Citrullinated calreticulin potentiates rheumatoid arthritis shared epitope signaling

Abstract

Objective

Citrullinated proteins are immunogenic in rheumatoid arthritis (RA), particularly in patients that carry shared epitope (SE)-coding HLA-DRB1 alleles. The mechanism underlying this association is unknown. We have previously identified SE as a ligand that interacts with cell surface calreticulin (CRT) and activates immune dysregulation. The objective of this study was to determine the effect of CRT citrullination on SE signaling.

Methods

CRT-SE binding affinity was measured by surface plasmon resonance. The role of individual CRT arginine residues was determined by site-directed mutagenesis. Nitric oxide levels were measured using a fluorochrome-based assay. CRT citrullination in synovial tissues and cell cultures was determined by 2-dimensional gel electrophoresis, immunoblotting and mass spectrometry techniques.

Results

Synovial tissues and fibroblast-like synoviocytes from RA patients were found to express higher abundance of citrullinated CRT compared to OA samples. Citrullinated CRT showed more robust interaction with the SE ligand, and transduced SE signaling at a 10,000-fold higher potency, compared to non-citrullinated CRT. Site-directed mutation analysis identified Arg²⁰⁵, which is spatially adjacent to the SE binding site in the CRT P-domain, as a dominant inhibitor of SE-CRT interaction and signaling, while a more remote arginine residue, Arg²⁶¹ was found to enhance these SE functions.

Conclusion

Citrullinated CRT is over-abundant in the RA synovium, and potentiates SE-activated signaling in vitro. These findings could introduce a new mechanistic model of gene-environment interaction in RA.

The shared epitope (SE), a five amino acid sequence motif in positions 70–74 of the HLA-DRβ chain, is the most significant genetic risk factor for rheumatoid arthritis (RA) (1,2). The disease in individuals carrying SE-coding HLA-DRB1 alleles is more erosive than in SE-negative individuals (3). The mechanism underlying the effect of the SE in RA is unknown. We have recently identified the SE as a ligand that activates innate signaling and Th17 polarization (4–7). Whether expressed in its native conformation on the cell surface; as a cell-free HLA-DR tetrameric molecule; engineered into large recombinant proteins; or as a short synthetic peptide, the SE activates nitric oxide (NO) production in a strictly allele-specific manner. SE-activated signaling is transduced via cell surface calreticulin (CRT) (6), an established innate immunity receptor. Using a point mutagenesis technique, we have recently mapped the SE binding site to amino acid residues Glu²¹⁷, Asp²²⁰ and Glu²²³ in the CRT P-domain (8).

Although genetic factors strongly influence disease risk, the concordance rate of RA in identical twins is only 15% (9). It has therefore been proposed that while susceptibility is determined genetically, disease onset may depend on environmentally triggered stochastic events, such as posttranslational protein modifications (reviewed in 10). One of the better-known protein modifications in RA involves deimination of arginine residues to citrulline by peptidyl arginine deiminase (PAD). Citrullinated proteins are overabundant in RA joints, as well as many other inflammatory tissues, such as the brain in multiple sclerosis, muscle of myositis patients, or the gut in Crohn’s disease (11,12).

Several citrullinated proteins have been identified in RA (13–15), and some of them have been found to be targets of anti-citrullinated protein antibodies (ACPA), which are useful biomarkers for RA (Reviewed in 16). Although the pathogenic role of ACPA in human RA remains an open question, it is well established that these antibodies are more commonly found in SE-positive RA patients (16). Furthermore, a SE gene-dose effect on RA risk has been documented in ACPA-positive RA patients (17).

Thus, RA development involves an intricate relationship between genetic (notably SE) and acquired, non-genetic (protein citrullination) factors. Several studies have identified synergism between SE and smoking in RA (18–20), and an association between smoking and pulmonary PAD-2 expression, supporting the hypothesis that smoking-induced PAD activation may trigger onset of a SE-driven disease (21).

