Isolation and characterization of cDNA clones encoding rat skeletal muscle peptidylarginine deiminase

Various mammalian tissues contain protein-arginine deiminases (EC 3.5.3.15), which convert the arginine residues in normal peptide bonds to the citrulline residues in calcium ion-dependent manners. Here, we describe the complete primary structure of rat skeletal muscle peptidylarginine deiminase deduced from the sequences of its cDNA clones isolated by recombinant DNA technology. We have isolated three overlapping cDNA clones which constitute a 4,507-base pair cDNA sequence including a 2,452-base pair 3'-untranslated region. The coding region consists of 1,995 base pairs encoding 665 amino acid residues. A potential N-linked glycosylation site is present at asparagine-534. The molecular weight of the enzyme calculated from the deduced amino acid sequence is 75,122. Direct repeat sequences resembling the rodent B2 type repetitive sequences appear in the 3'-untranslated region (nucleotides 3,090-3,198 and 3,270-3,391). Northern hybridization demonstrated the presence of its mRNA in poly(A)+ fractions of spinal cord, cerebrum, cerebellum, and submaxillary gland as well as skeletal muscle. The sizes of peptidylarginine deiminase mRNAs in these tissues were estimated to be 4.5-5.0 kilobases. No positive bands were detected on the blots of the corresponding RNA fractions of liver and kidney. Possible similarity of the amino acid sequence of peptidylarginine deiminase to those of other calcium binding proteins is discussed.     

Purification and characterization of peptidylarginine deiminase from rabbit skeletal muscle

The preceding paper described the identification and some properties of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle, kidney, brain, and lung. In the present work we purified peptidylarginine deiminase from rabbit skeletal muscle with a 16% yield by 7 steps. The purification involved ion-exchange chromatography on DEAE-Sephacel, gel filtration on Bio-Gel A-0.5 m, and affinity chromatography on soybean trypsin inhibitor-Sepharose 4B and aminohexyl-Sepharose 4B. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be about 83,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 130,000-140,000 by gel filtration on Sephadex G-200. The isoelectric point was 5.3 and the amino acid composition was also determined. The enzyme preferably catalyzed the formation of citrulline derivatives from arginine derivatives in which both the amino and carboxyl groups were substituted and showed the highest activity towards Bz-L-Arg-O-Et among the arginine derivatives tested. The Km value for Bz-L-Arg-O-Et was found to be 0.50 X 10(-3) M. The enzyme also showed marked activities towards native protein substrates, such as protamine sulfate, soybean trypsin inhibitor, histone and bovine serum albumin.     

Increased peptidylarginine deiminase type II in hypoxic astrocytes

Peptidylarginine deiminase type II (PAD 2) is the primary enzyme responsible for conversion of protein bound arginine to citrulline in the central nervous system. Evidence suggests that glial fibrillary acidic protein (GFAP), the main intermediate filament in astrocytes, is deiminated, but not much is known regarding factors that control this enzymatic reaction. The present study demonstrated that PAD 2 activity (as determined by Western blot analysis of citrullinated GFAP isoforms) was increased in human cultured astrocytes by hypoxic conditions. PAD 2 mRNA increased markedly during the first 2h of hypoxia, but using a single chain antibody against human PAD 2 produced from the ETH-2 phage library, it took approximately 8h of hypoxia to see marked increases in PAD 2 protein. Thus, this is the first report to demonstrate a measurable response in the amounts of PAD 2 mRNA, protein and activity in human astrocytes by prolonged hypoxic exposure.     

Expression pattern of peptidylarginine deiminase in rat and human Schwann cells

The peptidylarginine deiminase (PAD) family of enzymes are responsible for conversion of protein-bound arginine to citrulline in most tissues of the body and are garnering increased interest for their physiological and pathological roles. Although it has been shown that oligodendrocytes of the CNS express the PAD isoenzyme type 2, nothing is presently known about PAD expression in Schwann cells, the myelinating cells of the PNS. To evaluate PAD expression in the PNS, cultivated rat and human Schwann cells and slices of fetal, juvenile, and normal and regenerated adult sciatic nerves were examined with RT-PCR, Western blot, and immunohistochemical analysis. Samples from cerebellar cultures and skin served as positive controls. One of the principle findings was that cultivated Schwann cells expressed significant levels of mRNA and protein for the PAD isoenzymes 2 and 3. PAD1 and PAD4, however, were not expressed in any types of Schwann cells. Using double immunofluorescence, the majority of PAD2 staining was localized in immature cell stages. Moreover, increased amounts of PAD2, PAD3, and peptidyl-citrulline were also found in human fetal and rat juvenile and regenerated sciatic nerves as compared to similar normal adult specimens. Neuronal and inducible nitric oxide synthases, enzymes that convert free arginine to citrulline, were also expressed in Schwann cells; however, their massive induction by LPS/K(+), was not reflected in an enhanced peptidyl-citrulline immunosignal. These data suggest that, similar to the CNS, citrullination of proteins may also exert a specific role in thecourse of PNS development and repair.     

