The International Journal of Biochemistry & Cell Biology 38 (2006) 1662–1677

Review

Citrullination: A posttranslational modificationin health and disease

Bence Gyorgy b, Erzsebet Toth a, Edit Tarcsa c, Andras Falus b, Edit I. Buzas b,∗a Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary

b Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungaryc Abbott Bioresearch Center, Worcester, MA, USA

Received 22 December 2005; received in revised form 13 March 2006; accepted 14 March 2006Available online 30 March 2006

Abstract

Posttranslational modifications are chemical changes to proteins that take place after synthesis. One such modification, peptidy-larginine to peptidylcitrulline conversion, catalysed by peptidylarginine deiminases, has recently received significant interest inbiomedicine. Introduction of citrulline dramatically changes the structure and function of proteins. It has been implicated in sev-eral physiological and pathological processes. Physiological processes include epithelial terminal differentiation, gene expressionregulation, and apoptosis. Rheumatoid arthritis, multiple sclerosis, and Alzheimer’s disease are examples of human diseases whereprotein citrullination involvement has been demonstrated. In this review, we discuss our current understanding on the importanceof protein deimination in these processes. We describe the enzymes catalyzing the reaction, as well as their known protein sub-

strates. We review the citrullinated peptide epitopes that are proposed as disease markers, specifically recognized in certain humanautoimmune disorders. The potential autopathogenic role of citrullinated epitopes is also discussed.© 2006 Elsevier Ltd. All rights reserved.

Keywords: Biochemistry; Autoimmunity; Clinical; Cell Biology; Apoptosis

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16631.1. The citrullination reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16631.2. Enzymes catalysing citrullination: peptidylarginine deiminases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16631.3. General consequences of citrullination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1665

Abbreviations: ACPA, anti-citrullinated protein/peptide antibody; AD, Alzheimer’s disease; AKA, anti-keratin antibody; AFA, anti-filaggrin anti-body; APF, anti-perinuclear factor; cAMP, cyclic adenosine monophosphate; CIA, collagen induced arthritis; CNS, central nervous system; CARM-1,coactivator associated arginine methyltransferase-1; CIITA, major histocompatibility class II transactivator; DM, dismyelinating; EAE, experimentalautoimmune encephalomyelitis; GFAP, glial fibrillary acidic protein; FBG, fibrinogen; H, histone; Hcgp39, human cartilage glycoprotein-39; HGNC,HUGO Gene Nomenclature Committee; HUGO, Human Genome Organisation; K, keratin; MBP, myelin basic protein; NOS, nitrogen monoxidesynthase; NO, nitrogen monoxide; PAD, peptidylarginine deiminase (protein); PADI, peptidylarginie deiminase (gene); PRMT-1, protein argininemethyltransferase-1; PTM, posttranslational modification; RA, rheumatoid arthritis; SNP, single nucleotide polymorphism; THH, trichohyalin

∗ Corresponding author at: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Nagyvarad ter 4, H-1089,Hungary. Tel.: +36 1 2102930/6234; fax: +36 1 3036968.

E-mail address: buzedi@dgci.sote.hu (E.I. Buzas).

1357-2725/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.biocel.2006.03.008

B. Gyorgy et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1662–1677 1663

1.4. Substrate specificity of peptidylarginine deiminases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16651.5. Regulation of peptidylarginine deiminases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1665

2. Physiological roles of citrullination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16662.1. Cytoskeletal proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16662.2. Myelin basic protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16682.3. Nuclear substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16682.4. Other implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1669

3. Citrullination in inflammatory and other pathological conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16693.1. Rheumatoid arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16693.2. Autoantibodies against citrullinated proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16703.3. What about fibrin? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16703.4. Autoreactive T cells in rheumatoid arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16713.5. Environmental effects in rheumatoid arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16713.6. Peptidylarginine deiminase type 4 is associated with the disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16713.7. Citrullination in collagen-induced arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16723.8. Multiple sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16723.9. How can citrullination affect the stability of the myelin sheath? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16723.10. Alzheimer’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16733.11. Psoriasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16733.12. Peptidylarginine deiminase of a pathogen—a potential virulence factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1673

4. Closing remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1673Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1674References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1674

1. Introduction

Proteins are encoded by a surprisingly limited numberof genes in our genome. However, posttranslational mod-ifications (e.g. phosphorylation, glycosylation, citrulli-

changes the charge of the amino acid (Fig. 1). Argi-

nine is strongly basic (pI ∼ 10.76) due to the presenceof a guanidino group that could easily be protonated atphysiological pH. The removal of the imino moiety (atransformation referred to as deimination) is an enzy-matic reaction catalyzed by peptidylarginine deiminases

1p36.13; within a 300 kb region (Chavanas et al., 2004;

nation) can tremendously increase both the structural andfunctional diversity of the proteome. Posttranslationalmodifications (PTM) are common biological processesthat alter specific parts of a protein after synthesis. Nearlyall known proteins undergo some form of posttransla-tional modification, and almost all amino acids can bealtered by one or more of these processes. The modifiedproteins gain rare amino acids that can have critical influ-ence on the structure and function of the molecule. Thisreview will focus on the conversion of arginine to cit-rulline that was first described by Fearon (Fearon, 1939).

Peptide-bound arginine residues can undergo thismodification and the resulting citrulline remains part ofthe protein as peptidyl citrulline. Citrulline is not a natu-ral amino acid in proteins, therefore may induce immuneresponse. The importance of citrullinated autoantigensin autoimmune inflammatory diseases (e.g. rheumatoidarthritis, multiple sclerosis) has long been suspected.

1.1. The citrullination reaction

The conversion of arginine (Arg) to citrulline (Cit)

(PAD). The resulting citrulline lacks the strong basiccharacter; it is a neutral amino acid similar to Asn orGln.

At the protein level, the reaction leads to a 1 Da massreduction for each Arg modified. Basic charge(s) are lostthat will influence the overall charge, charge distribution,isoelectric point, as well as the ionic and hydrogen bondforming abilities of the protein (van Venrooij & Pruijn,2000; Tarcsa et al., 1996). The interactions of the proteinwith other proteins might also be altered.

1.2. Enzymes catalysing citrullination:peptidylarginine deiminases

The protein-deimination process is catalysed by afamily of calcium-binding enzymes, the peptidylargi-nine deiminases (EC 3.5.3.15.). To date, five isoenzymeshave been identified. Their genomic organisation, sub-cellular localisation and tissue-specific expression havebeen more or less determined (for review see Vossenaar,Zendman, van Venrooij, & Pruijn, 2003).

