Citrulline Peptides as Therapeutic Agents

Citrullinated peptides enable the study of autoimmune diseases, including Rheumatoid Arthritis (RA). In RA, the human body produces Anti-Citrullinated Protein Antibodies (ACPAs) that mistakenly attack citrulline-containing proteins. Synthetic citrullinated peptides enable the design and production of diagnostic assays needed to study how citrullinated proteins trigger immune responses.

Many recent studies have found connections between ubiquitous peptidylarginine deiminase (PAD) expression, citrullination, and numerous diseases, suggested to aid in the development and progression of rheumatoid arthritis (RA), prion disease, psoriasis, Alzheimer’s disease (AD), multiple sclerosis, various cancers, diabetes, and others.

Citrullination is a natural process; however, in RA, it is amplified, and several proteins are citrullinated in inflamed synovial tissue. Inflammation may denature proteins, making hidden arginine residues accessible to citrullination and potentially "seen" as non-self by the immune system. 

Citrulline is a non-essential amino acid that plays a physiological role in the human body, including the management of waste and the maintenance of blood flow. Unlike proteogenic amino acids, citrulline is not used for protein synthesis; instead, it functions as a key metabolic intermediate. The amino acids L-arginine (Arg) and L-citrulline (Cit) also play important physiological roles in the human body, including the production of nitric oxide (NO) and the removal of waste products during exercise. The non-essential amino acid citrulline can bypass hepatic metabolism to enhance arginine synthesis and improve NO bioavailability. Natural citrulline is found in watermelons.

Citrulline is produced from ornithine and carbamoyl phosphate, and it prevents the buildup of nitrogenous waste that can otherwise lead to fatigue or cognitive issues. Citrulline is a metabolite of the urea cycle produced by the mitochondrial enzyme ornithine carbamoyl transferase (EC 2.1.3.3). In the cytosol, L-Citrulline functions as a substrate for argininosuccinate synthase (EC 6.3.4.5). Arginine deiminase (EC 3.5.3.6) hydrolyzes free L-Arg to L-Cit by the concomitant release of NH3. Free L-Cit also occurs as a result of proteolytic degradation of post-translationally modified Arg residues by the enzyme protein arginine deiminase (EC 3.5.3.15). Another enzyme that converts L-Arg to L-Cit is the NO-producing enzyme nitric oxide synthase (NOS, EC 1.14.13.39). This pathway, together with the L-Arg recycling pathway over L-argininosuccinate, is known as the Arg-Cit cycle. Nitric oxide synthases are a family of enzymes that catalyze the conversion of L-arginine into NO and L-citrulline via the intermediate N-hydroxy-L-arginine. This conversion requires oxygen and nicotinamide adenine dinucleotide phosphate (NADPH) as additional substrates together with tetrahydrobiopterin (BH4), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and iron protoporphyrin IX as essential cofactors.

Citrullination is an important post-translational modification (PTM) of arginine, known to play a role in autoimmune disorders, the innate immune response, and the maintenance of stem cell potency. Citrullination involves the conversion of the amino acid arginine into citrulline. The family of peptidyl arginine deaminases (PADs) catalyzes the conversion of arginine to citrulline by replacing the nitrogen with an oxygen atom, removing the positive charge of arginine, and possibly altering the conformation of citrullinated peptides or proteins. Several proteins are known to be citrullinated, including histone proteins, fibronectin, and myelin basic protein (MBP), as well as other cellular proteins. Citrullinated peptides result from a post-translational modification called deimination. PAD converts the positively charged side chain of an arginine residue into the neutral side chain of citrulline. Arginine contains a guanidinium group (-NH-C(NH₂)=NH), whereas citrulline contains a ureido group (-NH-CO-NH₂).

Figure 1: Deimination reaction of arginyl residues within peptide bonds. Deimination of arginine results in neutral citrulline with the release of ammonia and the loss of one positive charge for each arginyl residue deiminated. The process is catalyzed by a calcium dependent peptidylarginine deiminase.

