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ABAP: Antibody-based assay for peptidylarginine deiminase activity

Members of the family of peptidylarginine deiminases (PADs, EC catalyze the posttranslational modification of peptidylarginine into peptidylcitrulline. Citrulline-containing epitopes have been shown to be major and specific targets of
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  ABAP: Antibody-based assay for peptidylarginine deiminase activity Albert J.W. Zendman  a , Reinout Raijmakers  a , Suzanne Nijenhuis  a , Erik R. Vossenaar  a ,Marloes van den Tillaart  a , Renato G.S. Chirivi  b , Jos M.H. Raats  b ,Walther J. van Venrooij  a , Jan W. Drijfhout  c , Ger J.M. Pruijn  a,* a Department of Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences, Institute for Molecules and Materials,Radboud University Nijmegen, Nijmegen NL-6500 HB, The Netherlands b ModiQuest B.V., Nijmegen NL-6525 EN, The Netherlands c Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden NL-2300 RC, The Netherlands Received 16 April 2007Available online 21 July 2007 Abstract Members of the family of peptidylarginine deiminases (PADs, EC catalyze the posttranslational modification of peptidylarg-inine into peptidylcitrulline. Citrulline-containing epitopes have been shown to be major and specific targets of autoantibodies producedby rheumatoid arthritis patients. Recently, the citrullination of histone proteins by PAD enzyme was reported to influence gene expres-sion levels. These findings greatly increase the interest in the PAD enzymes and their activities. A few procedures to monitor PAD activ-ity in biological samples have been described previously. However, these assays either have low sensitivity or are rather laborious. Herewe describe a reliable and reproducible method for the determination of PAD activity in both purified and crude samples. The method isbased on the quantification of PAD-dependent citrullination of peptides, immobilized in microtiter plates, using antibodies that areexclusively reactive with the reaction product(s). Our results demonstrate that this antibody-based assay for PAD activity, called ABAP,is very sensitive and can be applied to monitor PAD activity in biological samples.   2007 Elsevier Inc. All rights reserved. Keywords:  Peptidylarginine deiminase; Peptidylcitrulline; Citrullination; Rheumatoid arthritis; Enzymatic activity; In vitro activity assay; ELISA Peptidylarginine deiminases (PADs, 1 EC are afamily of Ca 2+ -dependent enzymes that posttranslationallyconvert peptidylarginine into peptidylcitrulline. PAD cata-lyzes the hydrolysis of the guanido group of arginine,resulting in the neutral ureido group of citrulline and therelease of ammonia, with concomitant loss of the positivecharge of the converted residue. This citrullination (deimin-ation) of peptidylarginine by PAD is highly dependent oncalcium [1], which on binding alters the conformation of  the catalytic domain of the enzyme to facilitate substrateconversion [2].In mammals, PAD enzymes are encoded by five distinctgenes [3]. These PAD isotypes are expressed tissue specifi- cally. During recent years, there has been increasing inter-est in the effects of protein citrullination srcinating fromdifferent fields of research. The finding that rheumatoidarthritis (RA) patients produce autoantibodies against cit-rulline-containing epitopes (anti-citrullinated protein anti-bodies [ACPAs]) greatly increased interest in the PADenzymes and their activities [4,5]. Recent studies have shown disease-stimulating effects of ACPAs in mouse 0003-2697/$ - see front matter    2007 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2007.07.009 * Corresponding author. Fax: +31 24 3540525. E-mail address: (G.J.M. Pruijn). 