All PADI genes are localized in one cluster at

1664 B. Gyorgy et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1662–1677

Fig. 1. The citrullination process involves enzymatic conversion of arginine to citrulline. The enzyme catalyzing this reaction is peptidylargininedeiminase (PAD). During the reaction, the arginine is attacked by the Cys residue of the enzyme establishing a tetrahedral adduct while ammoniais released. The adduct is then cleaved by the nucleophilic attack of a water molecule that regenerates the Cys residue and forms the keto-group.

Vossenaar et al., 2003). PADI-1, -3, -5 and -6 are in closeproximity, located within a 160 kb region. PADI-2 is theodd one out: the largest gene in the family, it is locatedfarther away from the other genes and transcribed inthe opposite direction. This peculiar genomic organisa-tion is conserved across species. The localisation anddirections of the genes, the intergenic sequence lengths,exon–intron boundaries and the coding sequences aresimilar between mouse and man (Chavanas et al., 2004;Vossenaar et al., 2003). Since human PADI5 proved tobe the human homologue of mouse PADI4, it has beenrecently approved to be renamed PADI4 by HUGO GeneNomenclature Committee (HGNC) (Vossenaar et al.,2003).

The PAD isozymes are widely distributed in mam-malian tissues. PAD1 is predominantly expressed inthe epidermis and the uterus, PAD3 in the hair folli-cles, while PAD4 in neutrophils and eosinophils. PAD6expression has been detected in eggs, ovaries and in

early embryos (Vossenaar et al., 2003). PAD2 is theubiquitous member of the family, expressed for exam-ple in skeletal muscles, spleen, brain, secretory glands,etc.

Arita et al. (Arita et al., 2004) proposed the followingcatalytic mechanism for peptidylarginine deiminases:the active site Cys residue of the enzyme attacks theguanidino group of the Arg, and establishes a tetrahedraladduct transition state intermediate while ammonia isreleased. The adduct is then cleaved by the nucleophilicattack of a water molecule. This latter step regeneratesthe Cys residue, and results in the formation of a keto-group, as well as a new primary amine (Fig. 1). Since theprimary amino group is the one attacked originally, thereaction could be considered deamination. However, theoverall process yields a deiminated amino acid, thereforeit is called deimiation.

This mechanism differs from the well-known nitro-gen monoxide synthase mediated arginine–citrulline

B. Gyorgy et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1662–1677 1665

conversion. In that pathway, the imino-group is oxidisedand released as NO.

One interesting consequence of the PAD reactionmechanism is, that not only Arg, but also methylatedarginine (with the methyl group on the primary amine)can be transformed to citrulline. In this case theby-product released is methyl-amine (Wang et al.,2004). Consequently, citrullination can antagonisewith arginine methyltransferases in two different ways.First, the methyl group can be directly removed fromthe arginine that undergoes citrullination. Second, themethyltransferase substrate, arginine can be abolished,as Cit cannot be methylated (Wang et al., 2004). Thephysiological role of this phenomenon will be discussedlater.

1.3. General consequences of citrullination

The deimination process could change the primary,secondary and tertiary structures of proteins. In vitroanalysis revealed that high degree of citrullination coulddenature proteins (Tarcsa et al., 1996). Experiments withtrichohyalin (THH) and filaggrin suggest that modifica-tion of 5% of the Arg residues starts to destroy the tertiarystructure, and modification of more than 10% of thearginines leads to a complete loss of ordered structure,causing denaturation of the protein. This phenomenoncould likely be attributed to the loss of basic residuesresulting in altered charge distribution, loss of ionic inter-aplpaotwop

1.4. Substrate specificity of peptidylargininedeiminases

A very important, but often neglected question is:what are the physiological substrates of PAD enzymes?Numerous in vitro studies suggest that in theory, anyArg residue could be deiminated. In fact, several Arg-containing proteins have been shown to be citrullinatedin vitro, albeit with different kinetic parameters (Kubilus,Waitkus, & Baden, 1980). Some of the factors that deter-mine the kinetics of deimination are summarised inTable 1 (Tarcsa et al., 1996).

1.5. Regulation of peptidylarginine deiminases

A key regulator of PAD enzymes is calcium. Bind-ing of calcium ions activates PADs (Arita et al., 2004).The different isoenzymes of the epidermis were shown toexhibit particular calcium and pH preferences (Mechinet al., 2005). In vitro citrullination is only observed inthe presence of high calcium concentration (Mechin etal., 2005; Vossenaar et al., 2003; Takahara, Okamoto,& Sugawara, 1986). These concentrations can only bereached in cells using calcium ionophores (ionomycin orA23187). Under physiological conditions, the calciumconcentration range is 10−8–10−6 M, therefore PADsare inactive (as demonstrated for PAD2 by Takahara etal. (1986)). This suggests that citrullination occurs onlyunder ‘extreme’ conditions, such as during apoptosis or

TS

P

N cyN ancyAN nates,

N

T the prima

ctions and H-bonds (Tarcsa et al., 1996). In vivo, underhysiological conditions we cannot assume that citrul-ination is a denaturation process. In general, we canresume that the modification alters the protein structurend results in a somewhat looser, less organised, morepen configuration (Tarcsa et al., 1996). The deimina-ion may also influence the interaction of the moleculeith other proteins. The wide array of primary effectsf citrullination leads to far-reaching physiological andathological consequences.

able 1ome ‘rules’ of deimination

rimary structure

-Arg-Glu-C Slowly citrullinates, up to 10% efficien-Arg-Asp-C Rapidly citrullinates, up to 100% efficirg close to N-terminus Barely citrullinated-Arg-Arg-C The closer to C-terminus barely citrulli

except the case of MBPa

-Pro-Arg-Pro-C Never citrullinates

his table reveals some preferences of peptidylarginine deiminase onnd C indicate the amino- and the carboxy-terminus of the protein.a Wood and Moscarello (1989).

terminal differentiation of the epidermis. Indeed, PADsare primarily involved in apoptotic and differentiationevents however; recently they have also been implicatedin some other cellular processes, such as gene regulation.This argues for the possible regulation of the enzymesby factors other than Ca(2+), that could enable PADs towork at physiological calcium concentrations in the cell.

Little is known about the gene regulation of theseenzymes. It has been shown that PADI2 can be inducedby 17�-oestradiol. During the menstrual cycle, the

Secondary structure

� Helix Hardly deiminated� Turn The most susceptible region for deiminationDisordered Rapidly citrullinates, up to 95% efficiency� Sheet No data available

ary and secondary structure of the substrate (Tarcsa et al., 1996). N

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activity of PAD2 changes in the pituitary gland andin the uterus (Senshu, Akiyama, Nagata, Watanabe, &Hikichi, 1989; Takahara et al., 1992).