Given the known association of SE with RA, its recently discovered signaling effect via CRT, the association of protein citrullination with the disease, and the interaction between SE and a citrullination-driven RA biomarker, we have sought to determine whether CRT citrullination affects SE signaling. Here we demonstrate that citrullinated CRT (Cit-CRT) has higher affinity for the SE ligand with resultant potentiation of SE-activated signaling. Using point mutagenesis, we have identified three arginine residues in CRT that modulate SE binding to CRT and, consequently the potency of its signaling. Finally, we demonstrate that Cit-CRT is over-abundant in RA synovial tissues. These results provide new insights into the functional effect of the SE and identify a candidate molecular basis for a mechanistic model of gene-environment interaction in RA.

MATERIALS AND METHODS

Cells and reagents

The mouse embryonic fibroblast (MEF) line K42, isolated from crt^−/− mice, has been previously described (22). Diaminofluorescein Diacetate (DAF-2 DA) was purchased from EMD-Calbiochem (Gibbstown, NJ). SE ligands were expressed as either 15mer synthetic peptides (65–79*0401 or 65–79*0402), or in the form of HLA-DR4 tetramers as previously described (4–8). All other chemicals were from Sigma (St. Louis, MO). Fibroblast-like synoviocytes (FLS) were prepared as previously described (22) from synovial tissues (ST), collected from RA and OA patients undergoing joint replacement surgery at the University of Michigan Health system. FLS were used between passages 4 and 8. Patients classified as RA satisfied 1987 ARA criteria for RA (23). The presence of OA was ascertained by clinical diagnosis and characteristic findings on radiographs. Procedures involving human subjects were conducted under an institutional IRB-approved protocol.

Generation of CRT point mutants

Site-directed mutagenesis was performed following the QuikChange protocol (Stratagene, La Jolla, CA). Sequences were verified by the University of Michigan DNA Sequencing Core. Mouse wild-type crt and its site-directed mutants were cloned into pBAD/glll A-His6 plasmid as described (24). Wild-type and mutant crt plasmids were transformed into GC10 cells for protein expression. The 6×His-tagged protein expression was induced by 0.002% L-arabinose for 4 hours and the protein was purified using a Ni-NTA resin following the manufacturer’s (QIAGEN, Valencia, CA) protocol.

In vitro citrullination of CRT

Purified mouse CRT was incubated with 2 units of PAD from rabbit skeleton muscle in the reaction buffer (0.1M Tris/HCL, pH7.6, 10mM CaCl2, 5mM dithioerythritol, 1X proteinase inhibitor cocktail) for 16hr at 37°C (25). At the end of incubation, the reaction mixture was loaded on 10% SDS-PAGE. The CRT band was in-gel digested by Glu-c. Then citrullination sites on CRT were confirmed by the LTQ-Oribtrap XL (see below) at the Proteomic Resource Facility in the Department of Pathology, University of Michigan.

Surface plasmon resonance (SPR)

A BIAcore2000 instrument (GE Healthcare, Piscataway, NJ) was used as previously described (6,8). All assays were performed at 25°C in a binding buffer containing 10 mM HEPES, pH 7.4, 50 mM KCl, 0.5 mM CaCl₂, 100 μM ZnCl₂, and 0.005% surfactant P-20. The analyte was injected at a flow rate of 10μl/min.

Chemical modification of citrulline residues

Cell lysates were chemically modified in reaction mixture with 8.3 mM antipyrine, 50% TFA and 8.3mM 2,3-butanedione, as previously described (26). The mixture was incubated for 2 hr at 37°C in the dark. At the end of modification, the reaction mixture leftover was removed by desalting column. The chemically modified cell lysates were processed in immunoprecipitation.

Mass spectrometry (MS)

