Role Of Citrullination Research Articles

Insights into peptidylarginine deiminase expression and citrullination pathways.

Peptidylarginine deiminases (PADs) are calcium-dependent enzymes that mediate citrullination, an irreversible post-translational modification (PTM). PAD enzymes have received increasing attention in (patho-)physiology since multi-omics analysis accelerated their expression profiling. Here, we provide a comprehensive overview of PAD expression at the RNA and protein levels, and a list of annotated substrates per PAD isozyme. We discuss novel roles of citrullination in cellular growth, epigenetic regulation, tissue remodeling, inflammation, and cancer in mouse models and humans. Additionally, we cluster similar effects of protein deimination to offer a different perspective and improve our understanding of citrullination in health and disease. Citrullination should no longer be considered as a rare PTM, but as an important regulatory mechanism in physiology and pathology.

State of the art: the role of citrulline as biomarker in patients with chemotherapy- or graft-versus-host-disease-induced mucositis.

Serum or plasma citrulline levels are used as biomarker for a broad spectrum of intestinal functions. During high-dose chemotherapy, citrulline levels are decreased due to mucositis, a common side effect of chemotherapy. This may decrease intestinal function and result in diarrhea. In this review, most recent studies investigating citrulline as biomarker for intestinal function are discussed, with focus on patients with oncological diseases, specifically hematological malignancies with chemotherapy- or Graft-versus-Host-disease (GVHD)-induced mucositis. Citrulline has recently been widely studied in relation to intestinal function and various clinical conditions. It seems therefore a promising noninvasive biomarker in clinical practice for more than intestinal function alone. The association between citrulline levels and intestinal function in patients with hematological malignancies, with or without mucositis remains unclear, as no other parameters of intestinal function for this purpose were assessed. In conclusion, citrulline seems to be a promising noninvasive biomarker for various intestinal conditions in general, and potentially for intestinal function in patients with chemotherapy- or GVHD-induced mucositis. It is unclear from recent literature whether high fecal volume or diarrhea as side effect, results in impaired intestinal function and severe malabsorption and if citrulline biomarkers can be useful to detect this.

The role of citrulline, intestinal fatty acid-binding protein, and D-dimer as potential biomarkers in the diagnosis of internal herniation after Roux-en-Y gastric bypass.

Diagnosing internal herniation (IH) in Roux-en-Y gastric bypass (RYGB) patients with acute abdominal pain poses a diagnostic challenge. Diagnostic laparoscopy is often required for a definitive diagnosis. We hypothesized that intestinal ischemia biomarkers would aid in the diagnosing of IH. To explore intestinal ischemia biomarkers in diagnosing IH. University Hospital, Sweden. Prospective inclusion of 46 RYGB patients admitted for acute abdominal pain between June 2015 and December 2017. Blood samples for analysis of citrulline, intestinal fatty acid-binding protein (I-FABP), and D-dimer were drawn <72 hours from admission and compared between patients with IH (n = 8), small bowel obstruction (SBO) (n = 5), other specified diagnoses (n = 12), or unspecified abdominal pain (n = 21). Levels of white blood cell count (WBC), C-reactive protein (CRP), and lactate at admission were compared. A prospective pain questionnaire for time of pain onset and level of pain at onset and at admission was analyzed. None of the investigated biomarkers differed significantly between diagnosis categories. Most patients with IH had normal CRP, WBC, and D-dimer levels while their lactate levels were significantly lower (P = .029) compared with the rest of the cohort. Neither pain level nor pain duration differed between the groups. This study shows that citrulline, I-FABP, and D-dimer cannot be used to diagnose IH and indicates that CRP, D-dimer, and lactate are rarely elevated by an IH. Furthermore, pain intensity and duration cannot differentiate patients with IH. A diagnostic laparoscopy remains the gold standard to diagnose and rule out an IH.

The Role of Citrullination in Inflammatory Bowel Disease: A Neglected Player in Triggering Inflammation and Fibrosis?

