Identification of Carbonic Anhydrase I Immunodominant Epitopes

Aug 3, 2010 - Academy of Sciences, Bratislava, Slovakia, Department of ... Marrow Transplantation Unit, National Cancer Institute, Bratislava, Slovaki...
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Identification of Carbonic Anhydrase I Immunodominant Epitopes Recognized by Specific Autoantibodies Which Indicate an Improved Prognosis in Patients with Malignancy after Autologous Stem Cell Transplantation Ludovit Skultety,†,‡ Barbora Jankovicova,§ Zuzana Svobodova,§ Pavel Mader,| Pavlina Rezacova,| Maria Dubrovcakova,⊥ Jan Lakota,*,⊥,# and Zuzana Bilkova*,§ Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovakia, Centre of Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia, Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic, Department of Structural Biology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic, Laboratory of Molecular Oncology, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia, and Bone Marrow Transplantation Unit, National Cancer Institute, Bratislava, Slovakia Received May 15, 2010

This work employs an epitope mapping of carbonic anhydrase (CA), isoform I (CA I), for detection of the main immunodominant epitopes. Our interest has arisen from an observed spontaneous tumor regression in patients who developed an aplastic anemia type syndrome after a high-dose therapy with autologous stem cell transplantation and whose sera contained high titer of anti carbonic anhydrase (anti-CA) autoantibodies. There are many indications that the presence of these autoantibodies may provide significant survival benefit for the patients. Western blot analysis confirmed strong immunoreactivity of the patients’ sera with several CA isoforms and the CA I has been selected for our study as a highly abundant and widely distributed isoform. The applied analytical approach consists of specific fragmentation of CA I protein followed by immunospecific isolation of peptides reacting with polyclonal anti-CA I autoantibodies of patients in spontaneous remission. We improved the standard epitope mapping schema by incorporating the benefits of magnetic carriers and biomagnetic separation techniques. Mass spectrometry has been applied for detection and identification of epitopes and the acquired results were verified by bioinformatic tools. The candidate epitopes of CA I (NVGHS, DGLAV, SSEQL, and SLKPI) are discussed herein as potential therapeutic targets. This work highlights the usefulness of the epitope mapping technique based on magnetic microspheres for effective and rapid determination of immunodominant epitopes of the target protein. Keywords: epitope mapping • carbonic anhydrase I • anti-CA autoantibodies • LC-MS/MS • bioinformatic tools • autologous stem cell transplantation • spontaneous remission

1. Introduction Mammalian carbonic anhydrases (CAs, EC 4.2.1.1) are widely distributed Zn(II) metalloenzymes that catalyze the reversible hydration of carbon dioxide to produce a bicarbonate anion and a proton (CO2 + H2O T HCO3- + H+).1 This reaction regulates a broad range of physiological procceses. In humans, 15 different CA isoenzymes are presently known, and many of them are quite recent discoveries compared with the physiologically abundant and widely distributed isoenzymes CA I * To whom correspondence should be addressed. E-mail: (Z.B., for analytical part) [email protected]; (J.L. for clinical part) Jan.Lakota@ nou.sk. † Institute of Virology, Slovak Academy of Sciences. ‡ Centre of Molecular Medicine, Slovak Academy of Sciences. § University of Pardubice. | Academy of Sciences of the Czech Republic. ⊥ Cancer Research Institute, Slovak Academy of Sciences. # Bone Marrow Transplantation Unit, National Cancer Institute. 10.1021/pr1004778

