The Deimination of Arginine to Citrulline
Andrew J. Creese and Helen J. Cooper
School of Biosciences, University of Birmingham,, Birmingham, UK
Overview of Arginine to Citrulline Conversion: Biological Importance
The nonstandard a-amino acid citrulline was first isolated in 1914 by Koga and Odake . In 1931 the conversion of arginine to citrulline, using alkaline hydrolysis to drive the reaction, was observed by Ackermann . Figure 7.1 shows schematically the conversion of arginine to citrulline; in vivo, the reaction is catalyzed by calcium and the peptidylarginine deiminase (PAD) enzymes. At physiological conditions, arginine has a charge of +1, and citrul- line is neutral; therefore the conversion of arginine to citrulline in proteins reduces the overall charge of the protein, which will affect the structure and function of the protein .
Citrullination has been linked to multiple autoimmune diseases including rheumatoid arthritis (RA) [4, 5], periodontitis , multiple sclerosis , and Alzheimer's disease . It has also been shown to play crucial roles in cancer [9, 10] and cardiovascular disease [11, 12]. Citrullination is essential in fetal development  and helps control gene expression . In 1998, Schellekens et al.  demonstrated that patients with RA produced antibodies against citrullinated proteins (anticitrullinated protein antibody (ACPA)). Since then, the study of the modification has grown with over 3500 publications on citrullinated proteins published (1998-2014, Web of Science) as shown in Figure 7.2.
As mentioned earlier, the deimination of arginine to citrulline is catalyzed in vivo by the PAD enzymes. In humans there are five PAD enzymes (PAD1-4 and PAD 6) . The family is highly homologous . Over 50% of the amino acid sequence is conserved across the five proteins . PAD3 is probably the least studied and has been localized to hair follicles. It is known to citrullinate
Analysis of Protein Post-Translational Modifications by Mass Spectrometry, First Edition. Edited by John R. Griffiths and Richard D. Unwin.
© 2017 John Wiley & Sons, Inc. Published 2017 by John Wiley & Sons, Inc.
Figure 7.1 The conversion of arginine to citrulline catalyzed by calcium and a peptidylarginine deiminase. The red dashed line indicates cleavage of isocyanic acid during collision-induced dissociation fragmentation.
Figure 7.2 The cumulative number of publications with "citrullinated" in the topic, Web of Science, 08/20/2015.
trichohyalin  and filaggrin , but there is no literature describing direct involvement in disease.
PAD1 is involved in late-stage epidermal differentiation. The enzyme citrul- linates keratin 1 , keratin 10 , and filaggrin . It has been shown that the citrullination of these three proteins maintains hydration of the stratum corneum , which in turn maintains cutaneous barrier function . PAD1 is predominantly expressed in the epidermis and the uterus .
Overexpression of PAD2 in mice has been shown to increase citrullination of myelin basic protein (MBP) in brain tissue leading to less stable myelin . Myelin forms a sheath around the axon of neurons and is therefore essential to the development of the nervous system. It has been shown that approximately
20% of MBP is significantly citrullinated (six arginine-citrulline conversions) in healthy white brain matter. In white matter from patients with multiple sclerosis, a greater percent of MBP was heavily citrullinated (45%) . Another study  demonstrated that overexpression of PAD2 in transgenic mice led to increased citrullination of MBP and destabilization of myelin. Similar to the results of Moscarello et al. , the levels of citrullination increased dramatically when comparing healthy to overexpressing samples. In this case, the increase in citrullination was observed when the mice were 3 months old.
Citrullination of vimentin by PAD2 is involved in the apoptosis of Jurkat T cells . It was observed that activated Jurkat cells expressed greater levels of PAD2 and more citrullinated protein was identified. Overexpression of PAD2 in Jurkat cells showed no difference in apoptosis from cells that did not overexpress PAD2; however, on activation, cell viability decreased significantly. This finding suggests that citrullination has a vital role in cell viability and increased citrullination causes cell death.
PAD2 has been identified in inflamed myofilaments from human hearts. Giles et al.  analyzed heart tissue from autopsy samples. Myocardial samples with rheumatic disease were compared to control samples (myocarditis and scleroderma). All five PAD enzymes were identified in the heart tissue. However, PAD2 and PAD4 were the dominant species in the inflamed heart tissue of the rheumatic samples. In a data-independent proteomics study of human heart tissue, Fert-Bober et al.  identified significant numbers of cit- rullinated proteins. They hypothesized that citrullination could regulate muscle contractile protein interactions. PAD2 is ubiquitously expressed; specifically, it has been localized in the brain , synovial tissues , and muscles .
The most studied PAD is PAD4 (over 430 publications to date, 07/20/2015, Web of Science, www.webofscience.com). It is expressed in hematopoietic progenitor cells , neutrophils , and eosinophils  and is associated with inflammatory autoimmune diseases . It has also been observed in carcinoma cells [9, 10]. Of all the PAD enzymes, PAD4 is unique in that it has been localized to the cell nucleus . In the nucleus, PAD4 is known to hypercitrul- linate histone, leading to chromatin decondensation and the formation of neutrophil extracellular traps (NETs) [32, 33], an essential part of the immune defense against pathogens.