Citrullination is a posttranslational modification of proteins mediated by a specific family of enzymes called peptidylarginine deiminases (PAD). Dysregulation of these enzymes is involved in the etiology of various diseases, from cancer to autoimmune disorders. In inflammatory bowel disease (IBD), data for a role of citrullination in the disease process are starting to accumulate at different experimental levels including gene expression analyses, RNA, and protein quantifications. Most data have been generated in ulcerative colitis, but data in Crohn disease are lacking so far. In addition, the citrullination of histones is the fundamental process promoting inflammation through the formation of neutrophil extracellular traps (NETs). Interestingly, NETs have also been shown to activate fibroblasts into myofibroblasts in fibrotic interstitial lung disease. Therefore, citrullination merits more thorough study in the bowel to determine its role in driving disease complications such as fibrosis. In this review we describe the process of citrullination and the different players in this pathway, the role of citrullination in autoimmunity with a special focus on IBD, the emerging role for citrullination and NETs in triggering fibrosis, and, finally, how this process could be therapeutically targeted.

Functional Relevance of Citrulline in the Vegetative Tissues of Watermelon During Abiotic Stresses.

A non-protein amino acid, citrulline, is a compatible solute involved in the maintenance of cellular osmolarity during abiotic stresses. Despite its significance, a coherent model indicating the role of citrulline during stress conditions has not yet emerged. We have used watermelon, naturally rich in citrulline, as a model to understand its accumulation during drought stress and nitrogen perturbation using transcriptomic and metabolomic analysis. Experiments were performed in the semi-controlled environment, and open field to study the accumulation of drought-induced citrulline in the vegetative tissues of watermelon by monitoring the stress treatments using physiological measurements. The amino acid profiling of leaves and stems in response to drought stress showed up to a 38 and 16-fold increase in citrulline content, respectively. Correlation between amino acids indicated a concomitant activation of a metabolic pathway that included citrulline, its precursor (ornithine), and catabolic product (arginine). Consistent with its accumulation, the gene expression analysis and RNA-Sequencing confirmed activation of citrulline biosynthesis-related genes – Ornithine carbamoyl-transferase (OTC), N-acetylornithine deacetylase (AOD) and Carbamoyl phosphate synthases (CPS), and down-regulation of catabolic genes; Arginosuccinate lyase (ASL) and Arginosuccinate synthases (ASS) in drought-stressed leaf tissues. Based on the relative abundance in the nitrogen-depleted vegetative tissues and down-regulation of genes involved in citrulline biosynthesis, we also demonstrated that the nitrogen status of the plant regulates citrulline. Taken together, these data provide further insights into the metabolic and molecular mechanisms underlying the amino acid metabolism under environmental stress and the significance of non-protein amino acid citrulline in plants.

Haplotype Networking of GWAS Hits for Citrulline Variation Associated with the Domestication of Watermelon.

Watermelon is a good source of citrulline, a non-protein amino acid. Citrulline has several therapeutic and clinical implications as it produces nitric oxide via arginine. In plants, citrulline plays a pivotal role in nitrogen transport and osmoprotection. The purpose of this study was to identify single nucleotide polymorphism (SNP) markers associated with citrulline metabolism using a genome-wide association study (GWAS) and understand the role of citrulline in watermelon domestication. A watermelon collection consisting of 187 wild, landraces, and cultivated accessions was used to estimate citrulline content. An association analysis involved a total of 12,125 SNPs with a minor allele frequency (MAF)>0.05 in understanding the population structure and phylogeny in light of citrulline accumulation. Wild egusi types and landraces contained low to medium citrulline content, whereas cultivars had higher content, which suggests that obtaining higher content of citrulline is a domesticated trait. GWAS analysis identified candidate genes (ferrochelatase and acetolactate synthase) showing a significant association of SNPs with citrulline content. Haplotype networking indicated positive selection from wild to domesticated watermelon. To our knowledge, this is the first study showing genetic regulation of citrulline variation in plants by using a GWAS strategy. These results provide new insights into the citrulline metabolism in plants and the possibility of incorporating high citrulline as a trait in watermelon breeding programs.

Citrullination in Cancer.