2. Physiological roles of citrullination

Protein citrullination is implicated in several physi-ological processes including terminal differentiation ofthe epidermis and apoptosis (Fig. 2).

2.1. Cytoskeletal proteins

Cytokeratin is an intermediate filament produced bykeratinocytes. It determines the consistency of skin, hairand nails. Cytokeratin consists of �-helical segments and�-turns (the latter can easily be deiminated) (Tarcsa et al.,1996). The keratinocytes also produce other importantstructural proteins like filaggrin and loricrin that organisethe keratin matrix. During terminal differentiation, ker-atinocytes travel from the basal layers of the epitheliumto the upper areas before undergoing cell death. Dur-ing this process the intracellular environment is exposedto increasing calcium concentrations that gradually acti-vate PAD1, 2 and 3 (these isoenzymes are expressed inthe epidermis and also in the hair follicle (Vossenaar etal., 2003)). To date, three molecules are known to bedeiminated in the epidermis: filaggrin and keratins K1and K10 (these are the suprabasal cytokeratin isoforms)(Senshu, Kan, Ogawa, Manabe, & Asaga, 1996; Senshu,Akiyama, & Nomura, 1999). Citrullination enables these

matrix (Pearton, Dale, & Presland, 2002; Vossenaar etal., 2003). (Pro)filaggrin readily undergoes citrullinationas it contains several �-turns. Some data suggest thathigh level citrullination of (pro)filaggrin helps proteasesto cleave it further, into amino acids that are osmoticallyactive and contribute to hydration of the skin (Harding& Scott, 1983; Scott, Harding, & Barrett, 1982).

Trichohyalin (THH) is an important structural pro-tein that bundles cytokeratin filaments. Therefore, it isalso referred to as keratin filament matrix protein. THHis mainly expressed in the inner root sheath cells andthe medulla of the hair follicle (Rogers & Powell, 1993;Tarcsa et al., 1997). After synthesis, trichohyalin formsinsoluble vacuoles that are stabilised by ionic inter-actions between �-helixes (Lee et al., 1993). Duringdifferentiation, the increasing Ca(2+) concentration acti-vates PAD3, and THH gets citrullinated. As a result,THH loses its highly organized �-helixes and forms amore open structure. These changes have two impor-tant consequences: THH becomes soluble and it becomesa substrate for crosslinking by transglutaminases. THHcan be cross-linked to both keratin and other THHs bytransglutaminase 3, for which citrullinated THH is apreferred substrate. Transglutaminases form isopeptide-bonds between protein bound Glu and Lys residues.Crosslinking will stabilise the organised keratin matrix,and THH will become insoluble again; but this timeas a functionally relevant molecule highly integratedwith keratin filaments (Tarcsa et al., 1997). Interestingly,in the medulla THH remains in vacuolar form and is

proteins to bind to each other by reducing their isoelec-tric point. Native cytokeratin and loricrin have very lowaffinity for each other, since both proteins are stronglybasic (interestingly, loricrin contains no Arg). Citrulli-nated keratin on the other hand, binds well to loricrinand possibly also to desmoplakin. Desmoplakin is adesmosomal protein that helps the keratin matrix toextend transcellularly (Ishida-Yamamoto et al., 2000).Keratin citrullination has also been implicated in thepathomechanism of psoriasis (as discussed later in thisreview).

Filaggrin is synthesised as a large molecule, calledprofilaggrin, and stored in the cells in a heavily phos-phorylated state. Upon terminal differentiation severalpostsynthetic modifications occur: profilaggrin is citrul-linated, dephosphorylated and then cleaved to filaggrinunits. Citrullination is very important in this process. Viadeimination of some of its arginines, filaggrin acquires amore open, disordered structure that is more accessible toproteolysis. Calpain, an intracellular Ca(2+)-dependentneutral protease then cleaves it to smaller units thatare able to bundle keratin filaments into an organised

involved in thermoregulation (Tarcsa et al., 1997).Vimentin is an intermediate filament expressed by

various cells. In contrast to actin microfilaments andmicrotubules, intermediate filaments form a very stable,less dynamic molecular network. Regulation of the poly-merisation and depolymerisation processes has not beenelucidated yet. Candidate mechanisms include phospho-rylation/dephosphorylation and citrullination. Inagaki etal. have shown that cAMP-dependent protein kinase andprotein kinase C enzymes can phosphorylate vimentin(Inagaki, Nishi, Nishizawa, Matsuyama, & Sato, 1987).The head domain of vimentin contains many �-turnsthat could easily be deiminated. Both phosphorylationand deimination reduces the isoelectric point of the headdomain of vimentin. As a result, the protein looses itsability to polymerise (soluble vimentin) or it under-goes depolymerisation (filamentous vimentin) (Asaga,Yamada, & Senshu, 1998; Inagaki, Takahara, Nishi,Sugawara, & Sato, 1989). This means that Arg-s areessential in maintaining the ability of vimentin to formfilaments. Modification of these Arg-s by 1,2 cyclo-hexadion inhibits polymerisation of vimentin (Traub et

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Fig. 2. Schematic overview of intracellular citrullination processes in non-apoptotic and apoptotic cells. The major processes are indicated by black rectangles: protein demethylation (Denman,2005); keratin matrix organization (Ishida-Yamamoto et al., 2000, 2002); desmoplakin–keratin association (Ishida-Yamamoto et al., 2000); gene regulation (Denman, 2005; Wang et al., 2004);disorganization of the nuclear lamina (Mizoguchi et al., 1998); disassembly of vimentin filaments (Inagaki et al., 1989); regulation of assembly–disassembly of vimentin (Inagaki et al., 1989),unfolding of nucleosomes (Vossenaar et al., 2003). Note that all events do not occur simultaneously within the same cell.

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Vorgias, 1984). During apoptosis, when the intracellularcalcium concentration is very high, activation of the PADenzymes results in the complete loss of vimentin inter-mediate filament network through the high rate of headdomain deimination. This mechanism could play a rolein the morphological changes associated with apoptosis(Asaga et al., 1998).