A continuous spectrophotometric assay method for peptidylarginine deiminase type 4 activity

A simple, continuous spectrophotometric assay for peptidylarginine deiminase (PAD) is described. Deimination of peptidylarginine results in the formation of peptidylcitrulline and ammonia. The ammonia released during peptidylarginine hydrolysis is coupled to the glutamate-dehydrogenase-catalyzed reductive amination of alpha-ketoglutarate to glutamate and reduced nicotinamide adenine dinucleotide (NADH) oxidation. The disappearance of absorbance at 340nm due to NADH oxidation is continuously measured. The specific activity obtained by this new protocol for highly purified human PAD is comparable to that obtained by a commonly used colorimetric procedure, which measures the ureido group of peptidylcitrulline by coupling with diacetyl monoxime. The present continuous spectrophotometric method is highly sensitive and accurate and is thus suitable for enzyme kinetic analysis of PAD. The Ca(2+) concentration for half-maximal activity of PAD obtained by this method is comparable to that previously obtained by the colorimetric procedure.     

Immunohistochemical demonstration of prostaglandins in various tissues of the rat

To demonstrate the tissue localization of prostaglandin (PG) E₂, PGF₂ and 6-keto-PGF₁ (a stable metabolite of PGI₂) various tissues, including decalcified periodontal tissue of 7-week-old male Wistar strain rats, were immunohistochemically examined using a streptavidin-biotin complex method. Besides tissue macrophages and endothelial cells in various tissues, hepatocytes, renal tubular cells, and parietal and chief cells in the gastric mucosa showed a positive reaction for the various PGs examined. PGs were demonstrated in the cytoplasm or in association with the cell membrane. We generally observed no difference between the localization patterns of PGE₂-, PGF₂-, and 6-keto-PGF₁-positive cells in these tissues. However, in the periodontal ligament and alveolar bone, 6-keto-PGF₁ was localized in the cytoplasm of osteocytes, osteoblasts, cementocytes, and cementoblasts, while no reaction for PGE₂ or PGF₂ was revealed in these cells. We demonstrated the immunohistochemical localization of PGs in various rat tissues including decalcified periodontal tissue and discuss the important roles of PGs in the modulation of their normal functions in these tissues.     

Affinity chromatography of peptidylarginine deiminase from rabbit skeletal muscle on a column of soybean trypsin inhibitor (Kunitz)-Sepharose

We designed a simple procedure for the purification of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle using substrate affinity chromatography. Of the immobilized substrate ligands tested, i.e. protamine and soybean trypsin inhibitor (Kunitz) (STI), STI-Sepharose was found to be an effective affinity adsorbent for purification of the enzyme. The specific binding of peptidylarginine deiminase to STI-Sepharose was observed in the presence of calcium ion, and the enzyme could be selectively eluted from the affinity adsorbent by washing with chelator. A 1,800-fold purification with a 50% yield was achieved in the three-step procedure, which involved DEAE-Sephacel ion-exchange and STI-Sepharose affinity chromatography. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity and the recovery were considerably higher than have been obtained by any procedures previously reported. The specific interaction of peptidylarginine deiminase with STI immobilized on Sepharose was also investigated quantitatively by frontal affinity chromatography. In this method, a peptidylarginine deiminase solution was applied continuously to an STI-Sepharose column and the retardation of the elution front was measured as a parameter of the strength of the interaction. The dissociation constant for the enzyme with STI was found to be 2.3 X 10(-7)M. This value was in good agreement with that obtained by kinetic analysis in our previous studies. Peptidylarginine deiminase required millimolar Ca2+ for the binding to STI-Sepharose. The Ca2+ dependence of the enzyme binding was quite similar to that of the enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen regulates peptidylarginine deiminase levels in a rat pituitary cell line in culture