 2010 American Chemical Society

and II.1-4 Association of two CA isoenzymes (CA IX and XII) with cancer has been confirmed and the involvement of others is not excluded and is a subject of a contemporary research. The expression profile of CA IX and XII was successfully applied for discrimination of cancerous cells (overexpression) from healthy cells (minimal expression).4 Clinical modulation of CA activity with inhibitors has proven a reliable treatment for a range of human disease states.5 Sulfonamide based CA inhibitors were also shown to inhibit the growth of several tumor cell lines in vitro and in vivo, representing thus interesting leads for development of novel antitumor therapies.6 High-dose therapy (HDT) with autologous stem cell transplantation (ASCT) is the standard treatment for patients with chemosensitive relapsed/refractory malignant diseases such as Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, multiple myeloma, and others.7-10 There is an increasing evidence that autoimmunity can inhibit growth of solid tumors and that Journal of Proteome Research 2010, 9, 5171–5179 5171 Published on Web 08/03/2010

research articles antitumor activity also operates in autoimmunity against hematopoetic stem cells in acquired aplastic anemia (AA).11 The presence of autoantibodies in the sera of patients who relapsed after HDT with ASCT was described by Lakota et al.12 In these patients, who developed aplastic anemia type syndrome after the therapy, spontaneous tumor regression was observed and blood serum analysis revealed high titer of antiCA autoantibodies. The specificity of the antibodies was confirmed by repeated screenings of the patients’ sera by Western blot analysis with human blood cell lysate. The presence of specific IgG molecules, the dynamics of their production, and the total amount in serum directly correlated with the course of the disease. The sera of patients in the disease remission contained specific autoantibodies in a sufficiently high titer and the decreased signal or even negative results of Western blot analysis corresponded with the relapse of the disease. These anti-CA autoantibodies produced after transplantation of autologous hematopoietic cells seem to be the main factor in the transition of the disease to remission and their presence in serum represents a significant survival benefit.12 The foregoing results of Western blot analysis confirmed the immunoreactivity of the patients’ sera autoantibodies with several CA isoforms (CA I, II, IX and XII). The mapping of immunodominant epitope(s) on CA I is a subject of this study. The knowledge of the epitope sequence is essential as this information has a potential to be applied consequently in clinical practice not only for diagnostic tests, but also for therapeutic purposes. The epitope mapping is an analytical approach used for identification of the immunodominant structures that are able to be recognized by the immune system, reactive T and B cells, or specific antibodies. A number of in vivo and in vitro methodologies can be used for epitope localization.13 In this work, we apply a traditional epitope mapping technique14 improved by the employment of magnetic carriers. Our strategy was based on exploitation of the benefits of magnetic microspheres, such as large specific surfaces for chemical binding, fast and simple handling, effective separation by permanent magnets, good flow characteristics, and mechanical and chemical stability.15 Furthermore, the use of biofunctionalized magnetic carriers can minimize the analysis time, and the amount of reagents and decreases the loss of analytes. This technique also eliminates contamination by autolytic fragments of proteases, increases the binding efficiency of desired proteins/ peptides, and overall lowers the cost of the analysis. Our novel approach consisted of two steps: (i) the specific fragmentation of the target protein with proteolytic enzymes covalently bound to magnetic microspheres and (ii) the immunomagnetic separation of generated peptides based on specific molecular recognition. Tandem mass spectrometry MS/MS was applied for detection and identification of the peptides. The effectiveness of such strategy has been suggested in our previous work.16 We are profiting from our longstanding experience with magnetic micro- or nanospheres, their surface modifications, and their application in biotechnology and protein analysis.16-22 To retain the highest biological activity, we have immobilized the ligands in an oriented manner enabling the steric accessibility of the majority of the active or binding sites. The identification of the main CA I epitopes binding specifically to the anti-CA autoantibodies and matching these results with data obtained by bioinformatic tools were the principal 5172

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Skultety et al. objectives of this research. We assume that these new data may reveal the etiology of the disease and may clarify the mechanism of tumor growth inhibition in relation to the appearance and dynamics of autoantibody formation and to the disease prognosis of the monitored patients. Tumor control in relation to autoimmunity has been observed in a variety of malignancies.11