MS analysis of in-vitro citrullinated CRT was performed by Liquid chromatography electrospray ionization (ESI/LC) MS/MS method at the Proteomic Resource Facility in the Department of Pathology, University of Michigan. Peptide samples were suspended in 1% acetic acid and 2% acetonitrile, and loaded onto an in-house packed reverse phase separation column (0.075 × 100mm, MAGIC C18 AQ particles, 5μm, Michrom Bioresources). The peptides were separated on a 1% acetic acid/acetonitrile gradient system (5–50% acetonitrile for 75min, followed by a 10min 95% acetonitrile wash) at a flow rate of ~300nl/min. Peptides were directly sprayed onto the MS using a nanospray source. An LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, MA) was run in automatic mode collecting a high resolution MS scan (FWHM 30,000) followed by data-dependent acquisition of MS/MS scans on the 9 most intense ions (relative collision energy ~35%). Dynamic exclusion was set to collect 2MS/MS scans on each ion and exclude it for an additional 2min. Charge state screening was enabled to exclude +1 and undetermined charge states (27). Raw data were searched against Uniprot mouse protein database (rel 2011/05) appended with reverse sequences using X!Tandem/TPP software suite. Carbamidomethylation of cysteine (57.0214 Da), oxidation of methionine (15.9949 Da) and citrullination of arginine (+0.9840 Da) were considered as potential modifications. Precursor mass tolerance of 50 ppm, and fragment mass tolerance of 0.5 Da were allowed. Peptides containing citrullinated arginine with a PeptideProphet probability of >0.9 were considered positive and manually verified.

To identify CRT citrullination sites in ST and FLS, Mass spectrometric analysis of chemical modified citrulline residues was performed in the University of Michigan Proteome Mapping Core. The MALDI TOF/TOF mass spectra of chymotrypsin-digested peptide were obtained using an Applied Biosystems 4800 Proteomics Analyzer, as previously described (8).

Immunoprecipitation analysis

Tissue or cell lysates were pre-cleared by incubation with protein G-agarose beads (Sigma, St. Louis, MO) for 15 min at 4°C overnight with beads and a 1:25 dilution of mouse anti-Calreticulin antibody (BD, Franklin Lakes, NJ). Beads were then washed once with RIPA and twice with PBS, and the immune complexes were released from the beads by boiling in sample buffer for 5 min. Following SDS gel electrophoresis, immunoprecipitated products were analyzed by Western blotting using anti-citrulline antibody (Abcam, Cambridge, UK). The CRT level on same blot was confirmed by rabbit anti-CRT antibody (Pierce, Rockford, IL).

Two-dimensional gel electrophoresis

RA and OA FLS were analyzed by western blot using 2-DE membranes. Proteins were focused at 20°C, with 11 cm immobilized pH 3 to 5.6 gradient IPG ReadyStrips (Bio-Rad Laboratories, Hercules, CA), which had been incubated in 200 μl protein extract mixed with rehydration buffer (8 M urea, 2% CHAPS, 1% DTT, trace of bromophenol blue, 0.2% Biolyte carrier ampholytes 3 to 5.6; Bio-Rad Laboratories) for 16 hours. The Protean IEF cell (BIO-RAD Laboratories) was used with fast-voltage ramping at a maximum voltage of 6,000 V for 20 hours. After the first dimension run, the strips were equilibrated by incubation in 6 M urea, 0.375 M Tris–HCl, pH 8.8, 2% SDS, 20% glycerol, 2.5% (w/v) DTT at 10 ml per strip for 20 minutes in room temperature, followed by an incubation for 30 minutes in the same buffer but in which DTT was replaced by 2.5% (w/v) iodoacetamide. Strips were then placed on the top of 4% to 12% Criterion^™ XT precast gels (11 cm × 8 cm × 1 mm) (Bio-Rad Laboratories, Hercules, CA) and migrated constantly at 200 V until the bromophenol blue dye front had reached the bottom of the gel. The BenchMark^™ prestained protein ladder (Invitrogen) was used as the MW standard in the second dimension step. Finally, gels were either stained with SYPRO Ruby Protein Gel Stain (Bio-Rad Laboratories, Hercules, CA) or were electroblotted onto nitrocellulose membranes. SYPRO Ruby-stained 2-D gels were scanned using a densitometer, and western blotting analyses were performed.

Signal transduction assays

To measure NO production, crt^−/− MEF were seeded in a 96-well plate at 30,000 cells per well. Cultures were loaded with recombinant WT or mutant CRT overnight. Cells were then labeled by 20 μM DAF-2 DA for 1 hour and stimulated with SE ligands. NO production rates were determined as previously described (4–8).

Statistical analysis

Data are expressed as mean ± SEM from triplicate samples. Statistical analyses were performed using a 2-tailed Student’s T-test.