A Nonidet P-40 extract of growth hormone-producing rat pituitary MtT/S cells was found to contain peptidylarginine deiminase (EC 3.5.3.15), which was indistinguishable from an enzyme preparation from rat muscle in Western immunoblotting and immunoprecipitation. This enzyme was immunocytochemically detected in the cytoplasm but was not secreted into the medium during the cultivation. When the cells were cultured for 2 days with various concentrations of 17 beta-estradiol (E2), the enzyme activity increased in a dose-dependent manner, reaching a maximum level (four- to fivefold higher than control) at about 10(-9) M. This increase in the enzyme activity was evident by 14 hr of culture and became relatively stable after 24 hr. It correlated well with the increase in the amount of the muscle type enzyme per cell as analyzed by Western immunoblotting. Estriol and a synthetic estrogen, diethylstilbestrol, also increased the enzyme activity, whereas testosterone, progesterone, and corticosterone were without effect. An antiestrogen, tamoxifen, which by itself was inactive, partially suppressed the effect of E2. Exposure of MtT/S cells for 14 hr to E2 increased incorporation of 35S-labeled amino acids into the immunoprecipitable peptidylarginine deiminase. This increase was dependent on the concentration of E2, attaining a maximum level (about tenfold higher than the control) at about 10(-9) M. These results indicate that estrogen effects the increase in peptidylarginine deiminase content in the pituitary cells by stimulating enzyme synthesis.     

Immunocytochemical and biochemical studies of GLUT4 in rat skeletal muscle.

In muscle and adipocytes, glucose transport is regulated by the translocation of insulin regulatable glucose transporters (GLUT4) between an intracellular compartment and the cell surface. In these studies we have characterized the cellular compartments containing GLUT4 in rat skeletal muscle. Immunocytochemical studies showed that in unstimulated muscle, GLUT4 was not present in surface membranes. Tubulo-vesicular structures clustered in the trans Golgi reticulum were enriched in GLUT4. GLUT4 underwent translocation to the sarcolemma in response to combined stimulation with insulin and exercise. Using immunoisolation, the intracellular GLUT4 vesicles (IRGTV) were purified 300-fold over the cell homogenate. IRGTV from unstimulated muscle were not enriched in markers specific for the sarcolemma, transverse tubules, sarcoplasmic reticulum or mitochondria; this was confirmed using gel filtration chromatography. Insulin resulted in a 40% decrease in GLUT4 levels in IRGTV confirming that this represents the intracellular compartment of GLUT4. GLUT4 is a major component of the IRGTV, constituting at least 5% of total vesicle protein. A subset of polypeptides are also markedly enriched in the muscle IRGTV. In conclusion, these data suggest that translocation of GLUT4 from intracellular tubulo-vesicular structures is the major mechanism by which insulin and exercise regulate muscle glucose transport.     

Histochemical demonstration of ATP: creatine phosphotransferase in rat skeletal muscle

Summary An attempt was made to locate the ATP: creatine phosphotransferase (creatine kinase, CKase) in rat skeletal muscle by a lead precipitation method. The muscle is not stained at all with creatine phosphate (CP), and only weakly with adenosine diphosphate (ADP) as substrate, while it hydrolyzes adenosine triphosphate (ATP) actively. Taking advantage of this fact, it is possible to demonstrate the CKase activity using both ADP and CP as substrate. The CKase activity thus obtained was located in various profiles of sarcoplasmic reticulum as well as in A bands, the staining being comparable to that obtained with ATP as substrate.A weak activity was found only in cisternal dilatations of sarcoplasmic reticulum when sections were incubated with ADP as substrate.     

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

Peptidylarginine deiminase (PAD) catalyzes the post-translational modification of protein through the conversion of arginine to citrulline in the presence of calcium ions. Human, similar to rodents, has four isoforms of PAD (type I, II, III and IV/V), each of which is distinct in substrate specificity and tissue specific expression. In our large-scale sequencing project, we identified a new human PAD cDNA from a human fetal brain cDNA library. The putative protein encoded by this cDNA is designated hPADVI. Expression analysis of hPADVI showed that it is mainly expressed in adult human ovary and peripheral blood leukocytes. We conclude that hPADVI may be orthologous to mouse ePAD, basing on sequence comparison, chromosome localization and exon-intron structure analysis. PAD-mediated deimination of epithelial cell keratin resulting in cytoskeletal remodeling suggests a possible role for hPADVI in cytoskeletal reorganization in the egg and in early embryo development. This study describes a new important member of the human PAD family.     