2. Experimental Section 2.1. Chemicals and Reagents. Carbonic anhydrase I, from human erythrocytes (CA I), trypsin (EC 3.4.21.4, 13 000 IU mg-1 solid), R-chymotrypsin, from bovine pancreas, Type II (EC 3.4.21.1, 83.9 IU mg-1 solid), DL-dithiothreitol (DTT), formic acid (FA), [Glu1]-fibrinopeptide B (GFP), iodoacetamide (IAA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride(EDAC), benzamidine (BA), NR-benzoyl-DL-arginine p-nitroanilide hydrochloride (BAPNA), N-succinyl-L-phenylalanine-p-nitroanilide (SUPHEPA), sodium cyanoborohydride (NaCNBH3), Ponceau S, and D-glyceraldehyde were products of Sigma-Aldrich (St. Louis, MO). N-Hydroxysulfosuccinimide sodium salt (Sulpho-NHS), ammonium hydrocarbonate, and trifluoroacetic acid (TFA) were from Fluka (Buchs, Switzerland). RapiGest SF was supplied by Waters (Milford, MA). Acetonitrile (LiChrosolv quality) and water (LiChrosolv quality) were from Merck (Darmstadt, Germany). Perloza MT 500 (80-100 µm) was produced by Severoceske chemicke zavody (Lovosice, Czech Republic). One micrometer silica superparamagnetic nonporous microparticles with carboxyl and hydrazide moieties (SiMAG-Carboxyl and SiMAG-Hydrazide) were supplied by Chemicell (Berlin, Germany). Albumin from bovine serum, fraction V, pH 7.0 (BSA) was obtained from AppliChem (Darmstadt, Germany). Blot Quantified BSA, anti-human IgG (H&L) AP conjugate, and BCIP-NBT reagent were produced by Promega (Madison, WI). Human patient serum was provided by Dr. Lakota (National Cancer Institute, Bratislava, Slovakia) after signed patient informed consent. 2.2. Instrumentation. Magnetic separator (Dynal, Carlsbad, CA), UV/vis spectrophotometer Libra S22 (Biochrom, Cambridge, U.K.), Mini-PROTEAN 3 electrophoresis system, Biorad Model Mini Trans-blot Cell, BioLogic LP chromatography system and 1 × 5 cm (10 cm) columns (Bio-Rad, Hercules, CA), electrophoresis system Mini-Vertical Units (Amersham, U.K.), MicroSpin G-25 Column (GE Healthcare, Buckinghamshire, U.K.), Millex Filter, 0.45 µm (Millipore, Billerica, MA), nitrocellulose membranes (Schleicher Schuell, Du ¨ ren, Germany), nanoAcquity UPLC (Waters, Milford, MA), and Q-Tof Premier (Waters, Milford, MA). 2.3. Patient’s Serum. The serum of a patient with multiple myeloma after HDT and ASCT treatment (performed according to standard European Bone Marrow Transplantation group protocols employed at the National Cancer Institute) was utilized in this work for isolation of specific anti-CA I antibodies. Written and informed consent was obtained from the patient according the institutional guidelines. 2.4. Immobilization of Proteolytic Enzymes onto Magnetic Microparticles with Carboxyl Moieties. A one-step carbodiimideprocedurebasedonEDAC/Sulpho-NHS-coupledreaction19,23 was used for the immobilization of proteolytic enzymes (9 mg), trypsin or R-chymotrypsin, onto SiMAG-Carboxyl microparticles (3 mg). Immobilization was carried out overnight at 4 °C under stirring, and in the case of trypsin in the presence of 3.8 mM benzamidine (BA). Resulting proteolytic activity was