RESULTS

CRT citrullination enhances SE-CRT interactionand potentiates SE-activated signaling

RA epidemiology and translational research findings strongly suggest that while RA susceptibility is determined genetically, disease onset may depend on environmentally triggered stochastic events, such as posttranslational protein modifications. Given the growing evidence for aberrant protein citrullination in RA, we have first undertaken to examine whether citrullination of CRT could affect the functional properties of the SE.

Recombinant CRT was citrullinated in vitro as reported (25). To determine the effect of global CRT citrullination on its interaction with SE, Cit-CRT or native CRT (N-CRT) proteins were immobilized on biosensor chips and their binding affinities toward SE peptides, or SE-expressing HLA-DR tetramers, were determined. As can be seen in Figure 1, the SE ligand 65–79*0401 (a 15mer peptide corresponding to the 65–79 region, coded by a SE-positive allele DRB1*04:01), interacted more potently with Cit-CRT than with N-CRT. A control peptide 65–79*0402 (corresponding to the 65–79 region, coded by a SE-negative allele DRB1*04:02) did not bind to either N-CRT or Cit-CRT. The SE-positive HLA-tetramer T-0401 (4 units of heterodimeric molecules, each made of a DRβ chain coded by allele HLA-DRB1*04:01, and a DRα chain coded by HLA-DRA1) showed markedly higher interaction with Cit-CRT, compared to N-CRT. A control, SE-negative tetramer T-1501 did not bind to N-CRT and did not show preferential binding to Cit-CRT (Fig. 1B). Kinetics analysis (Table 1) revealed that Cit-CRT had 100-fold higher affinity (K_D) to the SE ligand, compared with N-CRT. Importantly, the affinity of C1q, another CRT ligand, was unaffected by citrullination. Thus, we conclude that global citrullination of CRT potentiates its interaction with the SE ligand.

Figure 1. CRT citrullination enhances SE-CRT interaction and potentiates SE-activated signaling.

A. N-CRT or Cit-CRT proteins were immobilized on a biosensor chip at a 3000 RU level, and SE-positive peptidic ligand 65–79*041 or a control SE-negative peptide 65–79*0402, each at 330 μM, were applied in the analyte and binding interactions determined. B. SPR data showing binding interactions between SE-positive (T-0401) or −negative (T-1501) tetramers at a 250 nM concentration, applied in the analyte. C. CRT-deficient cells were incubated with various concentrations of N-CRT or Cit-CRT. SE-positive peptide 65–79*0401 was added at a concentration of 100 μg/ml and NO was measured. D.Crt^−/− mouse cells were incubated with 1 μg/ml of N-CRT or Cit-CRT. SE-positive peptide 65–79*0401 was added at various concentrations and NO was measured. Data shown are representative of at least two independent identical experiments.

Table 1.

Kinetics of CRT-ligand interactions

Surface	65–79*0401			C1q
	Ka (1/Ms)	Kd (1/s)	KD (M)	Ka (1/Ms)	Kd (1/s)	KD (M)
N-CRT	8.7±6.2	8.2±2.1 × 10−4	9.6±3.8 × 10−6	1.7±2.0 × 105	2.8±0.2 × 10−4	1.7±0.2 × 10−9
Cit-CRT	560±0.4	5.1±0.2 × 10−5	8.4±0.3 × 10−8	2.7±0.5 × 105	2.4±0.4 × 10−4	9.0±1.1 × 10−10

We next performed experiments to determine whether increased affinity in cell-free binding studies has functional correlates in signal transduction assays. We (6, 8) and others (28,29) have previously demonstrated that when soluble CRT is added to CRT-negative cells it attaches to the cell surface and restores CRT receptor-mediated signaling. Accordingly, to determine the effect of CRT citrullination on signal transduction, soluble N-CRT or Cit-CRT proteins were added to CRT-deficient cells, followed by stimulation with the SE ligand. As can be seen, Cit-CRT mediated signal transduction much more effectively. It transduced SE signaling at significantly lower receptor concentrations (Fig. 1C) and at a much lower ligand concentration threshold (Fig. 1D) than the N-CRT. The EC₅₀ of SE with Cit-CRT (2.45 × 10⁻⁷) was 10,000-fold lower than with N-CRT (2.05 × 10⁻³).