1 Abbreviations used:  PAD, peptidylarginine deiminase; RA, rheumatoidarthritis; ACPA, anti-citrullinated protein antibody; DAMO, diacetylmonoxime, BAEE,  N  - a -benzoyl- L -arginine ethyl ester; Bz- L -Arg,  N  - a -benzoyl- L -arginine; AMC, anti-modified citrulline; scFv, single-chainvariable fragment; cDNA, complementary DNA; hPAD, human PAD;RT, reverse transcription; mRNA, messenger RNA; IPTG, isopropyl- b - D -thiogalactopyranoside; NTA, nitrilotriacetate; BSA, bovine serum albu-min; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; IgG,immunoglobulin G; ABAP, antibody-based assay for PAD activity; DTE,dithioerythritol; rPAD, rabbit PAD; TMB, tetramethylbenzidine; ELISA,enzyme-linked immunosorbent assay. Analytical Biochemistry 369 (2007) 232–240 ANALYTICALBIOCHEMISTRY  models [6,7]. Characterization of the structure, function, and regulatory mechanisms of PADs will help to under-stand the process of citrullinated antigen production andthe etiology of RA. When hypothesizing a functional rolefor ACPAs and their corresponding antigens in RA, inter-fering with citrullination by administration of PAD inhib-itors might have a therapeutic effect in RA patients. Also inthe field of gene expression (regulation of histone modifica-tion), the PAD enzymes have drawn attention. Two studieshave suggested a PAD4-dependent demethylation of his-tones (by converting methylated peptidylarginine intopeptidylcitrulline) [8,9]; however, this reactivity has beenquestioned by other studies [10,11].To determine PAD activity in biological samples, theaccess to suitable and sensitive detection methods is crucial.The srcinal methods were based on visualization of thecitrulline moiety of the reaction product. These colorimet-ric methods are all based on the Fearon reaction firstdescribed in 1939 [12]. Since then, many researchers have tried to improve the procedure by testing different productstabilizers and color intensifiers [13]. These optimizations have culminated into a useful colorimetric assay to deter-mine ureido groups (e.g., urea, citrulline, carbamyl) asdescribed by Boyde and Rahmatullah [14]. The method uses diacetyl monoxime (DAMO) in an acidic environment(mixture of phosphoric and sulfuric acid), and the resultingstabilized product is detected by its absorbance at 530 nm.This procedure allows the detection of citrulline at approx-imately 0.1  l mol levels. Using a 96-well format, theDAMO reaction was fitted into a faster, lower volumemethod with increased sensitivity (nanomolar range) [15].Later, Sugawara and coworkers adapted this chemicalmodification method to detect citrullination in peptide-likesubstrates of PADs, namely,  N  - a -benzoyl- L -arginine ethylester (BAEE) and  N  - a -benzoyl- L -arginine (Bz- L -Arg) [16].Often these methods are inconvenient (because they includea 95   C incubation step), are lengthy, and have low sensi-tivity and reproducibility. Moreover, citrulline detectioncan be biased by the presence of urea and free citrullinein the samples to be analyzed.Only recently has the product of the citrulline modifica-tion reaction with 2,3-butanedione (reactive compoundafter hydrolyzation of DAMO) and anti-pyrine in the pres-ence of FeCl 3  been characterized [17]. The product has an additional mass of 238 Da and an absorption maximum at464 nm. This resulting bulky group was used by Senshuand coworkers to raise the anti-modified citrulline(AMC) rabbit polyclonal antibodies, which are routinelyused to detect protein-bound citrulline on Western blot[18]. This AMC method, with femtomolar sensitivity forcitrulline residues in proteins, was used by Nakashimaand coworkers to monitor citrullination after postlytic acti-vation of PAD-containing cells [19].In addition, several other ways to determine citrullinationproducts have been reported. Using an HPLC–fluorometricassay, the interference of endogenous ureido compoundscould be avoided and the sensitivity was increased to picom-olar ranges [20]. However, this assay is time-consuming andis not suited for parallel analyses of multiple samples. Incontrast, the spectrophotometric assay described by Liaoand coworkers allows continuous and parallel analysis withgood sensitivity [21]. In this approach, the liberated ammo- nia from the deimination reaction is used to generateNADH from NAD + in a two-step reaction, where conver-sion can be measured by absorbance at 340 nm. Theenzymes and components used, however, make it hard toadapt this method to biological