Posttranslational modifications of proteins have been implicated in pathogenesis of numerous diseases. Arginine deimination (also known as citrullination) has a principal role in progression of rheumatoid arthritis through generation of autoantibodies and exacerbation of the inflammatory response. Recently, multiple research groups provided solid evidence of citrullination being in control of cancer progression; however, there is no comprehensive overview of these findings. This article summarizes and critically reviews the influence of citrullination on different aspects of tumor biology, including (i) regulation of apoptosis and differentiation, (ii) promoting EMT and metastasis, and (iii) potential use of citrullinated antigens for immunotherapy. In addition, (iv) the role of citrullination as a cancer biomarker and (v) implication of neutrophil extracellular traps in tumorigenesis are discussed. In summary, current findings testify to the significance of arginine deimination in tumor biology and thus more basic and translational studies are needed to further explore this topic.

The function of citrulline in pediatric acute gastrointestinal injury

Objective To study the occurrence of acute gastrointestinal injury under different critical status in Pediatric Intensive Care Unit, and to assess the connection between citrulline and acute gastrointestinal injury.To analyze the correlation among serum citrulline and C-reaction protein(CRP), procalcitonin (PCT), creatinine, urea nitrogen, bilirubin, lactate, albumin.To study the role of citrulline in evaluating the children condition, prognosis and changes in critically ill children. Methods Ninety-six pediatric patients who were critically ill at the Pediatric Intensive Care Unit of the Second Hospital of Lanzhou University were selected, excluding those with primary gastrointestinal diseases and chronic kidney disease.Clinical features were recorded and CRP, PCT, creatinine, urea nitrogen, bilirubin, lactate, and albumin were measured.Serum citrulline concentrations were measured by high performance liquid chromatography in 65 patients with gastrointestinal injury, and sensitivity, specificity and area under the receiver operating characteristic(ROC) curve were calculated. Results The incidence of acute gastrointestinal injuries in non-critical group, critical group, reorganization critical group were 47.06%(16/34 cases), 75.00%(36/48 cases), 92.86%(13/14 cases) (χ2=11.848, P=0.003). The serum citrulline concentrations of gastrointestinal injury group and non- gastrointestinal injury group were (14.655±8.231) μmol/L, (23.522±11.079) μmol/L (t=4.398, P<0.05). The area under ROC of citrulline was 0.079 8.Serum citrulline concentrations increased significantly in infants with very severe gastrointestinal injury after 3 days of treatment compared with admission [(26.40±16.10) μmol/L vs.(14.40±6.82) μmol/L, t=5.524, P=0.029]. The lower serum citrulline levels the children had, the higher mortality was.There were negative correlations among serum citrullin and CRP, PCT and length of hospital stay(r=-0.319, -0.299, -0.364, P=0.003, 0.006, 0.001). There were no correlations among serum citrullin and blood urea nitrogen, lactic acid, albumin, and bilirubin levels. Conclusion The more critically ill children have a higher incidence of acute gastrointestinal injury and the worse the prognosis.Serum citrulline levels may well reflect the extent of acute gastrointestinal injury and the outcome.Perhaps it can be one of the reliable markers to evaluate the function of gastrointestinal and participate in assessing critical condition and prognosis in critical ill children ness. Key words: Citrullin; Acute gastrointestinal injury; Critical illness

Comment on "Synovial fibroblast-neutrophil interactions promote pathogenic adaptive immunity in rheumatoid arthritis".

Discrepancies on the role of citrullination in the induction of experimental arthritis by neutrophils extracellular traps.

Generation and characterisation of Porphyromonas gingivalis mutant lacking peptidylarginine deiminase activity

ABSTRACTPorphyromonas gingivalis peptidylarginine deiminase (PPAD) is the focus of several studies due to its ability to citrullinate in vitro human proteins, which have been linked to the aetiopathogenesis of rheumatoid arthritis (RA). The aim of this work was the generation by homologous recombination and characterisation of a P. gingivalis W50 mutant lacking pad gene (PG1424) to study the role of PPAD in RA. To confirm the absence of PPAD activity in P. gingivalis PG1424, cells were incubated with arginine-containing substrates and citrullination of L-arginine measured using a colorimetric assay and thin-layer chromatography. Furthermore, arginine and lysine protease (gingipain) activities were assessed and immunoblotting was performed using monoclonal antibody 1B5 (mAb1B5) and a commercial anti-modified citrulline antibody (AMC) to detect differences in virulence factor expression. The deletion of pad gene in P. gingivalis PG1424 completely abolished the ability to autocitrullinate P. gingivalis proteins in the mutant strain and also the citrullination of used substrates but not free L-arginine. Moreover, the wild-type and mutant strains had similar total gingipain activities and reactivity with mAb1B5. In conclusion, this work has produced a well-characterised PPAD-deleted P. gingivalis strain, which can be used to help determine the role of citrullination by this microorganism in RA.