The last cytoskeletal protein discussed is glial fibril-lary acidic protein (GFAP). GFAP is specific to astro-cytes, the major intermediate filament in these cells(Gopalan, Wilczynska, Konik, Bryan, & Kordula, 2006).GFAP exists in various isoforms, and some of themare citrullinated. These deiminated forms of GFAP arepresent in the brain of adult rats (Nicholas et al., 2003).The physiological role of GFAP deimination is stillunknown, however, there is some evidence that the cit-rullinated GFAP filaments are more likely to disassemble(Inagaki et al., 1989). During kainate acid (Asaga &Ishigami, 2001) or hypoxia (Asaga & Ishigami, 2000)induced neurodegeneration both the total amount ofGFAP and its level of citrullination increased in theastrocytes. In the central nervous system only PAD2 isexpressed (Kubilus & Baden, 1983; Sambandam et al.,2004; Watanabe et al., 1988). PAD4 is only found in theCNS when PAD4 containing leukocytes invade the brain(Sambandam et al., 2004). PAD2 protein was found invivo in astrocytes (Vincent, Leung, & Watanabe, 1992),microglial cells (Vincent et al., 1992) and was detectedin vitro in oligodendrocytes (Akiyama, Sakurai, Asou,& Senshu, 1999). Normally, PAD2 seems to become

point of these proteins can cause dramatic changes inthe interaction with lipids (Boggs et al., 1999).

MBP is synthesised in various isoforms that laterundergo several posttranslational modifications, suchas deamidation, deimination, sulphoxyde-formation,methylation and phosphorylation (Pritzker, Joshi,Gowan, Harauz, & Moscarello, 2000a). These modifica-tions produce numerous ‘secondary isoforms’ referredto as ‘charged isomers’, which have different isoelec-tric points. Citrullination of MBP (MBP-Cit) reducesthe interaction with the negatively charged phosphatidyl-serines due to the loss of basic residues. The ratio ofdeiminated MBP/total MBP is crucial in the physio-logical function of CNS. Native MBP contains sev-eral arginines (not deiminated), and forms very tightand compact myelin sheaths. These can reorganise onlyslowly. Citrullinated MBP is not able to form such com-pact sheaths (Beniac et al., 2000), on the other hand,the lipid complex formation is more rapid. The MBP-Cit/total MBP ratio changes amazingly in postnatal life:under 2 years of age nearly all MBP is deiminated.Above 4 years, the MBP-Cit/total MBP ratio is only18% (Moscarello, Wood, Ackerley, & Boulias, 1994;Vossenaar et al., 2003). This ratio remains constant inadults. Changes of MBP-Cit/total MBP correlate withthe high plasticity of the brain of a young child (theconductance of the myelin sheath can be lower as thedistance to be bridged is shorter). Similarly, the caudalareas of CNS with more rudimentary functions containmore citrullinated MBP (Nicholas et al., 2003).

activated during neurodegeneration (Asaga & Ishigami,2001). Under hypoxic conditions, the amount of PAD2mRNA is increased in type II astrocytes (Sambandam etal., 2004). GFAP deimination is characteristic for certaindiseases, such as Alzheimer’s in humans and experi-mental autoimmune encephalomyelitis (EAE) in mice(Ishigami et al., 2005; Nicholas, Sambandam, Echols, &Barnum, 2005; Raijmakers et al., 2005). This raises thepossibility that citrullination could be an early marker inneurodegenerative diseases.

2.2. Myelin basic protein

Myelin basic protein (MBP) is synthesised byoligodendroglial cells. The myelin sheath consists oftwo components: lipids and proteins. Proteins pack thelipid bilayers together and form a complex molecularstructure. A key element in sheath formation is thelipid–protein interaction. It is based on ionic interactionsbetween negatively charged (phosphatidyl-serine andsialic acid containing gangliosides) and the basic pro-teins (MBP, lipophilin). Any alteration of the isoelectric

Citrullination of MBP also affects the speed ofits degradation by the metalloproteinase cathepsin D(Pritzker et al., 2000a). This enzyme cleaves Phe–Phepeptide bonds, most of which are located in the internalregions of the protein, not exposed to solvent. Deimi-nation opens up the structure of MBP. As a result, thePhe–Phe linkages become accessible for cathepsin D,and it can be degraded five times faster than the nativeprotein. Citrullination of MBP has been implicated in thepathomechanism of multiple sclerosis (discussed later).

2.3. Nuclear substrates

In granulocytes, the intracellular localisation of PAD4is the nucleus (Nakashima, Hagiwara, & Yamada, 2002).The nuclear substrates identified include histones H2A,H3 and H4; as well as nucleophosmin/B23. One of themost important cellular effects of protein citrullinationis the deimination of histones, a mechanism involved ingene regulation (Wang et al., 2004).

It is well known that some oestrogen-dependent genesare induced by histone methylation (Bauer, Daujat,

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Nielsen, Nightingale, & Kouzarides, 2002; Metivier etal., 2003). During oestrogen signalling, 17�-oestradiolbinds to its cellular receptor. This complex activatesspecific co-activators with methyltransferase activity:CARM1 (coactivator associated arginine methyl trans-ferase 1) and PRMT1 (protein arginine methyl trans-ferase 1). These coactivators transfer methyl groupsfrom S-adenosyl-methionine to specific histone arginylresidues (H3: Arg17; H4: Arg3) (Wang et al., 2004).Methylated histones in turn, speed up gene expression.The importance of PAD4 lies in its demethylating activ-ity. As mentioned above, PAD can convert either ‘nor-mal’ or methylated Arg to Cit. This reaction is referredto as ‘demethylimination’. Demethylimination is strictlygene-specific, demanding high specificity from PAD4.Immunoprecipitation analysis of the chromatin revealed,that some PAD4 is attached to histones even beforetheir PRMT1/CARM1-dependent methylation. Thesehistones are localized at specific gene promoters. WhenMCF-7 (human, Caucasian, breast, adenocarcinoma)cells were treated with 17�-oestradiol, Arg-methylationof H3 and H4 histones increased dramatically within20 min. At the same time certain genes (observationswere made on the pS2 gene) were induced (Wang et al.,2004). After 40–60 min the amount of PAD4 enzymeassociated with histones on the pS2 promoter, showeda two-fold increase. In parallel the levels of methyl-arginine were reduced, and there was an increase inhistone citrullination. This data suggest that PAD4 islikely to function in reversing the transcriptional activa-tndalottatambcw

Cticdh

DNA. It has been suggested that citrullination of his-tones causes the nucleosomes to open up, and renderDNA more accessible to nucleases. However, this pro-cess has not been fully elucidated yet (Nakashima et al.,2002; Vossenaar et al., 2003).