It is worth noting that different from N-CRT, Cit-CRT transduced much more potent SE-activated signals when added to tissue cultures at lower concentrations (Fig. 1C). The reason for this ‘paradoxical’ dose-response curve is unclear. One possible explanation is that at high tissue culture concentration, there is saturation of the CRT-anchoring sites at the cell surface with resultant higher abundance of free Cit-CRT in the supernatant. Due to its higher affinity, unbound Cit-CRT could conceivably ‘intercept’ the SE ligand before it binds to the cell surface-anchored signal-transducing receptor.

The impact of individual arginine residues

Given that citrullination involves conversion of positively-charged arginine residues to neutrally-charged citrullines, we sought to determine whether the augmented interaction between SE and Cit-CRT shown above is contributed by a loss of positive charge near the SE binding site, previously mapped to the CRT P-domain (8). Of the 8 arginine residues in the mature CRT protein, only Arg²⁰⁵ and Arg²⁶¹ are located in its P-domain. Another remote, but possibly spatially relevant residue is Arg¹⁶⁰, located in the CRT N-domain, close to its junction with the P-domain. Mass spectrometry analysis of the Cit-CRT used in Figure 1 showed that these 3 arginine residues were all citrullinated (Table 2). We therefore theorized that one or more of them may have contributed to Cit-CRT effects. Since citrulline is not a genetically-coded residue, selective insertion of individual citrulline residues by recombinant DNA technology is not possible. We therefore adapted a site-directed citrulline-simulation approach, by mutating positively-charged arginine residues to neutrally-charged alanines. These substitutions did not introduce conformational changes in CRT, as evidenced by circular dichroism (Supplementary Figure 1).

Table 2.

MS analysis of CRT citrullination sites

Origin	Designation	Citrullinated sites
Rec. protein*	mCrt	Arg19, Arg160, Arg205 , Arg261
FLS**	RA1	Arg19, Arg56, Arg145
	RA2	Arg56, Arg160, Arg205
	OA1	Arg56, Arg205
	OA2	Arg56, Arg205
ST**	RA3	Arg56, Arg145, Arg205
	RA4	Arg19, Arg56, Arg145, Arg205
	RA5	Arg19, Arg205, Arg261
	OA3	Arg56, Arg145, Arg160, Arg205
	OA4	Arg19, Arg56, Arg145, Arg160, Arg205 , Arg261

^*Recombinant mouse CRT was citrullinated in vitro. CRT citrullination sites were identified by MALDI TOF/TOF

^**CRT citrullination sites in cell or tissue extracts were identified by ESI/LC-MS/MS

Wild-type CRT (WT-CRT) or its point mutant variants were immobilized on biosensor chips as above and their binding to the peptidic SE ligand, or HLA-DR tetrameric proteins was determined. As Figure 2 shows, a single arginine-to-alanine substitution in position 205 (R205A) augmented CRT-SE interaction to SE-expressing peptides (Fig. 2A) and tetrameric proteins (Fig. 2B), while R261A demonstrated diminished binding. R160A mutation did not have a significant impact on CRT-SE binding. None of the substitutions had a significant impact on CRT interaction with SE-negative peptides or tetramers, confirming the specificity of the findings. Interestingly, the two P-domain arginines, Arg²⁰⁵ and Arg²⁶¹ had a diametrically opposite effect on SE binding. To further characterize the relative role of these arginines, we generated a double-mutant expressing both substitutions (R205A/R261A). As can be seen in Figure 2, the double mutant R205A/R261A retained an enhanced interaction with the SE, indicating that Arg²⁰⁵ had a dominant effect.

Figure 2. Role of individual CRT arginine residues.