CA I Epitopes Recognized by Specific Autoantibodies determined using low-molecular weight substrates: BAPNA for trypsin and SUPHEPA for R-chymotrypsin. 2.5. Digestion of Unfolded CA I by Immobilized Proteolytic Enzymes. For improving the sensitivity to proteolysis, CA I was unfolded by RapiGest SF combined with DTT and IAA. Unfolded CA I (1.5 mL, 1 mg/mL) was added to SiMAGCarboxyl microparticles (2.9 mg) with immobilized proteolytic enzyme (see Section 2.4), the molar ratio E/S was 1:60. The mixture was incubated at room temperature (RT) under stirring. Efficiency of digestion was verified by Tris-Tricine-SDS-PAGE and LC-MS/MS analysis (see Section 2.9). 2.6. Preparation of Specific Anti-CA I Magnetic Immunosorbent. The preparation of anti-CA I magnetic immunosorbent for specific immunocapturing of CA I fragments comprising the immunodominant epitope(s) consists of three steps: (i) the isolation of the whole IgG fraction from the patient’s serum by Protein G bioaffinity chromatography, (ii) the consequent isolation of specific anti-CA I IgG antibodies from the IgG fraction by affinity chromatography with CA I as an immobilized ligand, and finally (iii) the covalent immobilization of isolated specific anti-CA I IgG antibodies onto SiMAGHydrazide magnetic microparticles. 2.6.1. Isolation of IgG Fraction from the Patient’s Serum. One milliliter of the patient’s serum (see Section 2.3) was diluted with 1 mL of 20 mM phosphate buffer, pH 7.0, filtered using 0.45 µm filter, and applied on Protein G Sepharose column (10 mL) equilibrated with 20 mM phosphate buffer pH 7.0. For capturing of IgG, the flow rate was decreased to 0.8 mL/min. Subsequently, IgG was eluted with 0.1 M glycine buffer pH 3.0 (flow rate 1 mL/min). Elution fractions (2 mL) were neutralized with Tris-HCl buffer pH 9.0 (50 µL), concentrated, homogenized, and immediately used for isolation of specific anti-CA I IgG antibodies. 2.6.2. Isolation of Specific Anti-CA I IgG. Preparation of the Carrier with Immobilized CA I. Perloza MT 500 (5 mL of the sediment) was washed with distilled water (10 × 5 mL) and oxidized with 0.2 M NaIO4 (5 mL) for 80 min at RT in the dark, under stirring. After washing with distilled water (10 × 5 mL), followed by 0.1 M phosphate buffer pH 7.0 with 0.15 M NaCl (10 × 5 mL), protein CA I (2 mg in 5 mL of 0.1 M phosphate buffer pH 7.0 with 0.15 M NaCl) was added and incubated for 10 min at RT under stirring. Then, 35 mg of NaCNBH3 was added and immobilization was carried out overnight at 4 °C under stirring. The binding efficiency was approximately determined from the difference of CA I solution absorbance at 280 nm before and after the immobilization. Isolation of the Specific Antibodies. The carrier with immobilized CA I (5 mL, see above) was packed into the chromatography column (1 × 5 cm) and equilibrated with 0.1 M phosphate buffer pH 7.5 with 0.15 M NaCl (flow rate 1.5 mL/min, 50 mL). The capturing of specific anti-CA I IgG from the total IgG fraction of the patient’s serum (see Section 2.6.1) was applied in 0.1 M phosphate buffer pH 7.5 with 0.15 M NaCl at a flow rate 0.25 mL/min. Specific captured antibodies were eluted with 0.1 M glycine buffer pH 2.8 with 0.15 M NaCl (flow rate 1 mL/min). Pooled elution fractions (1 mL), neutralized with 1 M Tris (50 µL), were concentrated to 1 mL, and immediately immobilized onto SiMAG-Hydrazide magnetic microparticles (see Section 2.6.3). 2.6.3. Preparation of Immunosorbent with Oriented Immobilized Anti-CA I IgG. Specific anti-CA I IgG antibodies (1 mL, see Section 2.6.2) were coupled through carbohydrates located on the Fc fragment of the antibody to SiMAG-Hydrazide