Mizoguchi et al. described a nuclear protein, attachedto the nuclear lamina, that is deiminated upon ionomycintreatment (Mizoguchi et al., 1998). Nuclear lamina isconsidered to play a role in determining the shape ofthe nucleus, organising pore complexes and establishingconnection with chromosomes. The 70 kDa nuclear pro-tein is part of the nuclear lamina and may contribute tonuclear disassembly upon citrullination. Citrullinationreduces the isoelectric point of the protein and thereforereduces its affinity to phosphoseryl residues at the innersurface of the nuclear membrane. All these effects couldcontribute to disintegration of the nuclear lamina duringapoptosis, when Ca(2+) concentration is high enoughto activate PAD enzymes. After disorganisation of thenuclear lamina, DNA can leak out into the cytoplasmand subsequently be degraded by nucleases.

2.4. Other implications

There are additional proteins shown to be substrates ofPAD in vitro or in vivo, however the physiological rolesof their deimination have not been elucidated yet. Deim-ination of glycogen phosphorylase in vitro reduces itsphosphorylation rate mediated by phosphorylase kinase(Luo, Martin, Senshu, & Graves, 1995). No in vivo data is

ion. No PAD4 association with histones was observedear control genes (glyceraldehyde-3-phosphate dehy-rogenase, CIITA genes). Cuthbert et al. have proposedmodel for the antagonism of histone arginine methy-

ation by PAD4 (Cuthbert et al., 2004). Deiminationf arginine can block methylation/activation, becausehe produced citrulline is not substrate for methyla-ion. The demethylimination of methyl-arginine (Fig. 1.)lso represses transcription, as PAD4 directly removeshe methyl-group of the histone protein. Dimethylatedrginines cannot be deiminated by PAD4. Hence, thisechanism induces transcriptional activation that cannot

e reversed by PAD4. Whether the citrullinated histonesan be reverted to arginine-containing histones, and byhat mechanism, have not been elucidated yet.During programmed cell death, the intranuclear

a(2+) concentrations are high enough to activate PAD4o its maximal activity. This results in non-specific deim-nation histones, dramatically reducing their positiveharges (Nakashima et al., 2002). High rate of histoneeimination affects nucleosomal stability, as less basicistones establish weaker connections with the acidic

available as yet related to this possible control process.Fibrin and antithrombin are proteins reported to con-tain citrulline in vivo, and their citrullinated forms havebeen implicated in pathological processes discussed later(rheumatoid arthritis, see below) (Chang et al., 2005;Masson-Bessiere et al., 2001).

3. Citrullination in inflammatory and otherpathological conditions

3.1. Rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmunedisease characterised by symmetric inflammation ofthe peripheral synovial joints (for review see Firestein,2003). Initially, the inflamed joints are painful andswollen. Later, if not treated, the inflammation may leadto cartilage and bone destruction and could result in dis-ability. The disease is quite common, about 1% of theadult population is affected. Women are affected twoor three times more frequently than men. The serumand synovial fluid samples of patients with RA contain

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high concentrations of autoantibodies against varioustargets, for example collagen type II, aggrecan, heatshock proteins, and some glycoproteins (e.g. Hcgp39)(Firestein, 2003). These autoantibodies are not highlyspecific to RA. However, there are two autoantibodyfamilies that seem to be highly characteristic of the dis-ease: Rheumatoid Factor (IgM antibody against humanIgG Fc portion); and the family of anti-citrullinated pro-tein antibodies (ACPA).

3.2. Autoantibodies against citrullinated proteins

Young et al. described these ‘mysterious’ antibodiesfirst in 1979 (Young, Mallya, Leslie, Clark, & Hamblin,1979), but he could not identify the antigen recog-nised by them. The immunoglobulins in the sera of RApatients reacted with the epithelium of the rat oesopha-gus; and were therefore referred to as anti-keratin anti-bodies (AKA). It was hypothesised that they recognisedcytokeratin. Later, it was shown that the AKA antibod-ies not only reacted with the rat oesophagus epithe-lium but also with human buccal mucosal cells, thusstrengthening the keratin hypothesis (Johnson, Carvalho,Holborow, Goddard, & Russell, 1981). In 1993 it becameobvious that the AKA antigen was not cytokeratin,but rather it was an associated molecule (pro)filaggrin(Simon et al., 1993). Therefore, the antibodies weregiven the new name, anti-filaggrin autoantibodies (AFA).These AFA/AKA molecules proved to be identical to theanti-perinuclear factor (Sebbag et al., 1995), originally

nised in the joints as deiminated �- and �-chainsof fibrin (Masson-Bessiere et al., 2001). ThereforeAKA/AFA/APF belongs to the ACPA family of anti-bodies, similar to the anti-Sa (anti-vimentin) antibodies,which will be discussed later. In RA, 99% specificity and65.2% sensitivity have been reported for ACPA. It is thebest diagnostic marker for the disease to date, and is cur-rently used in everyday clinical practice (Sebbag et al.,2004). Synovial citrullinated proteins seem to be specificfor RA (De Rycke et al., 2005). It has also been demon-strated that PAD2 expression is higher in RA joints thanin controls. However, not all PAD2 positive cells containcitrullinated proteins, suggesting that both up-regulationof PADI2 gene and activation of PAD2 are important inthe pathogenesis of RA (De Rycke et al., 2005).

3.3. What about fibrin?

Fibrin plaques are frequently found in the synovial tis-sue. Under physiological circumstances small amountsof fibrinogen (FBG) and other pro-coagulant proteinscan penetrate the capillary wall and travel to the inter-stitium, where they can be cleaved to fibrin peptides andfibrin monomers producing local plaques. These micro-plaques are then broken down by enzymes (such as plas-min) or may undergo endocytosis by macrophages (theCD11c/CD18 integrin complex contributes to this pro-cess). One can assume, that there is a sensitive balancebetween the presence and absence of fibrin molecules inthe interstitium (balance of coagulation and fibrinolysis),

described in 1965 by Nienhuis and Mandema (Nienhuis& Mandema, 1965).

Sera from patients with rheumatoid arthritis wereshown to react strongly with (pro)filaggrin in vitro. How-ever, in further studies using recombinant (from E. coli)or synthetic peptide fragments of (pro)filaggrin therewas no reactivity (Girbal-Neuhauser et al., 1999). Thissuggested that the immunogenicity of (pro)filaggrin wasrelated to its posttranslational modification(s).

In 1999 Girbal-Neuhauser et al. suggested that theantigen, recognised by the AKA/AFA/APF antibod-ies, was citrullinated (pro)filaggrin produced locally byautoreactive B-cells (Girbal-Neuhauser et al., 1999).However, detailed analysis of synovial tissues hasrevealed no (pro)filaggrin expression in vivo (Masson-Bessiere et al., 2001). This excluded the possibility thatthe antigen recognised in vivo by AKA/AFA/APF was(pro)filaggrin, since the inflammation only occurs inthe joint and not in the epidermis, where (pro)filaggrinis abundant (Masson-Bessiere et al., 2001). Severalexperiments carried out by Masson-Bessiere and col-leagues have ultimately identified the antigen recog-

which can be defective under pathological circumstancessuch as in RA (Rubin & Sonderstrup, 2004). Citrullina-tion appears to be the key element in shifting this balance.