A. N-CRT, Cit-CRT, or CRT with selected Arg-Ala substitutions were immobilized at a 3000 RU level on a biosensor chip and SE-positive peptidic ligand 65–79*041, or a control SE-negative peptide 65–79*0402, each at 330 μM, were applied in the analyte and binding interactions determined. B. SPR data showing binding interactions between the different CRT variants shown in A and SE-positive (T-0401) or −negative (T-1501; T-0301) tetramers, applied in the analyte at a 250 nM concentration. C. Representative sensorgrams of the data shown in B. D. CRT-deficient cells were incubated with CRT or its variants. SE-positive peptide 65–79*0401 was added and NO signaling was measured. Data shown are representative of at least two independent identical experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.005, as compared to N-CRT (Figs. 2A and 2B), or WT-CRT (Fig. 2D).

We next determined the relative role of individual P-domain arginines on SE-activated signaling. As can be seen in Figure 2D, the R205A point substitution significantly enhanced SE-activated signaling, while other mutations had no significant effect of SE signaling. Collectively, these findings identify the positive charge of Arg²⁰⁵ as a critical determinant of CRT function. Neutralization of this single charge led to markedly enhanced receptor-ligand interaction and signaling. Thus, deimination of Arg²⁰⁵ is the single most likely protein modification responsible for the effect of global CRT citrullination. Deimination of the 2 other arginines, Arg¹⁶⁰ and Arg²⁶¹, does not appear to contribute meaningfully to the net effect of global CRT citrullination on its SE receptor function.

Over-abundance of Cit-CRT in RA synovium

As a prelude to future investigation of the clinical relevance of the data discussed above, we sought to determine the abundance of Cit-CRT in vivo. Figure 3A shows 2-dimentional protein gels of FLS extracts from representative RA and OA patients blotted with anti-citrulline antibodies. A citrullinated acidic protein spot (PI = 4.3) fitting the expected CRT coordinates was found to be much more abundant in the RA sample compared to the OA sample. When membranes were stripped and blotted with anti-CRT, the identity of that spot was confirmed as CRT. The RA spot showed a unique acidic shift, a feature which is characteristic for citrullinated proteins.

Figure 3. Quantification of synovial Cit-CRT.

A. Representative 2-D immunoblot display of protein citrullination patterns in RA and OA FLS. As shown in the left panel, an acidic protein fitting CRT 2-D coordinates (boxed) was found to be over-expressed in RA. An enlarged image of the spot is shown next to the original image. Membranes were stripped and re-probed with anti-CRT antibodies. The identity of the spot was confirmed as CRT. Enlarged image of the CRT spot (boxed) shows acidic shift in the RA sample, consistent with citrullination. B. Synovial tissues of 4 RA patients and 5 OA patients were obtained during joint replacement surgery. Total protein extracts were separated by PAGE and blotted with anti-CRT and anti-βactin. As shown, the levels of total CRT relative to β-actin in crude ST extracts were not statistically different between RA and OA. C. Cit-CRT index (the ratio between Cit-CRT and total CRT) was determined in ST of 4 RA patients and 5 OA patients. As can be seen, abundance of Cit-CRT in RA ST was significantly higher than in OA samples. D. Abundance of Cit-CRT in RA and OA FLS. Quantification of Cit-CRT in FLS was performed as above. NS, not significant.

There was no statistically significant difference in total CRT abundance between 5 RA and 4 OA ST (Fig. 3B). However, as shown in Fig. 3C, RA ST had a significantly higher relative abundance of Cit-CRT compared with OA tissues. We performed similar analysis on 22 fibroblast-like synoviocyte (FLS) cell lines. As shown in Figure 3D, RA FLS had a significantly higher abundance of Cit-CRT compared to OA FLS.

We have also used MS analysis to determine citrullination changes. Deimination of 1 arginine to citrulline results in a mass shift of 1 Da, which is difficult to detect by standard MS. To overcome this limitation, we used a chemical modification method, which involves a reaction of the ureido group of citrulline with 2,3-butanedione in the presence of antipyrin (29). The results in a characteristic mass shift of +238 Da, which is easier to detect. MS analyses were performed on samples from 5 RA and 4 OA patients (Table 2). There was some variability among individual samples in the repertoire of citrullination sites; however, we could not identify qualitative differences between RA and OA samples in this early study. It remains possible that while there are no qualitative differences between RA and OA in terms of the repertoire of citrullinated sites, there could be a quantitative difference, namely differential abundance of site-specific deimination. Demonstration of such differences is likely to require analysis of a very large number of samples.