Downregulation of the Deiminase PADI2 Is an Early Event in Colorectal Carcinogenesis and Indicates Poor Prognosis.

Peptidyl arginine deiminases (PADI) are a family of enzymes that catalyze the poorly understood posttranslational modification converting arginine residues into citrullines. In this study, the role of PADIs in the pathogenesis of colorectal cancer was investigated. Specifically, RNA expression was analyzed and its association with survival in a cohort of 98 colorectal cancer patient specimens with matched adjacent mucosa and 50 controls from donors without cancer. Key results were validated in an independent collection of tumors with matched adjacent mucosa and by mining of a publicly available expression data set. Protein expression was analyzed by immunoblotting for cell lines or IHC for patient specimens that further included 24 cases of adenocarcinoma with adjacent dysplasia and 11 cases of active ulcerative colitis. The data indicate that PADI2 is the dominantly expressed PADI enzyme in colon mucosa and is upregulated during differentiation. PADI2 expression is low or absent in colorectal cancer. Frequently, this occurs already at the stage of low-grade dysplasia. Mucosal PADI2 expression is also low in ulcerative colitis. The expression level of PADI2 in tumor and adjacent mucosa correlates with differential survival: low levels associate with poor prognosis. Downregulation of PADI2 is an early event in the pathogenesis of colorectal cancer associated with poor prognosis and points toward a possible role of citrullination in modulating tumor cells and their microenvironment. Mol Cancer Res; 14(9); 841-8. ©2016 AACR.

Ureido group-specific antibodies are induced in rabbits immunized with citrulline- or homocitrulline-containing antigens

The specificities and cross-reactions of antibodies induced by citrulline- and homocitrulline-containing proteins may give implications on the role of citrulline- and homocitrulline-binding antibodies in the pathogenesis and progression of rheumatoid arthritis (RA). Here we use rabbits as an experimental model of antibody development in RA. Thirty-two animals were immunized with peptide antigens containing either homocitrulline or citrulline. The sera were tested for binding to CCP and MCV antigens and to peptide sequences related to carboxyterminal telopeptides of type I and II collagens and containing arginine, citrulline, or homocitrulline. The binding of CCP and MCV antigens to antisera against homocitrulline-containing immunogens could be inhibited by human serum albumin containing homocitrulline, whereas similar binding to sera against citrulline-containing immunogens was not inhibited. The antisera induced with citrulline-containing collagen telopeptides recognized type I collagen-related antigens in a sequence-specific manner, as antibody binding to both citrulline- and homocitrulline-containing peptides was inhibited by corresponding citrullinated and native peptides. In contrast, type II collagen-related peptides were recognized by the antisera in a ureido group-specific manner, as their binding to homocitrulline-containing peptide was inhibited by both citrulline- and homocitrulline-containing, but not native peptide. Binding of the citrullinated type II collagen peptide could only be inhibited by the similarly citrullinated peptide. In conclusion, antibodies induced with citrulline or homocitrulline-containing antigens bound antigens in a ureido group-specific manner, recognizing citrulline and homocitrulline also in other sequences than those used in the original immunization. In competitive situations the amino acid present in the immunization antigen was favored.

Gammaglobulins, autoantibodies, therapeutic antibodies and anti-drug antibodies in rheumatoid arthritis.

Gammaglobulins, autoantibodies, therapeutic antibodies and anti-drug antibodies in rheumatoid arthritis.

