumatoid arthritis (RA), which includes the so-called "antikeratin" Abs and antiperinuclear factor. AFA are the most specific serological markers of RA. We previously showed that they recognize human epidermal filaggrin and other profilaggrin-related proteins of various epithelial tissues. Here, we report further characterization of the protein Ags and epitopes targeted by AFA. All the Ags that exhibit numerous neutral/ acidic isoelectric variants were immunochemically demonstrated to be deiminated proteins. In vitro deimination of a recombinant human filaggrin by a peptidylarginine deiminase generated AFA epitopes on the protein. Moreover, two of three filaggrin-derived synthetic peptides with a citrulline in the central position were specifically and widely recognized by AFA affinity-purified from a series of RA sera. These results indicate that citrulline residues are constitutive of the AFA epitopes, but only in the context of specific amino acid sequences of filaggrin. In competition experiments, the two peptides abolished the AFA reactivity of RA sera, showing that they present major AFA epitopes. These data should help in the identification of a putative deiminated AFA-inducing or cross-reactive articular autoantigen and provide new insights into the pathogenesis of RA. They could also open the way toward specific immunosuppressive and/or preventive therapy of RA.

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.

Molecular cloning of two novel types of peptidylarginine deiminase cDNAs from retinoic acid-treated culture of a newborn rat keratinocyte cell line.

Peptidylarginine deiminases (PADs) are a group of enzymes which convert protein arginine residues to citrulline residues. Using rat muscle PAD cDNA as a probe, we obtained two novel cDNAs, PAD-R11 and PAD-R4, from immortalized rat keratinocytes treated with all-trans retinoic acid. Comparison of the deduced amino acid sequences with those of muscle and hair follicle enzymes showed high conservation in the C-terminal region. Recombinant proteins encoded by both PAD-R11 and PAD-R4 showed the enzyme activities. That of PAD-R11 showed a characteristic feature of the enzyme found in the epidermis.

Selective deimination of vimentin in calcium ionophore-induced apoptosis of mouse peritoneal macrophages.

We found citrulline-containing proteins in mouse peritoneal macrophages undergoing calcium ionophore-induced apoptosis. Such proteins were products of deimination of arginine residues catalyzed by endogenous peptidylarginine deiminase (EC 3.5.3.15) activated by calcium influx. Western blotting analyses of the extract from macrophages incubated with 1 microM ionomycin showed selective deimination of vimentin without detectable degradation. Double immunofluorescence staining of deiminated proteins and vimentin suggested localization of deiminated vimentin around the periphery of round-shaped nucleus, which was thought to be an early morphological sign of apoptosis. The biological implication of vimentin deimination in macrophage apoptosis is discussed.

Preferential deimination of keratin K1 and filaggrin during the terminal differentiation of human epidermis.

The upper layers of mammalian epidermis contain citrulline-containing proteins formed by enzymatic deimination of arginine residues. To study the role of protein deimination in epidermal differentiation, we identified deiminated proteins extracted from human epidermis. Major deiminated proteins were identified as partially degraded keratin K1, while those from keratin K10 and a highly heterogeneous mixture of deiminated filaggrin isomers were detected as minor components. Deiminated keratins were recovered in a fraction enriched with keratins from the cornified layers. The subsequent immunohistochemical study showed that deiminated proteins were localized mainly in the lowermost cornified layer, but not in the granular layer. These data suggested that partially degraded/disulfide-cross-linked keratin K1 was preferentially deiminated during the terminal stages of epidermal differentiation. We therefore speculated that the protein deimination might influence the interaction of basic K1 with its acidic partner K10, pre-existent K5/K14 networks or keratin-associated protein filaggrin.

All-trans retinoic acid increases peptidylarginine deiminases in a newborn rat keratinocyte cell line.

We studied the expression of peptidylarginine deiminases (EC 3.5.3.15) in an immortalized newborn rat keratinocyte cell line. No measurable enzyme activities were noted in either growing or confluent cultures. The enzyme activity was increased by all-trans retinoic acid in dose- and time-dependent manners. The enzyme activity was resolved into two peaks by anion exchange chromatography. The minor peak resembled enzyme preparations obtained from the epidermis in earlier studies. The major peak was indistinguishable from rat muscle peptidylarginine deiminase in the chromatographic and Western blotting profiles. Northern blot hybridization showed a major band in retinoic acid-treated cells migrating slightly behind muscle peptidylarginine deiminase mRNA.

Detection of deiminated proteins in rat skin: probing with a monospecific antibody after modification of citrulline residues.