Citrullination occurs during epigenetic regulation. Other post-translational modifications include acetylation, methylation, phosphorylation, ubiquitination and citrullination among others. Histone modifications induce changes to the struc­ture of chromatin, thereby affecting the accessibility of the DNA strand to transcriptional enzymes, resulting in activation or repression of genes associated with modified histones. Citrulline modifications have related to autoimmune disorders such as multiple sclerosis and rheumatoid arthritis. However, the full role of citrullination at the cellular level is presently not well understood. Citrullination is known to play important physiological roles in maintaining stem cell potency, during the innate immune response, and in maintaining the skin barrier. Dysregulation of citrullination is a driving factor in rheumatoid arthritis (RA), psoriasis, and cancer.

Several proteins are known to contain citrulline as a result of posttranslational modification. Peptidyl arginine deiminases (PADs) generate citrulline residues by converting arginine into citrulline in a process called citrullination or deimination. Citrullination is a natural physiological process that occurs in many dying cells. The PAD enzyme has several isoforms. PAD2 and PAD4 are expressed in inflammatory leukocytes. The release of PAD from dying cells citrullinates extracellular proteins that contain arginine. Production of anticitrullinated protein antibody (ACPA) depends on the genetic background of a patient.

PAD4 regulates the citrullination of three histones: H2A, H3, and H4. PAD4 is the only PAD enzyme found within the cell nucleus. Histones are the primary component of chromatin, regulating gene expression. PAD4 targets specific arginine residues within each histone and converts them to citrulline. Citrullination appears to repress gene expression. PAD4 represses hormone-dependent transcriptional activation without disrupting activation by other enzymatic activators, allowing for multiple checkpoints to control gene expression. Ca2+ concentration levels in the cytoplasm regulate enzyme activity. PAD4 has been implicated in the development of rheumatoid arthritis.

A diagnostic assay utilizes a cyclic citrullinated peptide as an antigen. The anti-citrullinated peptide antibody (ACPA) response enables detection of elevated ACPA levels before clinical manifestations. Senshu et al. (1992) (Pubmed) developed a method for the colorimetric detection of deiminated proteins on polyvinylidene difluoride membranes. The method allowed the detection of 2 to10 fmol citrulline residues in enzymatically deiminated histones by incubating with diacetyl monoxime and antipyrine in a strong acid mixture.

Rabier & Kamoun (1995) reviewed the metabolism of citrulline in man, describing its pathways, their regulation, and variations across different tissues. Knipp & Vasak (2000) developed a colorimetric 96-well microtiter plate assay for the detection of enzymatically generated citrulline. This photometric method for the determination of L-Citrulline utilizes the reaction of citrulline with oximes, such as 3-hydroxyimino-2-butanone (diacetyl monoxime, DAMO), in strong acid, where the dye is formed. 
Van Gaalen et al. (2004) performed a study to assess the predictive value of RA-specific autoantibodies to cyclic citrullinated peptides (CCPs) in patients with undifferentiated arthritis (UA).

Schwarz et al. (2005) developed an HPLC-based method for the analysis of amino acids in plasma. Primary amino acids were derivatized with o-phthalaldehyde 3-mercaptopropionic acid (OPA) and detected by a diode array detector. Secondary amino acids were derivatized with 9-fluorenylmethyl chloroformate (FMOC) and detected fluorometrically. Chromatographic separation is achieved by two gradient elutions (two injections per sample), starting at different pHs, on a reverse phase Agilent Zorbax Eclipse C18 column AAA (4.6 x 150 mm). This method also allows the detection of citrulline.

To characterize citrullinated proteins in inflamed joints of RA patients, Holm et al. (2006) developed a chemical tag that enables the enrichment and subsequent detection of citrulline-containing protein fragments by mass spectrometry. The research group reported that the ureido group of citrulline reacts with 2,3-butanedione in the presence of antipyrine and found that this reaction is specific to citrulline residues. The modified product resulted in a characteristic mass shift of +238 Da, as observed by mass spectrometry. The product also absorbs UV–Vis radiation at 464 nm, enabling selective monitoring of citrulline-containing peptides in protein digests. 

A)

B)

Figure 2: (A) Suggested mechanism for the reaction of antipyrine via its enamine functionality with the tagging compound. The resulting product led to a mass shift of [M + 238 + H]+ for the modified peptide. The proposed mechanism for the reaction of N-butylurea with 2,3-butanedione illustrated the formation of the reactive imidazolone intermediate according to Liepa and co-workers. (B) Suggested mechanism for the reaction of antipyrine via its enamine. The resulting product is observed at [M + 238 + H]+, with a mass shift also observed in a citrullinated peptide.