Mitochondria: Role of citrulline and arginine supplementation in MELAS syndrome

Mitochondria are found in all nucleated human cells and generate most of the cellular energy. Mitochondrial disorders result from dysfunctional mitochondria that are unable to generate sufficient ATP to meet the energy needs of various organs. Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a frequent maternally inherited mitochondrial disorder. There is growing evidence that nitric oxide (NO) deficiency occurs in MELAS syndrome and results in impaired blood perfusion that contributes significantly to several complications including stroke-like episodes, myopathy, and lactic acidosis. Both arginine and citrulline act as NO precursors and their administration results in increased NO production and hence can potentially have therapeutic utility in MELAS syndrome. Citrulline raises NO production to a greater extent than arginine, therefore, citrulline may have a better therapeutic effect. Controlled studies assessing the effects of arginine or citrulline supplementation on different clinical aspects of MELAS syndrome are needed.

Citrullination of fibrinogen: generation of neoepitopes and enhancement of immunostimulatory properties

Rheumatoid arthritis (RA) affects approximately 0.5% of the world population, yet the mechanisms underlying the development and progression of RA remain poorly understood. We are investigating the role of citrullinated fibrinogen as a pathogenic antigen in RA. Using arthritis antigen arrays we demonstrate that citrullinated fibrinogen is one of the earliest targets of the autoantibody response in RA, with autoantibodies against citrullinated fibrinogen appearing up to 10 years prior to the development of clinical arthritis. We further demonstrate that approximately 50% of CCP+ RA patients possess circulating immune complexes containing citrullinated fibrinogen, and that citrullinated fibrinogen containing immune complexes are deposited in human RA synovial tissues. To determine whether citrullinated fibrinogen can induce inflammatory arthritis in mice, we immunized mice with citrullinated fibrinogen and demonstrated that an inflammatory arthritis results and that both T cells and serum can transfer arthritis to naive mice. Fibrinogen is an endogenous ligand for the innate immune receptor TLR4, and to determine whether citrullination might alter the ability of fibrinogen to bind TLR4 we performed in vitro macrophage stimulation assays with native and citrullinated fibrinogen. We found that citrullinated fibrinogen was ten-fold more potent than native fibrinogen at stimulating macrophage TNF release. Further, macrophage derived from mice deficient for TLR4 or MyD88 did not produce TNF in response to citrullinated fibrinogen. Thus, our results demonstrate a novel mechanism by which anti-citrullinated protein antibodies (ACPA) specifically targeting citrullinated fibrinogen may directly stimulate macrophage TNF production, via co-ligation of TLR4 and Fc-gamma-R. Our findings demonstrate a role for citrullination both in creating neoantigens targeted by the adaptive immune response in RA as well as by increasing the potency of fibrinogen as an endogenous innate immune ligand. These results provide insights into the mechanisms by which anti-citrulline autoimmunity, and specifically the citrullination of fibrinogen, may contribute to both the onset and propagation of inflammation in RA.

Plasma citrulline as surrogate marker of intestinal inflammation in pediatric and adolescent with Crohn’s disease: preliminary report

Several researchers have found that plasma citrulline could be a marker of reduced enterocyte mass. The aim of this study was to assess the relationship between plasma citrulline and bowel inflammation and/or disease location in pediatric and adolescent Crohn's disease (CD) patients. Between January 2008 and January 2010, 31 CD patients and 44 controls were included in our study, and 15 out of the 31 CD patients continued a prospective survey. We evaluated the differences between groups, at baseline, in plasma citrulline and glutamine and between their baseline and final values during the prospective survey, and correlation between baseline values of citrulline and duration of disease, C-reactive protein, and fecal calprotectin. Mean citrulline value was 33.0 ± 7.5μmol/L in controls and 23.5 ± 8.4μmol/L in CD patients (P < 0.0001). Plasma citrulline was significantly lower in patients with small bowel (SB) location than in patient with only ileo-colon disease (14.2 ± 5.5 and 24.7 ± 8.0, respectively; P = 0.0037). Citrulline ≤22μmol/L reached sensitivity of 100% (95% confidence interval (CI) 54-100) and specificity of 98% (CI 89-99) in differentiating control subjects from CD with SB location. CD patients have reduced concentration of plasma citrulline than controls. Intestinal damage rather than inflammation seems to be responsible for the reduced biosynthesis of citrulline, which decreases particularly in CD patients with SB location. This finding suggests the potential role of citrulline as marker of disease location, but future works will be needed to confirm this suggestion.