We performed a systematic study on deiminated proteins present in rat epidermis. Proteins extracted from various epidermal samples were resolved by either one- or two-dimensional gel electrophoresis and Western blotted to nitrocellulose membranes. Deiminated proteins were detected by modification of citrulline residues followed by probing with an anti-modified citrulline monospecific antibody. The cornified layer of adult plantar skin gave multiple series of isoelectric variants, most of which were found to be differentially deiminated type II keratins (60 kDa, and 67 kDa or above). The whole epidermis of 5-day-old rat back skin showed isoelectric variants of 60-kDa keratin as major deiminated components, and deiminated 55-kDa keratin and deiminated filaggrin as minor spots. In addition, we found highly deiminated proteins (200-220 kDa) thought to be derived from trichohyalin. The immunoreactivity of deiminated proteins was mainly localized in the granular and cornified layers of epidermis. Co-localization of deiminated filaggrin and keratins in the granular layer suggests the possible role of protein deimination during the terminal stage of epidermal differentiation.

Age-related increase in peptidylarginine deiminase in the male rat pituitary.

We have measured the activity of peptidylarginine deiminase (EC 3.5.3.15) in male Wistar rat pituitaries at various ages. Pituitaries obtained from 3- and 9-month-old rats showed negligible activities. The mean enzyme activity increased appreciably by 18 months and markedly by 24 months accompanied with actual increases in the enzyme content. The peptidylarginine deiminase mRNA content showed a similar but more gradual increase appreciable from 9 months. Many enzyme-positive cells were present in the pars distalis of 24-month-old male pituitaries. Most of the enzyme-positive cells coincided with lactotrophs. The pituitary prolactin content showed a gradually increasing profile resembling that of the enzyme mRNA, but the serum prolactin concentration did not increase significantly. Neither the serum 17 beta-estradiol content nor the pituitary estrogen receptor content showed significant variation that could account for the marked increase in the pituitary enzyme content between 18 and 24 months of age. These data suggest possible presence of other factors regulating the enzyme content in old male pituitaries.

Enzymatic deimination of glycogen phosphorylase and a peptide of the phosphorylation site: identification of modification and roles in phosphorylation and activity.

The functional role of arginine residues in glycogen phosphorylase b was probed by enzymatic modification by using peptidylarginine deiminase, which converts arginine residues to citrulline. A peptide with sequence LysArgLysGlnIleSerValArgGlyLeu, corresponding to the phosphorylation site of serine-14 in phosphorylase, was a substrate for the deiminase. Although both arginine residues could be converted to citrulline, modification of arginine-16 occurred more rapidly than modification of arginine-10. Previous studies have implicated a role for arginine, notably arginine-16, in determining phosphorylase kinase activity with the peptide. Deimination altered the phosphorylation of the peptide. Monodeimination of the peptide at arginine-16 slowed down the phosphorylation reaction, but did not diminish the total amount of phosphorylation that could be obtained. Deimination of both arginines produced a peptide that could not be phosphorylated. Modification of phosphorylase b resulted in activation or inactivation of enzyme activity depending on the extent of reaction with peptidylarginine deiminase. A low level of deiminase causes inactivation initially, but after prolonged incubation activation occurs. With high level of deiminase only activation can be observed. Because changes in activity are seen only at subsaturating AMP concentrations (50-100 microM), inactivation and activation are likely due to changes in affinity of the enzyme for AMP. The protein modified with the high level of deiminase has multiple sites of deimination. Arginine 16 was established as a major site of modification. Only protein modified with the high level of deiminase showed modification of arginine-16 and effects on the phosphorylation of phosphorylation b. As with the modified peptide substrate, the reaction was slower with modified phosphorylase in comparison with native phosphorylase b. The results show the importance of the guanidino group of arginine-16 of the protein substrate in modulating the phosphorylase kinase reaction.

Studies of calcineurin-calmodulin interaction: probing the role of arginine residues using peptidylarginine deiminase.

We have used an enzyme, peptidylarginine deiminase, to convert certain arginyl groups in calcineurin to citrulline. Amino acid analysis shows that only 3 of 34 arginines in calcineurin were deiminated; citrulline seems to be localized only in the calcineurin A (CaN A) subunit. Upon incubation with deiminase, the Mn2+/calmodulin-stimulated phosphatase activity decreases to 20-40% of the original activity within 1 h. However, the reduction in enzyme activity is fully protected by addition of calmodulin to the deimination reaction, and only 1.5 mol citrulline/mol calcineurin is found in this case. Removal of the calmodulin binding domain of the deiminated CaN A by limited proteolysis results in the reactivation of the phosphatase to the same level as digested native calcineurin and also results in the loss of all citrulline residues. The calmodulin activation curve of the deiminated enzyme is significantly shifted; the calculated apparent Kact using native calmodulin is 15-fold higher than that of native calcineurin while the apparent Kact using a fluorescent derivative of calmodulin, dansyl-calmodulin, is 10-fold higher. However, the Vm of deiminated calcineurin is similar to that of native if highly elevated levels of calmodulin are used to activate the modified calcineurin. To determine directly if the binding of calmodulin to calcineurin is affected upon deimination, fluorescence titrations using dansyl-calmodulin were performed. The Kd of deiminated calcineurin determined from these titrations is 10-fold higher than that of unmodified calcineurin, indicating that calmodulin binding is indeed affected. These data indicate that at least one arginine is important for calmodulin binding and is likely located at the calmodulin binding site of the CaN A subunit.