PAD4 citrullination is considered one of the most important factors in RA. One of the major outcomes of RA is bone loss; this occurs by two mechanisms, inflammation and autoimmunity . It has been shown that ACPAs are specific to RA  and the presence of ACPAs generally leads to more destructive bone loss . In RA, increased abundance of citrullinated proteins is linked with increased joint destruction. Hill et al.  developed transgenic mice with citrullinated fibrinogen; they found that arthritis was induced in 35% of the mice with citrullinated fibrinogen compared to 0% of mice without citrullinated fibrinogen.
The nuclear localization of PAD4 and the citrullination of histones have been shown recently to be key regulators in gene expression. The tails of histones are unstructured, arginine rich, and heavily post-translationally modified . The modification of arginine by histone methyltransferase results in monomethyla- tion and dimethylation. Methylation of histone can increase and decrease the expression of specific genes . It has recently been observed that PAD4 can demethylate monomethylarginine converting it to citrulline  regulating the expression of proteins. The citrullination is nonreversible.
Nakashima et al.  have shown that citrullination of histone H3 by PAD4 is involved in the regulation and proliferation of multipotent hematopoietic stem cells. Citrullination of histone H3 affects the expression of c-myc. The regulation of c-myc is essential for normal differentiation of hematopoietic stem cells (source of all blood cells). Decrease in c-myc leads to accumulation of hematopoietic stem cells, resulting in cytopenia (a reduction in the number of blood cells) .
PAD4 has been shown to citrullinate antithrombin by Chang et al. , who observed that the thrombin inhibitory activity of antithrombin could be hindered by PAD4 citrullination. Thrombin activates coagulation through the formation of fibrin clots, a prominent feature of RA . Increased thrombin activity is also indicative of cancer; it is an activator of angiogenesis, which leads to metastasis . PAD4 was identified as a corepressor of p53 and, together with the histone deacetylase HDAC2, supresses the expression of tumor repressor genes [14, 45].
PAD6 is expressed in the ovaries, oocytes, sperm cells, and the early embryo. It is essential for fertility and embryonic development. Esposita et al.  investigated the role of PAD6 in folliculogenesis. By breeding mice deficient in PAD6, they were able to observe the function of the enzyme in vivo. It was observed that female PAD6-deficient mice were infertile whereas males were not. On further investigation, it was noted that female mice ovulated and could be fertilized; however the embryo had arrested development at the two-cell stage. This observation is due to the lack of cytoskeletal sheet formation, which the authors attribute to the lack of citrullination by PAD6.
Due to the prevalence of citrullination in disease, there are a variety of PAD inhibitors in development. Wang et al.  developed a PAD4 inhibitor to target cancer cells that overexpressed PAD4. They found that the compound YW3-56, which they designed and synthesized, inhibited cancerous cell growth in both human osteosarcoma cells (U2OS) and mouse sarcoma cells (S-180). Wang et al. note that PAD4 may not be a suitable cancer therapy target as it is expressed in multiple cell types and is essential in the innate immune response. Another PAD inhibitor is Cl-amidine , which is an irreversible inhibitor of all PAD enzymes. Treatment of mice with collagen-induced arthritis showed a decrease in clinical disease activity and also in the levels of detected citrulli- nated proteins. Cl-amidine has also been tested on lupus-prone mice . The formation of NETs in lupus-prone mice can result in vascular damage and decreased life expectancy . Treatment with Cl-amidine resulted in less NET formation and protected against other lupus-related damage. Cl-amidine has a lethal effect on mouse embryos . Two-cell mouse embryos cultured in 200 pM Cl-amidine showed complete arrested development at eight cells.
Currently the clinical method for identification of citrullination as part of disease diagnosis is to use synthetic cyclic citrullinated peptides (CCPs) . ACPAs bind to the CCP in an enzyme-linked immunosorbent assay (ELISA), resulting in detection. The body produces ACPAs when proteins are citrulli- nated. The protein tertiary structure changes sufficiently, such that it is detected as an antigen. ACPAs were first associated with RA in the 1970s when antibody activity against keratin  was investigated. It was determined that the keratin was citrullinated. In 2007, a study by Coenen et al.  compared the sensitivity and specificity of ACPA detection for RA. From six commercially available antibody tests, it was found that the sensitivity was between 69.6% and 77.5% and the specificity was between 87.8% and 96.4%. A systematic review in 2011 by Taylor et al.  highlighted the variability in results from CCP and ACPA tests. Compiling analyses from 56 studies, CCP was found to have 68% sensitivity and 95% specificity. From 29 similar studies of ACPA tests, 60% specificity and 79% sensitivity were calculated.