Figure 3: Under acidic conditions, the ureido group of L-citrulline reacts with diacetyl monoxime in the presence of antipyrine, leading to the formation of a stable compound, which can be monitored at 450 nm.

The reported reaction, combined with an efficient tagging method, enables the direct in situ analysis of the citrullinated proteome and can be used for an MS-based proteomics study of citrullinated proteins and peptides as potential targets of the immune response in RA.

Curis et al. (2005) reviewed the citrulline metabolism in mammals, divided into two parts: free citrulline and citrullinated proteins, and suggested citrulline as a potential therapeutic agent in various pathologies.

Koivula et al. (2006) studied how soluble citrullinated telopeptides of type I and II collagens inhibit the binding of autoantibodies to their antigens immobilized on a solid support, showing that it is possible to measure autoantibody binding specifically to citrullinated telopeptides of type I and II collagens. Normal or citrullinated carboxy-telopeptides inhibited the binding of autoantibodies as determined using enzyme-linked immunosorbent assay (ELISA) methods. 

Pérez et al. (2006) developed an enzyme-linked immunosorbent assay test to detect autoantibodies in the sera of rheumatoid arthritis patients with high sensitivity and specificity using synthetic citrullinated peptides of fibrin. Fibrin is abundant in rheumatoid synovium. The research group selected antigenic peptides from α- and β-fibrin chains via computer-aided prediction of antigenicity and epitope mapping for synthesis.

Table 1. Citrullinated fibrin peptides selected by Perez et al.

Luban and Li (2010), in an evidence-based clinical review, summarized published data on peptide citrullination and ACPA and evaluated anti-cyclic citrullinated peptide (anti-CCP) antibodies for the diagnosis of RA. The review found that citrullinated vimentin and fibrin are likely just two of multiple citrullinated antigens in RA.

Since the substrate selectivity of PAD4 is poorly defined and its influence on other pathways is also poorly understood, Guo et al. (2011) designed a high-density protein array screen to identify 40 previously unreported PAD4 substrates. The most prominent hits, human 40S ribosomal protein S2 (RPS2), were found to be citrullinated by PAD4 at the Arg-Gly repeat region of RPS2. This sequence region is also an established site for arginine methylation by protein arginine methyltransferase 3 (PRMT3). As a result, the observed crosstalk between citrullination and methylation modifications is antagonistic, suggesting a conserved posttranslational regulatory pathway. RPS2 is citrullinated in a calcium-dependent manner at its N-terminal RG repeat region. This modification antagonizes Arg methylation at the same region by PRMT3. The citrullination protein array screen also showed that the following proteins are also citrullinated by PAD4: Cold-inducible RNA-binding protein (CIRP), Probable rRNA-processing protein (EBP2), Fasciculation and elongation protein zeta 1 (FEZ ), Ubiquitin-like protein FUBI (Fau), Heterogeneous nuclear ribonucleoprotein A1 (HNRPA1), Inhibitor of growth protein 4 (ING4), Protein arginine N-methyltransferase 1 (PRMT1), Proteasome subunit beta type 4 (PSBT4), ribosomal protein S2 (RPS2), Ufm-1-conjugating enzyme 1 (UFC).

Swart et al. (2012) tested 295 patient sera for ACPA using the QUANTA Lite® CCP 3 (INOVA Diagnostics, Inc., San Diego) and the EliA® CCP (CCP, Phadia, Germany) test kits. The rheumatoid factor (RF) was determined using Quantex RF(II) (Biokit, Spain). However, the undisclosed sequences of the cyclic peptides used were reported to be proprietary.

Moita et al. (2013) reported an integrated analysis method for inflammatory mediators. Quantitation of L-citrulline was possible using a modified method that employed diacetylmonoxime and antipyrine in acidic conditions.

Lewallen et al. (2015) developed a chemical proteomics platform to identify citrullinated proteins in cells. The method utilized biotin-conjugated phenylglyoxal (biotin-PG.

To identify these citrullinated proteins, we developed biotin-conjugated phenylglyoxal (biotin-PG). Using this probe and our platform technology, we identified >50 intracellular citrullinated proteins.         