samples. Alternatively, cit-rullination levels can also be monitored by mass spectrome-try (MS) [11]. Although rather laborious, an advantage of  MS is that sometimes the exact position of citrulline in aconverted substrate can be deduced. This can either be onthe basis of a +1 Da mass shift in combination with lossof a trypsin digestion site or be facilitated by the incorpora-tion of oxygen-stable isotopes (using H 218 O) [22].Here we describe a reliable and reproducible method forthe simultaneous analysis of multiple PAD activity-con-taining samples. This method is based on the specific recog-nition of a citrullinated epitope in a defined peptideresulting from conversion by PAD by an anti-peptidylcit-rulline antibody. We show that this method is as sensitive(picomolar range) as the HPLC method and can be appliedto monitor PAD activity in biological samples and to studyPAD inhibitory compounds. Materials and methods Anti-citrulline antibodies Single-chain variable fragments (scFvs) against citrullylepitopes (citrulline in protein context) have been generatedpreviously [23]. In the current study, we used the RA3 scFv, which is known to be reactive with filaggrin-derivedcitrullinated peptides. The RA3 scFv complementaryDNA (cDNA) was fused to a sequence encoding a humanC-kappa domain and cloned in pUC119. Bacterially pro-duced periplasmic RA3 scFvs were isolated as describedpreviously [23]. Rabbit polyclonal AMC antibodies were a kind gift from T. Senshu [18]. Expression and purification of recombinant PAD in prokaryotic cells Human PAD2 (hPAD2, AB023211 kindly provided bythe Kazusa Institute, Japan) and human PAD4 (hPAD4,cloned by reverse transcription [RT]–PCR from spleenmessenger RNA [mRNA]) coding sequences, cloned intothe pET16b expression vector (Novagen), were expressedas N-terminally His-tagged proteins in  Escherichia coli  BL21(DE3) pLysS overnight at 25   C after induction with0.4 mM isopropyl- b - L -thiogalactopyranoside (IPTG). Allsubsequent steps were performed at 4   C. Recombinantproteins were purified by Ni 2+ /nitrilotriacetate (NTA)affinity purification following the manufacturer’s protocol(Novagen). After binding, columns were washed extensively Antibody-based assay for PAD activity / A.J.W. Zendman et al. / Anal. Biochem. 369 (2007) 232–240  233  (buffer containing 20 mM imidazole) and subsequentlyeluted with 250 mM imidazole (in washing buffer). Proteinpurity was checked by SDS–PAGE and staining with Coo-massie Brilliant Blue. Enzyme samples were stored in ali-quots at   70   C in storage buffer (20 mM Tris–HCl [pH7.4], 10 mM  b -mercaptoethanol, 100 mM NaCl, and 10%glycerol) until use. Tissue sample extract Murine (C57BL/6-129S) tissue samples were obtainedfrom the animal facility at Radboud University Nijmegen.Fresh tissue samples, kept on ice, were homogenized in100  l l of lysis buffer (20 mM Tris–HCl [pH 7.4], 10 mM b -mercaptoethanol, 100 mM NaCl, and 10% glycerol, sup-plemented with Complete EDTA-free protease inhibitormix [Roche Biochemicals, one tablet/50 ml]) per 100 mgtissue using a 100  l m cell strainer (BD Falcon). After son-ication, samples were centrifuged for 30 min at 13,000  g   at4   C. Supernatants were used immediately in the activityassay. Protein detection Sample protein concentrations were determined withBradford reagent (Bio-Rad) using a calibration curve of bovine serum albumin (BSA). In brief, 200  l l of reagentwas added to a 25  l l sample and incubated for 15 min atroom temperature. Subsequently, the absorbance at595 nm was measured using a Tecan Sunrise absorbancereader. Western blotting  Proteins were separated by SDS–PAGE (13% gels), fol-lowed by electroblotting to Hybond C extra nitrocellulosemembranes (Amersham Biosciences). Blotting and loadingwere checked by Ponceau S staining. The blots were chem-ically modified prior to immunostaining as described previ-ously [18]. After a 2 h blocking step (phosphate-buffered saline [PBS] containing 5% nonfat dried milk and 0.1%Nonidet P-40), the blots were incubated for 3 h with theAMC polyclonal antibody (1:1600 in blocking buffer). Fol-lowing extensive washing in blocking buffer, bound