Polymerised fibrin is degraded by plasmin, a serine-protease that cleaves near basic amino acid residues(Lys and Arg). The disappearance of the arginine bydeimination reduces the number of cleavage sites, henceincreasing the quantity of polymer fibrin (Sebbag et al.,2004). Citrullination of antithrombin reduces its abilityto inhibit thrombin, leading to a higher speed of coagu-lation (Chang et al., 2005).

Citrullination of fibrin also renders the molecule anti-genic, recognised by the above-mentioned ACPA. Theimmune response results in increased plasma exudationand endothelial cell contraction. This in turn, increasesthe amount of FBG and other procoagulant proteins inthe interstitium (Rubin & Sonderstrup, 2004).

The most important question is whether RA is causedby the presence of fibrin plaques or not. This questioncould be approached in two ways.

Possible non-antigen-specific effects: it is certainthat fibrinogen can activate innate immunity by parallel

B. Gyorgy et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1662–1677 1671

stimulation of the CD11c/CD18 complex, Toll-likereceptor 4 and CD14 on the macrophages. These cellsthen produce various pro-inflammatory cytokines, likeIL-1, IL-8, IL-12 and TNF-�, causing local inflam-mation and increased plasma outflow from capillaries(vicious cycle) (Rubin & Sonderstrup, 2004).

Antigen-specific effects: the citrullinated �- and�-chains of fibrin are immunogenic, and stimulatethe adaptive immunity causing inflammation (Masson-Bessiere et al., 2001). ACPA belong to the IgG class;this suggests a role of autoreactive T-cells supportingthe isotype-switch.

Rubin and Sonderstrup have found that neither nativehuman FBG nor deiminated FBG induced arthritis.Although ACPA, cross-reactive with mouse FBG, wereelicited (Rubin & Sonderstrup, 2004). It still remains tobe answered, whether deiminated FBG plays a role inthe aetiology of RA.

3.4. Autoreactive T cells in rheumatoid arthritis

During T-cell response, the MHC has to bind theepitope. Thus, MHC has to interact with citrullinatedpeptides with a certain affinity in order to elicit anantigen-specific immune response. In 1987 a ‘sharedepitope hypothesis’ was postulated for the associationof particular MHC-II molecules and RA (Gregersen,Silver, & Winchester, 1987). Certain MHC-II moleculescontain a positively charged motif, the shared epitope,70QKRAA74 or 70QRRAA74 sequence in the HLA-DIesrMnPctDrRnm2

3

sd

al., 2006; Olsson, Skogh, Axelson, & Wingren, 2004).These environmental effects often induce cell death,thus necrosis could possibly play a role in the aeti-ology of RA (van Venrooij & Pruijn, 2000). Duringnecrosis the integrity of the cell membrane is lost,and the cytoplasm containing enzymes like PAD, isreleased from the cell. The high interstitial concentra-tion of Ca(2+) can initiate the citrullination process. Ithas been shown that vimentin can be deiminated underthese circumstances (Asaga et al., 1998). Its peptidefragments could be presented to T-helper cells by HLAmolecules containing the shared-epitope inducing a spe-cific immune response (Hill et al., 2003). Anti-vimentinantibodies have been detected in sera of patients withRA, these are called anti-Sa antibodies and belong tothe ACPA family (Menard, Lapointe, Rochdi, & Zhou,2000).

3.6. Peptidylarginine deiminase type 4 is associatedwith the disease

The λsib value for RA is 2–17 in different populationsshowing the importance of genetic factors in the aetiol-ogy of the disease (Seldin, Amos, Ward, & Gregersen,1999).

HLA is considered to be the major genetic factordetermining disease susceptibility. Recently, Suzuki etal. described an association of functional haplotypes ofPADI4 with the disease (p = 0.000008) (Suzuki et al.,2003). By analysing SNPs, four different haplotypes

R �-chain. Those who have this motif in the MHCI molecule are highly susceptible to RA. The sharedpitope is located in the P4 pocket, one of the sub-trate binding sites of the MHC-II. Further studiesevealed that citrulline fits easily into the P4 pocket of

HC-II containing the shared epitope, because of theon-refusing ionic interactions (Hill et al., 2003). The70-74 peptide can be either negatively or positivelyharged. Studies revealed that individuals carrying posi-ively charged P4 pockets (with shared epitope of HLA-RB*0101, *0401 or *0404) can mount an immune

esponse to citrullinated peptides and are susceptible toA (Hill et al., 2003). In contrast, those who expressegatively charged P4 pockets (HLA-DRB*0402)ight be protected from the disease (Reviron et al.,

001).

.5. Environmental effects in rheumatoid arthritis

It has been suggested that smoking, vibration, expo-ure to mineral dust, and injury increase the risk ofeveloping RA (Aho & Heliovaara, 2004; Klareskog et

of PADI4 have been described in a Japanese popula-tion. Two of these haplotypes (haplotype 1 and 2) arefound in 82–85% of the population, while haplotypes3 and 4 are in the remaining 15–18%. Haplotype 2 ofPADI4 gene was found to strongly correlate with RA.It has been shown that 32% of patients with RA havethis haplotype, while this number is only 25% amongcontrols. Why haplotype 2 of PADI4 may increase thesusceptibility to RA? The half-life of haplotype 2 mRNAis 11.6 min, while that of the non-susceptible haplo-type 1 is only 2.1 min. Therefore, susceptibility couldbe explained by the higher chance for PAD translation,resulting in more enzyme, leading to more protein (fib-rin or vimentin) deimination, which can induce adaptiveand innate immune responses leading to chronic inflam-mation. This study has evoked a great amount of interest,but other investigations have failed to detect any associa-tion of PADI4 with RA in patient populations in France,the United Kingdom and in Spain (Barton et al., 2004;Caponi et al., 2005; Martinez et al., 2005). Although, areplication study in Japan and also a Korean investiga-tion confirmed the association (Ikari et al., 2005; Kang

1672 B. Gyorgy et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1662–1677

et al., 2006). It is noteworthy that the two susceptibilitygenes to RA (PADI4 and HLA-DRB1) are both relatedto citrullination.