Kumari et al. (2017) studied the role of two peptide markers (anti-CCP and CRP) in an autoimmune skin disorder and their association with arthritis in this disorder. In 50 patients, anti-CCP was elevated in 36.37% with arthritis and in 12.82% without arthritis, whereas CRP was elevated in 63.63% with arthritis and in 35.89% without arthritis. Mean serum anti-CCP levels in patients with arthritis were 15.78±13.94 U/ml, and in patients without arthritis were 7.56±7.68 U/ml (p=0.01), which was statistically significant. Mean serum CRP in arthritis was 21.11±15.51 mg/l, and CRP without arthritis was 13.14±12.27 mg/l, with p=0.07, which was statistically not significant. These results revealed that both anti-CCP and CRP are valuable markers for autoimmune skin disorder, but anti-CCP appears to show a significant association with arthritis.

Lee et al. (2018) reported the construction of a library of reference spectra for ~2,200 citrullinated and 1,300 deamidated peptides. The research group validated 375 citrullination sites across 209 human proteins and found that >80% of the identified sites were novel. For 56% of the proteins, citrullination was detected for the first time. Their sequence motif analysis revealed a strong preference for Asp and Gly residues around the citrullination site. The study also found that these modifications were mostly detected in highly abundant proteins, indicating that the development of specific enrichment methods may be required to study the full extent of cellular protein citrullination. Ru et al. (2022) reported a new pattern of citrullinated peptides to improve the sensitivity of RA tests. All selected peptides were cyclized via disulfide bonds and, as shown in Table 1, biotinylated to allow the design of an ELISA assay. Park et al. (2023) reviewed the available literature on citrulline supplements. They found that 2.4 to 6 g of Citrulline taken per day for 7 to 16 days across various nutritional supplements showed a positive impact, increasing NO synthesis, enhancing athletic performance indicators, and reducing feelings of exertion.

Rebak et al. (2023) reviewed contemporary methods and challenges for studying citrullination by MS. They discussed how the development of modern citrullination-specific proteomics approaches may improve our understanding of citrullination networks. A big challenge is that the mass shift caused by citrullination is very similar to the naturally occurring shift caused by carbon-13 (13C) and nitrogen-15 substitution of the arginine residue. As a result, low mass accuracy could lead to the incorrect assignment of 13C-containing arginine residues as citrullinated residues, thereby yielding false-positive identifications. The research group suggested that with needed advances in mass spectrometry-based methods and protocols, large-scale citrullination analysis may soon be possible in the clinic.

Since dysregulation of protein citrullination is associated with disease development and progression, and the identification and characterization of citrullinated proteins is highly challenging and complicated by the low cellular abundance of citrullinated proteins, Wang et al. (2024) reviewed recent advancements of citrullination-specific mass spectrometry methods and their integration into methodology for improved citrullination identification and characterization. Another review by Vitorino et al. (2024) focused on key mechanisms in disease pathogenesis mediated by the protein post-translational modification citrullination. The research group discussed these processes in the context of complex diseases such as rheumatoid arthritis, cancer, central nervous system disorders, and cardiovascular disease.

Bustos et al. (2025) reviewed citrullinated proteins and peptides for their potential to reestablish immune tolerance in RA. This review explored the role of anti-citrullinated protein antibodies (ACPAs) in disease pathology and how targeting specific citrullinated antigens could modulate immune responses. The review also highlights the therapeutic relevance of altering T and B cell function to regulate the immune state.

Pitter and Zou (2025) reviewed PAD2- and PAD4-mediated citrullination in immune cell subsets within the tumor microenvironment. This review discussed how citrullination regulates immune cell function and tumor immunity and explored the potential of targeting citrullination as a strategy for cancer immunotherapy. Further, this report showed that in neutrophils, PAD4-mediated histone citrullination facilitates NET formation, thereby enhancing tumor progression and metastasis. In macrophages, PAD-dependent citrullination regulates antigen presentation and inflammatory cytokine production. However, the roles of PAD2 and PAD4 in other immune subsets, including T cells, B cells, and DCs, remain understudied. For example, PAD4 directly citrullinates and activates NF-κB in neutrophils, suggesting an opportunity to explore the roles of NF-κB citrullination in other innate and adaptive immune cells whose effector functions are governed by NF-κB signaling.