anti-bodies were visualized by incubation with horseradishperoxidase (HRP)-conjugated swine–anti-rabbit immuno-globulin G (IgG) antibodies (1:1000, Dako), washing (threetimes PBS with 0.1% Nonidet P-40 and one time with PBS),and chemiluminescence. ABAP: Antibody-based assay for PAD activity Filaggrin-based synthetic peptides, containing arginine(R) (Arg-peptide: SHQESTRGKSKGKAAAAA) or citrul-line (X) (Cit-peptide: SHQESTXGKSKGKAAAAA), werebound to 96-well microtiter plates. The incubation volumein all steps was 100  l l per well. Unbound peptide wasremoved by washing two times with 50 mM Tris–HCl (pH7.6) in the presence of 500 mM NaCl (TBS 500 ) and one timewith TBS (same as TBS 500  except with 150 mM NaCl). TBSwas used instead of PBS to avoid calcium phosphate precip-itation during subsequent incubation with Ca 2+ -containingsamples. The immobilized peptides were incubated withPAD-containing samples in citrullination buffer (40 mMTris–HCl [pH 7.6], 5 mM CaCl 2 , and 1 mM dithioerythritol[DTE]) or in control buffer lacking calcium (40 mM Tris– HCl [pH 7.6], 2 mM EDTA, and 1 mM DTE). The PADsample volume never exceeded 25  l l to limit buffer varia-tions. Purified rabbit muscle PAD enzyme, rPAD (Sigma),was used in PAD calibration curves and for initial optimiza-tion of the procedure. One unit of PAD activity was definedpreviously as the amount of enzyme that produces 1  l mol of  N  - a -benzoyl-citrulline ethyl ester from BAEE per hour at55   C at pH 7.2 [24]. After 1 h incubation at 37   C, plateswere washed five times with TBS 500  and 0.5% Tween 20(TBS 500 T). The plates were then incubated (either for 1 hat 37   C or overnight at 4   C) with anti-citrulline scFvRA3 diluted in PBS containing 0.5% Tween 20 and 1%BSA, followed by extensive washing in PBST (PBS contain-ing 0.05% Tween 20). Subsequently, the plates were incu-bated for 1 h at 37   C with rabbit anti-human Ig-kappaconjugated to HRP (1:2000 in PBS/0.5% Tween 20/1%BSA, Dako). After thorough washing with PBST, boundantibodies were visualized by the conversion of tetramethyl-benzidine (TMB) substrate according to standard proce-dures. After 5 min, the reaction was stopped by theaddition of an equal volume of 2 M H 2 SO 4 . The absorbanceat 450 nm (Tecan Sunrise Absorbance Reader) was used tomonitor product formation. All experiments were per-formed at least in duplicate unless specified otherwise.For the PAD inhibition experiment, recombinant hPAD4(0.1  l l, corresponding to 12 mU PAD activity) was preincu-batedfor 30 min at37   C in100  l l of PAD incubation bufferwith  N  - a -benzoyl-N 5 -(2-chloro-1-iminoethyl)- L -ornithine ethylester, a molecule similar to a known PAD-inhibiting com-pound,  N  - a -benzoyl-N 5 -(2-chloro-1-iminoethyl)- L -orni-thine amide [25]. This mixture was then applied to the coated enzyme-linked immunosorbent assay (ELISA) wellsand further processed as described above. Results ABAP optimization Because the available methods for the determination of PAD activity are either relatively insensitive or laborious,we developed a new method that is based on the recogni-tion of peptidylcitrulline by anti-citrulline scFv antibodies.An arginine-containing peptide, derived from a citrullinat-ed filaggrin epitope that is recognized by the scFv RA3 [23],is immobilized on a microtiter plate and incubated withPAD. The conversion of this peptide subsequently isdetected with the citrulline-specific scFv RA3. Bound 234  Antibody-based assay for PAD activity / A.J.W. Zendman et al. / Anal. Biochem. 