research articles magnetic microparticles (5 mg) in an oriented way. Before immobilization, antibodies were oxidized with 20 mM NaIO4 in the dark at RT for 30 min and then transferred by MicroSpin G-25 Column into a coupling buffer (i.e., 0.1 M phosphate buffer pH 7.0). The binding of IgG to the microparticles was achieved overnight at RT under stirring. For blockage of the remaining reactive groups, 0.1 M D-glyceraldehyde in a coupling buffer (0.5 mL) was applied for 1 h at RT under stirring. The binding efficiency was estimated from the difference of antibody solution absorbance at 280 nm before and after immobilization and also by Western blot analysis (see Section 2.8). 2.7. Immunomagnetic Isolation of CA I Specific Fragment(s). A sample of CA I digested by trypsin or R-chymotrypsin (0.5 mL, 1 mg/mL) (see Section 2.5) was applied to the anti-CA I immunosorbent (5 mg, see Section 2.6.3), equilibrated in 0.1 M phosphate buffer pH 7.0. After incubation for 1 h at RT under stirring, immunosorbent was washed first with 0.1 M phosphate buffer pH 7.0 with 0.2 M NaCl (10 × 1.5 mL), then 0.1 M phosphate buffer pH 7.0 with 1 M NaCl (5 × 1.5 mL), and finally with 0.01 M phosphate buffer pH 7.0 (5 × 1.5 mL). Consequently, we performed 3 repeated elutions with 0.05% (v/v) TFA (500 µL, pH 2.4) for 15 min at RT under stirring, followed by 2 elution steps with 0.05% (v/v) TFA (2 mL, pH 2.4) for regeneration of the immunosorbent. Separate elution fractions concentrated to 20 µL were analyzed by LC-MS/MS technique (see Section 2.9). 2.8. Western Blot Analysis. CA I was dissolved in a solution containing 8% (w/v) SDS, 20 mM Tris-HCl buffer pH 6.8, 40% (v/v) glycerol, and 10 mM EDTA. After the electrophoretic separation in 12.5% SDS-PAGE slab gel, the pure protein was transferred to the nitrocellulose membrane. The quality of protein transfer was controlled by staining with 0.1% Ponceau S in 5% acetic acid. The membranes blocked with 1% Blot Quantified BSA were incubated overnight with diluted serum fractions. After washing, the membranes were incubated with anti-human IgG (H&L) AP conjugate at a 5000-fold dilution and anti-CA I antibodies were detected with BCIP-NBT reagent. 2.9. Mass Spectrometric Analysis. Peptide mixtures eluted from the immunosorbent were analyzed by automated nanoflow RP-UPLC system coupled to a Q-Tof Premier (Waters, Milford, MA) tandem mass spectrometer (LC-MS/MS). Peptides injected onto a reverse-phase column (nanoAcquity UPLC column BEH 130 C18, 100 µm × 150 mm, 1.7 µm particle size) were separated using the acetonitrile gradient (3-50% B in 15 min; A ) water with 0.1% (v/v) formic acid, B ) acetonitrile containing 0.1% (v/v) formic acid) at a flow rate of 350 nL/ min. The column was directly connected to the PicoTip emitters (New Objective) mounted into the nanospray source of a Q-TOF Premier instrument (Waters, U.K.). A nanoelectrospray voltage of 3.5 kV was applied, with the source temperature set to 70 °C. For protein identification, a novel multiplex approach called MSE was used.24 This method uses an integrated approach of parallel, alternating scans at low-collision energy, to obtain precursor ion information, and high-collision energy, to obtain full-scan accurate mass data in a single run. The spectral acquisition scan rate was 0.6 s with a 0.1 s interscan delay. In the low energy MS mode, data were collected at constant collision energy of 3 eV. In elevated energy MS mode, the collision energy was ramped from 20 to 35 eV during each integration. The MS spectra obtained at different collision energies were stored separately. During data acquisition, the quadrupole analyzer was not mass selective, but Journal of Proteome Research • Vol. 9, No. 10, 2010 5173