Meelker Gonzales et al. (2026) developed a high-throughput chemical workflow for global citrullinome profiling with high sensitivity and reproducibility to address current limitations. The research group developed a clickable derivatization strategy utilizing cleavable derivatization for selective peptide labeling and clean release under mild conditions amenable to mass spectrometry-based analysis. Control peptides were SAVRA(Cit)SSVPGVR and SAVRARSSVPGVR. The researchers identified >50 intracellular citrullinated proteins, many of which are involved in RNA splicing. These observations suggest that citrullination modulates RNA biology.

Excessive citrullination promotes protein autophagy and subsequent presentation by dendritic cells (DCs), macrophages, and thymic DCs, driving CD4+ T cell activation. Citrullination can also enhance peptide binding affinity to MHC-II, thereby activating CD4+ T cells and contributing to tolerance breakdown.

Currently, the pharmacologic treatment options for RA are steroids and disease-modifying antirheumatic drugs (DMARDs). However, the treatment with steroids is only symptomatic and is not able to change the long-term course of the disease; therefore, the European League Against Rheumatology (EULAR) recommendations for RA treatment utilize conventional synthetic DMARDs (csDMARDs), such as methotrexate (MTX), as initial treatment, eventually in combination with short-term glucocorticoids during disease flares. If csDMARDs are ineffective, biological DMARDs (bDMARDs) targeting cytokine pathways or targeted synthetic DMARDs (tsDMARDs), such as Janus Kinase (JAK) inhibitors, are used.

Restoring immune tolerance to citrullinated proteins is a promising strategy to stop disease progression and establish lasting remission. 

Vimentin is an intermediate filament widely expressed in mesenchymal cells and macrophages. During apoptosis, deimination occurs in macrophages, and inadequate clearance of apoptotic material leads to the production of citrullinated vimentin.

Table 2: Citrullinated peptides as drug candidates for rheumatoid arthritis.

Solid-Phase Peptide Synthesis (SPPS) allows the synthesis of citrullinated peptides.

There are two primary synthesis strategies to use:

(1) The Building Block Approach, in which Fmoc-based SPPS utilized a pre-manufactured Fmoc-Citrulline amino acid building block. The citrulline residue is inserted into the peptide sequence during automated synthesis.

(2) Global Post-Synthetic Modification. In this approach, the peptide is first synthesized with an arginine residue at specific positions. After the peptide chain is complete, PAD enzymes or chemical reagents convert arginine to citrulline. However, this approach often yields a "partial" conversion, resulting in a mixture of peptides that may be difficult to purify.

Reference

Bustos AH, Brüner M, Kragstrup TW, Astakhova K. Citrullinated peptides as drug candidates for rheumatoid arthritis. Front Immunol. 2025 Nov 25;16:1648913. [PMC]

Guo Q, Bedford MT, Fast W. Discovery of peptidylarginine deiminase-4 substrates by protein array: antagonistic citrullination and methylation of human ribosomal protein S2. Mol Biosyst. 2011 Jul;7(7):2286-95. [PMC]

Holm A, Rise F, Sessler N, Sollid LM, Undheim K, Fleckenstein B. Specific modification of peptide-bound citrulline residues. Anal Biochem. 2006 May 1;352(1):68-76. Mass spec analysis! [Sciencedirect]

Koivula MK, Aman S, Alasaarela E, Karjalainen A, Hakala M, Risteli J. Inhibitory characteristics of citrullinated telopeptides of type I and II collagens for autoantibody binding in patients with rheumatoid arthritis. Rheumatology (Oxford). 2006 Nov;45(11):1364-9. [Rheumatology]

Kumari B, Kumar P, Chaudhary RKP. Evaluation of Anti-Cyclic Citrullinated Peptide Autoantibodies and C-Reactive Protein in Common Autoimmune Skin Diseases with and without Arthritis. J Clin Diagn Res. 2017 Jul;11(7):BC06-BC08.[Article]

Liepa, A.j., Wright, D.M.J., The reaction of 1,2-diones with ureas: a synthesis of 4-(1-substituted)alkylimidazol-2-ones, Aust. J. Chem. 53 (2000) 73–76.