3.7. Citrullination in collagen-induced arthritis

Recent studies suggest that citrullinated collagentypes I and II are also targets for ACPA (Burkhardt etal., 2005; Suzuki et al., 2005). Immunisation of geneti-cally susceptible mice or rats with type II collagen resultsin the development of collagen-induced arthritis (CIA).In the rat model of CIA Lundberg et al. (Lundberget al., 2005) described ACPA to be reactive with cit-rullinated type II collagen and also cross-reactive withnative collagen. This means that citrullination of a self-antigen breaks immune tolerance. Collagen, as a keyjoint antigen, contributes to RA pathogenesis, and thisstudy revealed that citrullinated forms of collagen type IIhave increased immunogenicity and arthritogenicity. Itwas also shown that the severity of the arthritis correlateswith PAD4 expression (in the infiltrating mononuclearcells) and with the amount of citrullinated collagen. Mostimportantly, it was also demonstrated that clinical signsof arthritis preceded the presence of citrullinated proteinsand PAD4 expression. This strongly suggests that citrul-lination is more likely a consequence rather than a causeof joint inflammation. However, the pronounced anti-body response against citrullinated joint antigens couldcontribute to the progression of the autoimmune inflam-mation.

human MS patients (Mastronardi, Mak, Ackerley, Roots,& Moscarello, 1996).

3.9. How can citrullination affect the stability of themyelin sheath?

Citrullination reduces the positive charge of the pro-tein, lowering its affinity to the negatively chargedmyelin phosphatidyl-serine residues. It has been shownin vitro that the amount of citrulline residues in MBPnegatively correlates with its lipid-aggregating abil-ity (Mastronardi et al., 1996; Wood & Moscarello,1989).

Cathepsin D can degrade MBP more easily if itis citrullinated, as MBP-Cit has a more open struc-ture (Pritzker et al., 2000a). The protease can releasean immunodominant peptide, the 44Phe–Phe89 peptide,and this way an autoimmune response can be elicited(Whitaker, Bashir, Chou, & Kibler, 1980). Lymphocytesand other immune cells infiltrate the nervous tissue andcause local inflammation, oxidative stress and nerve celldeath or myelin sheath destruction. However, the mech-anism that allows immune cells to penetrate the blood-brain barrier has not been uncovered yet.

Genetic association of MS with PADI has notbeen shown. Pritzker et al. proposed a mechanism forthe increased citrullination in MS patients, claimingthat methylation affects citrullination (Pritzker, Joshi,Harauz, & Moscarello, 2000b). MBP is a methylatedprotein, and if it is not methylated, no myelin sheath

3.8. Multiple sclerosis

Multiple sclerosis (MS) is a severe autoimmune dis-ease that affects myelin sheaths in the CNS. The neu-rons of the CNS gradually lose their myelin sheathsynthesised by oligodendroglial cells. As a result, elec-trical conduction is disturbed. The disease eventuallycauses paralysis and death. Citrullination plays a keyrole in the pathogenesis of MS. Current knowledgeattests that MS is caused mainly by overcitrullinationof the MBP (Moscarello et al., 1994; Vossenaar etal., 2003; Wood, Bilbao, O’Connors, & Moscarello,1996). There is an increase in both the overall ratioof MBP-Cit/total MBP (see earlier) and the numberof citrullines within the MBP-Cit. Therefore, we cansay that in MS the brain resembles an ontogeneticallyearlier state, since young children have similar MBP-Cit/total MBP ratios (see above). In DM20 transgenicmice (DM = dismyelinating) containing 2–70 copies ofmyelin proteolipid protein DM20, MBP citrullinationis increased and the mice show the same symptoms as

is formed. Methylation increases the hydrophobicity ofMBP and reduces its ability to become citrullinated, asmethylated MBP has a more condense structure thanthe unmodified version. It could be speculated that areduced methyltransferase activity is responsible for theenhanced citrullination of MBP in MS.

GFAP (glial fibrillary acidic protein), an astrocyte-specific intermediate filament, also undergoes deimi-nation. Nicholas et al. suggested that GFAP could beovercitrullinated in MS (Nicholas, Sambandam, Echols,& Tourtellotte, 2004). Some recent reports have demon-strated similar deiminated proteins in experimentalautoimmune encephalomyelitis (EAE), an animal modelof MS (Raijmakers et al., 2005). Several patches contain-ing citrullinated peptides were found in the CNS of micewith EAE induced by the injection of myelin oligoden-drocyte glycoprotein (Nicholas et al., 2005). The deim-inated proteins found in these patches were MBP andGFAP. It’s not surprising that citrullinated MBP inducesEAE in mice (Cao, Sun, & Whitaker, 1998), supportingthe hypothesis that citrullination plays a cardinal role inthe development of EAE and MS.

B. Gyorgy et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1662–1677 1673

As citrullination seems to be a key process in MS,a PAD2 inhibitor therapy trial is currently underway(http://www.mssociety.ca/entxt/research/PT991213.htm).A cytotoxic, chemotherapeutic taxol derivative, pacli-taxel inhibits human PAD2 in vivo and in vitro (Pritzker& Moscarello, 1998). Administering paclitaxel toDM-20 mice, the demyelinisation process slows downand the CNS symptoms abate (Moscarello, Pritzker,Mastronardi, & Wood, 2002).

3.10. Alzheimer’s disease

A recent report claims that patients with Alzheimer’sdisease (AD) have significantly elevated rate of citrulli-nation in their CNS, mainly in the hippocampus, whichis the region of the brain most affected by the dis-ease (Ishigami et al., 2005). Comparison of data frompatients with AD with those obtained from control indi-viduals has revealed that it was the activity of PAD2rather than its total amount that was increased in thepatients. One can hypothesise that during neurodegener-ation a higher concentration of Ca(2+) activates the cit-rullination process. The predominant over-citrullinatedproteins include vimentin, MBP and GFAP.

3.11. Psoriasis

This disease is characterised by an enormous mitoticactivity in the human epidermis and the rapid cell prolif-eration results in abnormal cornification. Typical symp-tpocurda(ds

3p

dToPlh

arginine or soluble arginine substrates (in a Ca(2+)-independent manner). The enzyme plays a key role in thepathogenesis of periodontitis, a common disease causedby this bacterium (McGraw et al., 1999). During Argconversion, ammonia is produced that contributes to theneutralisation of the local pH. The enzyme can inac-tivate anaphylatoxins, produced locally during comple-ment activation, or by the cleavage of C5 by the bacterialRGPs protease (Wingrove et al., 1992). It can inactivatebradikinine and can regulate plasma-outflow (Imamura,Pike, Potempa, & Travis, 1994). It can also inactivatespecial anti-adhesive molecules produced by the host.For these reasons, bacterial PAD could be regarded as apotential virulence factor.