Reactive oxygen species inhibit catalytic activity of peptidylarginine deiminase

Protein citrullination catalysed by peptidylarginine deiminase (PAD) may play an important pathogenic role in several chronic inflammatory diseases and malignancies. PAD2, PAD4, and citrullinated proteins are found in the synovium of rheumatoid arthritis patients. PAD activity is dependent on calcium and reducing conditions. However, reactive oxygen species (ROS) have been shown to induce citrullination of histones in granulocytes. Here we examine the ability of H₂O₂ and leukocyte-derived ROS to regulate PAD activity using citrullination of fibrinogen as read-out. H₂O₂ at concentrations above 40?µM inhibited the catalytic activity of PAD2 and PAD4 in a dose-dependent manner. PMA-stimulated leukocytes citrullinated fibrinogen and this citrullination was markedly enhanced when ROS formation was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI). In contrast, PAD released from stimulated leukocytes was unaffected by exogenously added H₂O₂ at concentrations up to 1000?µM. The role of ROS in regulating PAD activity may play an important part in preventing hypercitrullination of proteins.     

Immunocytochemical demonstration of vasopressin binding in rat kidney

We investigated the immunoperoxidase demonstration of vasopressin (VSP) bound to paraffin-embedded sections of rat kidney and the effects of various fixatives. Slices of rat kidney from normal and 4-day water-deprived rats were incubated with 10(-7) M VSP, fixed, and embedded in paraffin. Hydrated sections of these tissues were again incubated with 10(-7) M VSP or 10(-7) M VSP and 10(-5) M oxytocin (OXY). VSP bound to the sections was demonstrated using rabbit anti-Arg8 VSP antiserum and peroxidase-labeled second antibody. In sections of kidney from both normal and water-deprived rats, immunoperoxidase labeling was most intense in the renal papilla and was restricted to the cells of the ducts of Bellini and loops of Henle. In the medulla, the collecting ducts and medullary thick ascending limbs of Henle were moderately stained. In the normal kidney sections there was no staining of the proximal tubules, distal convoluted tubules (DCT), and only slight staining of the cortical collecting ducts (CCD). However, in the water-deprived rats there was a considerable increase in the staining of the DCT and CCD. Simultaneous incubation in OXY and VSP resulted in reduced immunoperoxidase labeling of the tubules. Omission of VSP incubation led to a similar decrease in stain intensity, indicating a specificity for the sites of VSP binding. This technique allows the identification of cells responsible for the binding of VSP in the kidney.     

Human skeletal muscle fiber type adaptability to various workloads

Muscle biopsy specimens were removed from the vastus lateralis muscles of three groups of human subjects: controls, weight lifters, and distance runners. The runners proved to be a unique group with respect to the variables measured (low body weight and percentage body fat, and high VO2 max). Additionally, a histochemical analysis of the biopsy specimens revealed that the runners had a significantly higher percentage of fiber types I and IIC than either the controls or the weight lifters. Using a cryostatic retrieval method, each of the fibers identified histochemically was then analyzed morphometrically using electron microscopy. The results of volume-percent mitochondria demonstrated a strong relationship between the ATPase activity and oxidative potential of the fiber types for all three groups such that the oxidative activity would be ranked I greater than IIA greater than IIB. Irrespective of fiber type, there were significant differences between the groups with regard to muscle-fiber mitochondrial (runners greater than lifters greater than controls) and lipid content (runners greater than controls greater than lifters). The lifters had a significantly greater content of mitochondria than the controls, which may suggest that inactivity rather than the lifting exercise contributes to a low volume-percent mitochondria and a high percentage of type IIB fibers.     

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.     

Nuclear localization of peptidylarginine deiminase V and histone deimination in granulocytes

Peptidylarginine deiminase (PAD) deiminates arginine residues in proteins to citrulline residues Ca(2+) dependently. There are four types of PADs, I, II, III, and V, in humans. We studied the subcellular distribution of PAD V in HL-60 granulocytes and peripheral blood granulocytes. Expression of green fluorescent protein-tagged PADs in HeLa cells revealed that PAD V is localized in the nucleus, whereas PAD I, II, and III are localized in the cytoplasm. PAD V deletion mutants indicated that the sequence residues 45-74 have a nuclear localization signal (NLS). A sequence feature of this NLS is a three-lysine residue cluster preceded by a proline residue and is not found in the three other PADs. Substitution of the lysine cluster by an alanine cluster abrogated the nuclear import activity. These results suggested that the NLS is a classical monopartite NLS. HL-60 granulocytes, neutrophils, and eosinophils stained with antibody specific for PAD V exhibited distinct positive signals in the nucleus. Subcellular fractionation of HL-60 granulocytes also showed the nuclear localization of the enzyme. When neutrophils were stimulated with calcium ionophore, protein deimination occurred in the nucleus. The major deiminated proteins were identified as histones H2A, H3, and H4. The implication of PAD V in histone modifications is discussed.