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Figure 1. Schematic of analytical approach used in this work. (A) fragmentation of CA I after the initial unfolding using RapiGest SF, DTT, and IAA with immobilized trypsin or R-chymotrypsin; (B) preparation of anti-CA I magnetic immunosorbent consisting of the isolation of whole IgG fraction from the patient’s serum using bioaffinity chromatography with Protein G, consequent isolation of the specific anti-CA I IgG antibodies by CA I affinity chromatography, and the final immobilization of isolated specific anti-CA I IgG antibodies onto magnetic microparticles with hydrazide moieties; (C) specific immunocapturing of CA I fragments comprising the immunodominant epitope(s) using an anti-CA I carrier followed by mass spectrometric analysis.

operated in the radio frequency only mode. Thus, all ions were passed to the TOF analyzer. This yielded exact mass fragment ions that were potentially observed for every peptide precursor ion present in the low-energy TOF data set. The obtained MS/MS data were processed using the ProteinLynx Global Server v. 2.4 (Waters, U.K.) that provided background-subtraction, smoothing, centroiding and deisotoping. All data were lockspray calibrated against [Glu1]fibrinopeptide (GFP) using data collected from the reference line during acquisition. The lockmass-corrected, centroided and deisotoped data were charge-state reduced to produce a single accurately mass measured monoisotopic mass for each peptide and the associated fragment ion. The initial correlation of a precursor and possible fragment ion was achieved by means of time alignment. The resulting data were searched against human CA I database (http://www.uniprot.org/uniprot/P00915). Variable modifications of carbamidomethyl-C, oxidation M, deamidation Q, deamidation N, acetylation N-terminus were specified. One missed cleavage site was allowed. Protein search parameters also included a 50 ppm tolerance against the database-generated theoretical peptide ion masses, detection of at least three fragment ions per peptide, minimum of 1 matched peptide, and the identification of the protein in at least 2 out of 3 technical replicates. 2.10. Bioinformatic Analysis. The multiple sequence alignment of human isoenzymes CA I, CA II, CA IX, and CA XII was performed using ClustalX.25 To analyze the position and accessibility of experimentally detected peptides, protein structure of CA I, deposited in the Protein Data Bank (PDB) under the accession code (PDB code 1AZM)26 was used. To quantify the solvent accessibility of the peptides, for each residue, its solvent accessible surface area (ASA) within the protein structure was calculated using the PISA server27 and expressed as a percentage of a residue total ASA. 5174

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3. Results and Discussion This study performs epitope mapping of carbonic anhydrase I (CA I, EC 4.2.1.1) with the intention to find its immunodominant epitope(s). It follows the results of the research, which has repeatedly verified the presence of high titers of anti-CA autoantibodies in the sera of patients with spontaneous regression after HDT and ASCT therapy. Immunoreaction in Western blot analysis with the CA isoforms differed in sera obtained from patients with various types of the disease. Sera of Hodgkin’s disease patients reacted with CA I, II, and XII, while sera of multiple myeloma patients reacted with the CA I, II, XII, and IX. Presence of these antibodies in sera represented a significant survival benefit for patients.12 We have utilized the patients’ antibodies for the identification of immunodominant epitopes of CA I using an epitope mapping algorithm composed of the effective fragmentation of CA I using magnetic microspheres with covalently bound trypsin and subsequent bioaffinity capturing of signature peptides corresponding to prospective immunodominant epitope(s) of CA I using a magnetic carrier with the patients’ anti-CA I IgG molecules. Schematic representation in Figure 1 describes all steps in the sequence as they were realized. Mass spectrometry has been applied for detection and identification of epitopes and the acquired results were verified by bioinformatic tools. In comparison with other methods used for the characterization of epitopes, such as random phage epitope library screening, site-directed mutagenesis or utilization of overlapping peptides in combination with competitive ELISA, the mentioned technique is considered to be rapid and it can be applied also for the identification and characterization of discontinuous epitopes.28 Within the framework of this strategy, there are several critical steps. First, it is necessary to obtain a sufficiently pure antigen sample. The enzymatic fragmentation of this antigen