DISCUSSION

The basis of RA-SE association and the role of protein citrullination in the disease are both unknown. We have previously demonstrated that the SE is a ligand that interacts with cell surface CRT, at a specific binding site in the receptor P-domain, and transduces innate immune signals. Here we demonstrate that CRT citrullination increased its affinity for the SE ligand and enhanced its signaling potency. By introducing citrullination-mimicking site-specific mutations, we were able to demonstrate that particular arginine residues in the vicinity of the SE binding site play distinct modulatory roles in SE-CRT interaction and signal transduction. Further we demonstrate for the first time that Cit-CRT is overabundant in RA synovial tissues and synovial tissue-derived FLS.

The findings of this study have dual significance: first, they provide important insights into the molecular topology and tri-dimensional confines of SE-CRT interaction; second, they offer a plausible model that could explain the pathogenic role of the SE in RA and the interplay between genetic and non-genetic factors in the disease.

The impact of citrullination on the SE-CRT interaction observed here is consistent with previous reports concerning structural and functional effects of this posttranslational protein modification. Citrullination involves deimination of arginine with resultant conversion to citrulline. Arginine is strongly basic, whereas citrulline is a charge-neutral residue. Citrullination therefore introduces net loss in the protein charge, and consequently could affect interaction with proteins other biomolecules (30–33). In addition to the effect of deimination on the polypeptide primary sequence, citrullinated proteins can undergo secondary conformational changes that influence their access to other molecules (34).

Of the 2 mechanisms by which protein citrullination can affect interaction with other molecules we tend to favor loss of charge as an underlying mechanism. As CD spectra indicate (Supplementary Figure 1), CRT citrullination did not produce a significant conformational change. Further, the R205A mutant, with a single substitution could reproduce the effect of global citrullination, suggesting that a charged moiety near the SE binding site accounts for most of the citrullination effect on CRT-SE interaction. Based on the finding that single substitutions at Arg¹⁶⁰ and Arg²⁶¹ had a neutral or an opposite effect, respectively, we propose that citrullinated Arg²⁰⁵ may have a dominant SE binding enhancing effect, which overrides an opposite effect by the more remote Arg²⁶¹. A proposed model of the spatial role of P-domain arginine residues on SE binding is depicted in Figure 4.

Figure 4. A model of CRT-SE interaction.

The SE ligand (cyan) interacts with Glu²¹⁷, Asp²²⁰ and Glu²²³ (pink), previously identified as the SE binding site in the CRT P-domain (Orange) (8). P-domain Arg²⁰⁵ and Arg²⁶¹ are shown in green. As can be seen, CRT Arg²⁰⁵ side chain points towards the SE, and reaches a distance of 3.1 Å from the SE. The model proposed here predicts that this positively-charged moiety exerts an inhibitory effect on CRT-SE interaction. Citrullination of Arg²⁰⁵ may therefore decrease a putative hindrance and increase binding affinity. Arg²⁶¹, on the other hand, is pointing away from the SE binding site at a predicted distance of 12.9 Å, in an orientation that cannot inhibit SE-CRT interaction. We propose that the enhancing effect of Arg²⁶¹ on SE-CRT binding could be related to charge-based interaction with other parts of the HLA-DR molecule, or stabilizing effect on CRT spatial conformation. The functionally inert residue Arg¹⁶⁰ is located in the N-domain at a distance of 24.7 Å from the SE binding site (not shown).

Consistent with the SE ligand hypothesis (35–37), the affinity of SE-CRT interaction correlated with signaling potency. Cit-CRT which displayed higher affinity for the SE ligand in a cell-free binding assay transduced a markedly more potent signal for production of NO than did N-CRT. Likewise, CRT mutant R205A, which demonstrated higher SE binding, transduced more potent SE signaling, while R160A, which did not significantly affect the interaction with the SE, also did not produce significant signaling effect. An interesting exception to this rule was seen with CRT mutant R261A, which despite its significantly weaker interaction with the SE ligand in cell-free binding assays, maintained signal transduction potency comparable to that of WT-CRT. The reason is unclear, but could conceivably be related to the fact that Arg²⁶¹ side chain is directed away from the SE binding site (Fig. 4). This dichotomy suggests that citrullination of Arg²⁶¹ is less likely to impact SE biologic effects in physiologic conditions. The specificity of the impact of citrullination changes around the SE binding site indicate that augmented SE-CRT binding, rather than non-specific increased signaling by Cit-CRT, is responsible for the observed effect on SE-activated signaling.