Concomitant apolipoprotein A-IV and citrulline plasma changes during short-term parenteral nutrition in surgical patients

Summary Background & aims Apolipoprotein A-IV (apo A-IV) and citrulline are non-invasive biomarkers that can be used to monitor nutritional and intestinal changes in patients receiving parenteral nutrition (PN). Yet, while the role of citrulline has been well established, further research is needed to understand apo A-IV’s utility. Methods Twenty-eight patients who underwent gastrointestinal surgery by laparotomy, either a subtotal gastrectomy or a hemicolectomy, and were placed on short-term PN, were selected and assigned on a Parenteral-Oral (4-day PN and 4-day oral, 8 patients) or a Parenteral-Only (7-day PN, 20 patients) nutritional regime. They were delivered PN solutions (PN1 to PN4) containing varying amounts of amino acids, fat and carbohydrates. Results Pre-operative apo A-IV and citrulline values were within those of a western population. On day 4 in the Parenteral-Oral regime, apo A-IV and citrulline levels were 70% and 60% lower than pre-operative levels, respectively. When enteral feeding was resumed, citrulline rose faster than apo A-IV and was close to pre-operative values on day 8. In the Parenteral-Only regime the PN solution composition had no influence on the parameters. While Apo A-IV levels decreased with time, citrulline started increasing on day 2. Conclusions Both biomarkers can be used complementarily to monitor intestinal function. While apo A-IV may be more sensitive than citrulline in measuring the impact of a lack of oral intake, citrulline reacts faster to the onset of enteral nutrition.

Abstract 1102: Regulation of protein citrullination through p53/PADI4 network in DNA damage response

Abstract Upon wide range of cellular stresses, p53 is activated and inhibits malignant transformation through the transcriptional regulation of its target genes related to apoptosis, cell cycle arrest, and DNA repair. However its involvement in post-translational modifications of proteins has not been well characterized so far. We here report the novel role of p53 in the regulation of protein citrullination. Citrullination (also referred as deimination) is one form of PTMs converting an arginine residue in protein to a citrulline residue. This reaction is mediated by a Ca2+-dependent enzyme called as peptidylarginine deiminase. Citrullination of proteins results in a loss of positive charge and causes a significant biochemical change. On the other hand, citrullinated peptides/proteins are recognized as a non-self protein and subsequently activate immune systems. Hence, protein citrullination is now considered to play a pivotal role in various human diseases, but any roles of citrullination in carcinogenesis were not reported so far. We found p53 transactivated peptidylarginine deiminase type 4 (PADI4) through intronic p53-binding site and ectopic expression of p53 or PADI4 induced protein citrullination. In addition, various proteins were citrullinated in response to DNA damage, but knockdown of PADI4 or p53 remarkably inhibited their citrullination, indicating the regulation of protein citrullination in a p53/PADI4 dependent manner. We identified that PADI4 citrullinated histone chaperon protein, nucleophosmin (NPM1) at arginine 197 residue in vivo under physiological condition. NPM1 was shown to continuously shuttle between the nucleus and cytoplasm and translocate from nucleoli to nucleoplasm as a response to DNA damage. Therefore, we investigated the effect of protein citrullination on subcellular localization of NPM1. Immunocytochemical analysis revealed that citrullination of NPM1 by PADI4 resulted in its translocation from nucleoli to nucleoplasm, while PADI4 did not alter the localization of mutant NPM1 (R197K). Furthermore, ectopic expression of PADI4 inhibited tumor cell growth, and concordantly knockdown of PADI4 attenuated p53-mediated growth inhibitory activity, demonstrating the significance of PADI4-mediated protein citrullination in the p53 signaling pathway. Most of previous studies assessing enzymatic activities of PADIs were conducted under non-physiological condition using Ca ionophore or at higher Ca concentration. To our knowledge, this is the first report demonstrating the physiological regulatory mechanism of protein citrullination. Thus, p53 that can directly or indirectly modulate biological property of various proteins through the protein citrullination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1102.

The Role of Citrulline in Amino Acid Metabolism and the Ornithine Cycle in Cryptomeria japonica Seedlings

The Role of Citrulline in Amino Acid Metabolism and the Ornithine Cycle in Cryptomeria japonica Seedlings

Role of citrulline in rejection monitoring after bowel transplantation

Role of citrulline in rejection monitoring after bowel transplantation