4. Closing remarks

Although in vitro citrullination of proteins can beeasily detected using various substrates (Kubilus et al.,1980), as yet we do not know how in vitro data reflectthe in vivo situation. It seems logical to hypothesise atwo-level regulation model: at an extremely high con-centration of Ca(2+) PAD is fully active and may losesubstrate specificity. This may occur in vitro, where,in fact, all arginine-containing proteins could be deimi-nated, albeit with different kinetic parameters (Kubiluset al., 1980). This could also happen extracellularly, ifthe concentration of Ca(2+) were high enough.

However, it is possible that there is a physiologicalregulatory mechanism at low concentrations of Ca(2+),

oms are itching, sensitivity of affected skin and theresence of red patches covered with silvery-white scalesf dead skin. Citrullination is implicated in the pathome-hanism of psoriasis, however the exact mechanism isnknown. Cytokeratin K1 has been shown to containeduced amount of citrullyl residues in the psoriatic epi-ermis (Ishida-Yamamoto et al., 2000). Interestingly, inrecent phase II pilot trial the PAD2 inhibitor paclitaxel

also implicated as a therapeutic agent in MS) has beenemonstrated to have therapeutic activity in patients withevere psoriasis (Ehrlich et al., 2004).

.12. Peptidylarginine deiminase of a pathogen—aotential virulence factor

Peptidylarginine deiminases are ancient enzymes thateveloped early in evolution (Vossenaar et al., 2003).his has been verified by the identification of a prokary-te PAD enzyme in Porphyromonas gingivalis (McGraw,otempa, Farley, & Travis, 1999). This enzyme shows

ittle sequence similarity to the human PAD enzymes,owever, it can efficiently deiminate either peptidyl

possibly mediated by other proteins or PAD proteininteractions. We propose that when PAD is active at a‘low-level’ of Ca(2+), it has the higher substrate- andarginine-specificity required for example, for gene reg-ulation. Further studies would be required to elicit the invivo substrates of PAD, and to elucidate how PAD mightwork at low calcium concentrations.

At a ‘high-level’ of Ca(2+), PAD can deiminate nearlyany arginines of any proteins. This could lead to autoim-munity, since the highly variable deiminated proteins (inwhich arginines are citrullinated), may be recognised asneoantigens by the immune system. Emerging insightsinto the pathomechanism of RA or MS allowed the test-ing of drug compounds against PAD enzymes, althoughfurther studies are required to investigate the PAD poly-morphisms in MS, RA or even AD.

It was an important finding that citrullination couldantagonise methylation in signalling (Denman, 2005).However, we do not know how a citrullinated pro-tein can thereafter revert back to its original, arginine-containing form. It does not seem likely that during generegulation citrullinated histones are ‘discarded as junk

1674 B. Gyorgy et al. / The International Journal of Biochemistry & Cell Biology 38 (2006) 1662–1677

molecules’, and are not converted back. However, a ‘pep-tidyl citrulline-iminase’ enzyme has not been discoveredyet, if it exists at all. Therefore, we refer to methylationat present as an irreversible PTM process.

At present there is no experimental proof for the incor-poration of citrulline into a newly synthesised polypep-tide chain. Just as there is no evidence for the existenceof a tRNA capable of transporting citrulline. However,given the high activity of nitric oxide synthase (NOS; anenzyme producing nitric oxide and citrulline from argi-nine) in inflammatory sites, it is tempting to speculateas to some association between local autoimmune reac-tions to citrullinated antigens and NOS activity. Furtherinvestigations are required to clarify this question.

Citrullination of proteins, among other posttransla-tional modifications, significantly broadens the spectrumof autoepitopes that the immune system has to recogniseand tolerate.

Current evidences strongly suggest that immune tol-erance to citrullinated proteins may be lost in autoim-mune diseases and thus, this small posttranslationalmodification is in the focus of a growing interest inbiomedicine.

Acknowledgement

This work has been supported by grants OTKAT046468 IB2, OTKA TS/2 044707, ETT 287/2003 andETT 134/2003.

Bauer, U. M., Daujat, S., Nielsen, S. J., Nightingale, K., & Kouzarides,T. (2002). Methylation at arginine 17 of histone H3 is linked to geneactivation. EMBO Rep., 3(1), 39–44.

Beniac, D. R., Wood, D. D., Palaniyar, N., Ottensmeyer, F. P.,Moscarello, M. A., & Harauz, G. (2000). Cryoelectron microscopyof protein–lipid complexes of human myelin basic protein chargeisomers differing in degree of citrullination. J. Struct. Biol., 129(1),80–95.

Boggs, J. M., Rangaraj, G., Koshy, K. M., Ackerley, C., Wood, D. D.,& Moscarello, M. A. (1999). Highly deiminated isoform of myelinbasic protein from multiple sclerosis brain causes fragmentation oflipid vesicles. J. Neurosci. Res., 57(4), 529–535.

Burkhardt, H., Sehnert, B., Bockermann, R., Engstrom, A., Kalden, J.R., & Holmdahl, R. (2005). Humoral immune response to citrul-linated collagen type II determinants in early rheumatoid arthritis.Eur. J. Immunol., 35(5), 1643–1652.

Caponi, L., Petit-Teixeira, E., Sebbag, M., Bongiorni, F., Moscato, S.,Pratesi, F., et al. (2005). A family based study shows no associationbetween rheumatoid arthritis and the PADI4 gene in a white Frenchpopulation. Ann. Rheum. Dis., 64(4), 587–593.

Cao, L., Sun, D., & Whitaker, J. N. (1998). Citrullinated myelin basicprotein induces experimental autoimmune encephalomyelitis inLewis rats through a diverse T cell repertoire. J. Neuroimmunol.,88, 21–29.9.

Chang, X., Yamada, R., Sawada, T., Suzuki, A., Kochi, Y., &Yamamoto, K. (2005). The inhibition of antithrombin by peptidy-larginine deiminase 4 may contribute to pathogenesis of rheuma-toid arthritis. Rheumatology (Oxford), 44(3), 293–298.

Chavanas, S., Mechin, M.-C., Takahara, H., Kawada, A., Nachat, R.,Serre, G., et al. (2004). Comparative analysis of the mouse andhuman peptidylarginine deiminase gene cluster reveals highly con-served non-coding segments and a new human gene, PADI6. Gene,330, 19–27.

Cuthbert, G. L., Daujat, S., Snowden, A. W., Erdjument-Bromage,H., Hagiwara, T., Yamada, M., et al. (2004). Histone deimination

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