369 (2007) 232–240  RA3 is finally visualized with standard ELISA proceduresfollowed by spectrophotometric quantification.The first parameters to be optimized were the amount of peptide bound to the well and the dilution of the scFv RA3to be used for detection. The plates were incubated withincreasing amounts of the Cit-peptide, varying from 1 to1000 ng per well. After coupling and washing, the plateswere incubated with a dilution series of RA3. The results,depicted in Fig. 1, show that a maximal signal is reachedat 50 ng Cit-peptide in combination with a 10-fold dilutionof RA3. The detection limit ranged between 5 and 10 ng of Cit-peptide. Note that Fig. 1 does not represent a substratesaturation curve; rather, it shows only the maximal amountof citrullinated peptide that can be detected in this system.For all subsequent experiments, we coated 100 ng Arg-pep-tide (two times coating capacity to ensure maximal coating)and used 10-fold diluted RA3.To determine whether the Arg-peptide could be con-verted by PAD to detectable levels of Cit-peptide, we incu-bated the Arg-peptide-coated plates with increasingamounts of purified rPAD in the presence of either calciumor EDTA (as a negative control). The subsequent incuba-tion with RA3 was performed either overnight at 4   C orfor 1 h at 37   C. The results showed that the Arg-peptidewas efficiently converted by rPAD in a dose- andCa 2+ -dependent manner (Fig. 2). A comparison of the Fig. 1. Detection of the citrullinated peptide by scFv RA3. Increasing amounts of citrullinated peptide were immobilized and detected with a dilutionseries of scFv RA3 (indicated with different symbols). Bound RA3 was detected with an HRP-conjugated secondary antibody using TMB as a substrateand absorbance measurement at 450 nm. 01234 0 0.11 10 rPAD (mU)       O      D      4     5     0 1 h 37 ° C Calcium overnight 4 ° C Calcium1 h 37 ° C EDTAovernight 4 ° C EDTA Fig. 2. Optimization of PAD activity detection. Increasing amounts of purified rPAD were used to convert coated arginine-containing peptide. Theproduct (citrullinated peptide) was detected using either 1 h (37   C) or overnight (4   C) incubation with the RA3 antibody (indicated by different shades of gray). As a negative control, incubations in the presence of EDTA were performed in parallel. Antibody-based assay for PAD activity / A.J.W. Zendman et al. / Anal. Biochem. 369 (2007) 232–240  235  efficiency of Cit-peptide detection with the two differentRA3 incubation conditions revealed that the overnightincubation was at least 10 times more efficient than the1 h incubation. In all subsequent experiments, we usedthe overnight RA3 incubation at 4   C.For quantitative enzyme activity assays, it is importantto know the minimal amount of enzyme required and theconcentration range that leads to reliable quantitativedata. To establish the sensitivity of the ABAP, a dilutionseries of rPAD was analyzed. The results in Fig. 3 showthat a minimum of 0.01 mU rPAD is required for thedetectable conversion of the Arg-peptide and that themaximal level of conversion is reached when incubatingwith approximately 0.5 mU rPAD. Within this range, alinear relation was observed between the OD 450  and thelogarithm of the concentration of PAD enzyme (Fig. 3:OD 450  = 0.1487 * log(rPAD) + 1.9698). ABAP applications PAD enzymes require calcium as an essential cofactorfor their activity [24]. To study this dependence, an incubation with hPAD2, hPAD4, and rPAD was per-formed with a concentration range of calcium varyingbetween 2 and 5000  l M. The results in Fig. 4 show thatthe activity of rPAD was half-maximal at approximately70  l M Ca 2+ . The CaCl 2  concentration for half-maximalactivity of hPAD2 was comparable to that of rPAD(80  l M) and somewhat higher than that of hPAD4(40  l M). Fig. 3. Dynamic range of PAD activity in ABAP test. Varying amounts of rPAD were used to determine the minimal detectable amount of PAD enzyme.Incubations were performed in the presence of either calcium (squares) or EDTA (triangles). The PAD activity detected (OD 450 ) when using between 0.02and 0.5 mU enzyme was linear when plotted against the amount of enzyme on a log scale (OD 450  = 0.1487 * log (rPAD) + 1.9698).Fig. 4. Calcium-dependent activity of PAD enzymes. Three different PAD enzymes—rPAD (squares), hPAD2 (triangles), and hPAD4 (circles)—wereincubated in the ABAP test in the presence of increasing concentrations of CaCl 2 . Half-maximal activities were obtained (dotted lines) using the averageOD 450  value of the background and maximal signals (both indicated with arrows).236  Antibody-based assay for PAD activity / A.J.W. Zendman et al. / Anal. Biochem. 369 (2007) 232–240
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