CA I Epitopes Recognized by Specific Autoantibodies

research articles enzymes, in comparison to the standard procedure using a soluble form, offer several benefits such as high proteolytic activity, high operational and storage stability, reduction in digestion time, absence of autolytic fragments in the peptide mixture, and low frequency of missed cleavages.19

Figure 2. Tris-Tricine-SDS-PAGE. Gel, 16.5% T, 3% C (MW 1-70 kDa, %T, % concentration of both monomers, acrylamide and bis-acrylamide; %C, % concentration of cross-linker relative to the concentration of T), detection with silver staining. Lanes: (1) nondigested native CA I (5 µg), (2) nondigested unfolded CA I (5 µg), (3) unfolded CA I digested 3 h by immobilized trypsin (5 µg), (4) molecular marker (10-250 kDa).

has to be effective and specific. In the next steps, the isolation of polyclonal anti-CA I autoantibodies with desired affinity and their proper immobilization to the carrier are important for obtaining efficient immunosorbent for subsequent capturing of CA I peptides. To carry out an effective and reproducible fragmentation of the native antigen (first step), the proteolytic enzyme trypsin, which digests at the carboxyl side of the amino acids Lys or Arg, was immobilized to magnetic microspheres SiMAGCarboxyl via a 1-step carbodiimide method, which is a binary covalent binding system guaranteeing a good reproducibility. The reversible inhibitor benzamidine, present in the binding mixture (see Section 2.4), temporarily protects the enzyme against autoproteolysis. The final activity of the immobilized trypsin (1.24 × 103 IU/mg of carrier) was estimated with lowmolecular weight substrate BAPNA. This carrier with defined proteolytic activity was used for digestion of folded or unfolded CA I as the target protein. The efficiency and specificity of fragmentation was controlled by MS/MS analysis. Immobilized

The time of digestion required optimization; the intervals 15, 30, 45, 60, 90, 120, 150, 180 min and 24 h were tested and the efficiency of cleavage was confirmed by Tris-Tricine-SDSPAGE (Figure 2) followed by LC-MS/MS analysis (Figure 3). We observed high resistance of native CA I to enzymatic digestion. The results of Tris-Tricine-SDS-PAGE demonstrated only partial fragmentation of native CA I even after 24 h. It can be explained by a compact globular structure of the native protein. On the basis of these findings, CA I was unfolded by reductive alkylation using DTT and IAA in combination with RapiGest SF reagent to make it more sensitive to enzymatic proteolysis (see Section 2.5). For denatured molecules of CA I, the intermediate peptides were found as early as after 15 min of digestion. On the basis of the MS/MS analysis, the period of 3 h was estimated as sufficient for efficient fragmentation and production of peptides of appropriate length. Eleven specific peptides out of the 15 peptides (i.e., 70.9% coverage), which can be theoretically produced by trypsin digestion, were confirmed in the range of MW 600-5000 (Figure 3A). To perform the last step of peptide mapping by means of bioaffinity capturing of peptides corresponding to potential epitopes of CA I (Figure 1C), a magnetic carrier with immobilized anti-CA I IgG molecules was prepared. The IgG molecules were isolated from the human patient’s serum using Protein G-Sepharose affinity chromatography (Figure 4A). Elution fractions were pooled, concentrated to 10 mL, and applied for a specific isolation of anti-CA I antibodies using macroporous bead cellulose (Perloza MT 500) carrier with immobilized CA I as an affinity ligand (Figure 4B). The immobilization of CA I on the beads was performed by method described previously by Turkova et al.29 This method is based on periodate oxidation of the carrier and reduction of the Schiff base to a secondary amine by sodium cyanoborohydride (see Section 2.6.2). The binding capacity of prepared bioaffinity carrier, estimated from a depletion of protein from solution after immobilization, was 11 nmol/mL. Control by SDS-PAGE using 10% separating gel was also performed.