Lee CY, Wang D, Wilhelm M, Zolg DP, Schmidt T, Schnatbaum K, Reimer U, Pontén F, Uhlén M, Hahne H, Kuster B. Mining the Human Tissue Proteome for Protein Citrullination. Mol Cell Proteomics. 2018 Jul;17(7):1378-1391. [PMC]

Lewallen DM, Bicker KL, Subramanian V, Clancy KW, Slade DJ, Martell J, Dreyton CJ, Sokolove J, Weerapana E, Thompson PR. Chemical Proteomic Platform To Identify Citrullinated Proteins. ACS Chem Biol. 2015 Nov 20;10(11):2520-8. [PMC]

Luban S, Li ZG. Citrullinated peptide and its relevance to rheumatoid arthritis: an update. Int J Rheum Dis. 2010 Oct;13(4):284-7. [Wiley]

Meelker González, R., Laposchan, S., Riedel, E. et al. High-throughput chemical proteomics workflow for profiling protein citrullination dynamics. Nat Commun 17, 1982 (2026). [Nature]

Moita E, Gil-Izquierdo A, Sousa C, Ferreres F, Silva LR, Valentão P, et al. Integrated analysis of COX-2 and iNOS derived inflammatory mediators in LPS-stimulated RAW macrophages pre-exposed to Echium plantagineum L. bee pollen extract. PLoS One 2013;8(3):e59131. [PMC]

Park HY, Kim SW, Seo J, Jung YP, Kim H, Kim AJ, Kim S, Lim K. Dietary Arginine and Citrulline Supplements for Cardiovascular Health and Athletic Performance: A Narrative Review. Nutrients. 2023 Mar 3;15(5):1268. [PMC]

Pérez ML, Gómara MJ, Kasi D, Alonso A, Viñas O, Ercilla G, Sanmartí R, Haro I. Synthesis of overlapping fibrin citrullinated peptides and their use for diagnosing rheumatoid arthritis. Chem Biol Drug Des. 2006 Oct;68(4):194-200. [Wiley]

Pitter MR, Zou W. Citrullination in tumor immunity and therapy. J Clin Invest. 2025 Oct 15;135(20):e196348. [CJI]

Rabier, D. and Kamoun, P.; metabolism of citrulline in man. Amino Acids (1995) 9: 299-316.

Rebak AS, Hendriks IA, Nielsen ML. Characterizing citrullination by mass spectrometry-based proteomics. Philos Trans R Soc Lond B Biol Sci. 2023 Nov 20;378(1890):20220237. [PMC]

Ru Z, Zhang H, Huang X, Lou J, Liao J, Chen Z, Yang X. A new pattern of citrullinated peptides improves the sensitivity for diagnosing rheumatoid arthritis. Clin Biochem. 2022 Jul-Aug;105-106:87-93. [Sciencedirect]

Senshu T, Sato T, Inoue T, Akiyama K, Asaga H. Detection of citrulline residues in deiminated proteins on polyvinylidene difluoride membrane. Anal Biochem 1992;203(1):94-100. [PubMed]

Schwarz EL, Roberts WL, Pasquali M. Analysis of plasma amino acids by HPLC with photodiode array and fluorescence detection. Clin Chim Acta. 2005 Apr;354(1-2):83-90. [Sciencedirect]

Swart A, Burlingame RW, Gürtler I, Mahler M. Third generation anti-citrullinated peptide antibody assay is a sensitive marker in rheumatoid factor negative rheumatoid arthritis. Clin Chim Acta. 2012 Dec 24;414:266-72. [Sciencedirect]

van Gaalen FA, Linn-Rasker SP, van Venrooij WJ, de Jong BA, Breedveld FC, Verweij CL, Toes RE, Huizinga TW. Autoantibodies to cyclic citrullinated peptides predict progression to rheumatoid arthritis in patients with undifferentiated arthritis: a prospective cohort study. Arthritis Rheum. 2004 Mar;50(3):709-15. [Wiley]

Vitorino R, Guedes S, Vitorino C, Ferreira R, Amado F, Van Eyk JE. Elucidating Citrullination by Mass Spectrometry and Its Role in Disease Pathogenesis. J Proteome Res. 2021 Jan 1;20(1):38-48. [PMC]

Wang B, Fields L, Li L. Recent advances in characterization of citrullination and its implication in human disease research: From method development to network integration. Proteomics. 2023 Nov;23(21-22):e2200286. [PMC]

---...---