We found no measurable differences in the abundance of total CRT between OA and RA samples. However, the fraction of Cit-CRT out of total CRT was significantly higher in RA. These data corroborate and expand on a brief prior observation that Cit-CRT is over-expressed in the serum of patients with early RA (38). The data shown here are the first indication that the synovial tissue itself, the primary disease focus in RA, over-expresses this posttranslationally-modified protein. It is important to note that abundance of citrullinated proteins in pathologic tissue is not unique to RA, and has been reported in many other conditions, such as multiple sclerosis, Alzheimer’s disease and Crohn’s disease (11,39). The cause of excessive protein citrullination in pathologic tissues is unclear, but could be a consequence of cell death (40) or oxidative stress (41).

Citrullinated proteins may contribute directly to the disease process, rather than be only a consequence of inflammation (42). However, different from all other conditions in which citrullinated proteins have been found, in RA these proteins act as neo-antigens and trigger an autoantibody response (ACPA). The strong association between ACPA and RA, the appearance of these autoantibodies prior to disease onset and their association with SE and smoking have led many to suggest that ACPA may play a direct pathogenic role in RA (43). Some evidence in support of this hypothesis has been documented in experimental murine models of arthritis (44, 45).

It has been previously reported that SE-expressing HLA-DR molecules can bind, and present citrullinated peptides (46, 47). Given that observation, could Cit-CRT peptides be preferentially presented by SE-expressing DR molecules in RA and generate ACPA? The data shown here cannot refute or substantiate that scenario. However, it is important to point out that presentation of Cit-CRT peptides is not the underlying mechanism of our findings for several reasons. First we used different allele-specific tetramers, all of which contain a uniform, covalently bound groove CLIP peptide. It is highly unlikely that these tetramers would be able to present other peptides. Likewise, synthetic SE peptides in solution are incapable of antigen presentation. Second, we are not aware of reports of autoantibodies to Cit-CRT, while there are many other Cit-peptides that are known to be immunogenic in RA (citrullinated-fibrinogen, citrullinated-vimentin, etc). We therefore believe it is not necessary to postulate that Cit-CRT might be the autoantigen that drives the anti-CCP response.

This study did not set out to explain the genesis of ACPA, and therefore we cannot determine the cause-effect relationship between SE-activated signaling and ACPA. However, it should be pointed out that in a recent study we demonstrated that the SE-CRT pathway leads to immune dysregulation that diminishes Treg numbers, and reciprocally enhances Th17 expansion (7). It remains to be determined whether SE-mediated immune dysregulatory effect can facilitate autoimmune responses, such as ACPA.

Independent of the putative pathogenic role of ACPA, the data shown here could help to better understand the interaction between inherited and acquired factors in RA. Epidemiologic data strongly suggest that an interaction between genetic (SE) and non-genetic factors may be involved. Here we identified an acquired, possibly environmentally-induced event that amplified substantially the functional effect of a genetically-determined factor, the SE. It is therefore tempting to propose that the SE functional effect in RA may be facilitated by site-specific citrullinated CRT. Thus, in healthy individuals carrying SE-encoding HLA-DRB1 alleles, the SE ligand interacts at low affinity with cell surface CRT that is largely un-citrullinated. This interaction could be beneficial by activating low-grade Th17 polarization (7), which could be advantageous against pathogens (48). As a result of environmental stresses (for example, long-term exposure to cigarette smoke) there could be a gradual activation of PAD with resultant citrullination of many proteins, including CRT. As the abundance of Cit-CRT gradually increases, it could reach a critical threshold, when the affinity to the SE results in exaggerated signaling with resultant pathology. In individuals with the right constellation of genetic risk factors, the combined scenario described above could trigger disease onset.