Figure 3. The coverage map of CA I peptides (A) using trypsin for its digestion, (B) using R-chymotrypsin for its digestion; detected peptides are highlighted. Journal of Proteome Research • Vol. 9, No. 10, 2010 5175

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Figure 4. Procedure of anti-CA I immunosorbent preparation. (A) LC chromatogram of IgG isolation from 1 mL of the human patient’s serum by affinity chromatography with Protein G bound on Sepharose carrier (10 mL), BioLogic low-pressure chromatography system, and chromatography column 1 × 10 cm (Bio-Rad, Hercules, CA) were used. Detection: absorbance profile at 280 nm (upper line), conductivity profile (bottom line). Steps: (I) preliminary equilibration, (II) binding, (III) elution, (IV) regeneration, and (V) final equilibration (see Section 2.6.1). (B) LC chromatogram of specific anti-CA I IgG isolation by affinity chromatografy with CA I bound on Perloza MT 500 carrier (5 mL), BioLogic low-pressure chromatography system and chromatography column 1 × 5 cm (Bio-Rad, Hercules, CA) were used. Dtection: absorbance profile at 280 nm (upper line), conductivity profile (bottom line). Steps: (I) preliminary equilibration, (II) binding, (III) elution, (IV) regeneration, and (V) final equilibration (see Section 2.6.2). (C) Western blot analysis: immobilization of specific anti-CA I antibodies onto SiMAG-Hydrazide microparticles.Lanes: (1) molecular marker, (2) initial fraction of antibodies before immobilization, (3) fraction of antibodies after immobilization, (4) first, (5) second,and (6) third washing fraction after immobilization, (7) patient’s serum; dilution, (2-6) 100×, (7) 500×.

Isolation of specific anti-CA I IgG from the total IgG fraction using carrier with immobilized CA I was performed in lowpressure column arrangement (Figure 4B, see Section 2.6.2). Eluted polyclonal anti-CA I IgGs were immediately immobilized onto magnetic microparticles SiMAG-Hydrazide (see Section 2.6.3) via their oligosaccharide chains located in the Fc region of the antibodies far from their binding sites, thus, allowing an optimal steric accessibility for interaction with antigen or antigenic peptides.30 The binding capacity of prepared immunoaffinity carrier (0.47 nmol/mg) was estimated by highly sensitive Western blot analysis of solution before and after immobilization (see Section 2.8). Nondigested CA I was transferred from 12.5% SDS-PAGE gel to the nitrocellulose membrane by Western blotting and strips of the membrane were incubated with respective fractions from anti-CA I IgG immobilization. As shown in Figure 4C, almost all specific antibodies were immobilized onto the carrier. This carrier with immobilized anti-CA I IgG molecules had been repeatedly utilized for epitope extraction (Figure 1). The mixture of CA I tryptic fragments was applied to the immunosorbent, unbound peptides were washed out by the equilibration buffer (pH 7.0), and the same buffer enriched by NaCl (0.2 and 1 M) was applied to remove possible nonspecifically sorbed peptides. Specifically, captured peptides were eluted with 0.05% TFA. All binding and elution fractions were subsequently analyzed by LC-MS/MS. The MS/MS spectra of the elution fractions repeatedly demonstrated the presence of 4 tryptic peptides only: EIINVGHSFHVNFEDNDNR, HDTSLKPISVSYNPATAK, ESISVSSEQLAQFR, and ADGLAVIGVLMK (Table 1). It was necessary to confirm that capturing of these peptides was through a specific interaction with anti-CA I antibodies, and that they were not sorbed nonspecifically on the carrier or the ligand. The specificity of binding of CA I tryptic fragments was tested in the presence of a large excess of tryptic 5176

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Table 1. Tryptic Fragments of CA I Repeatedly Identified in Elution Fractions by LC-MS/MS mass

position

MCa

peptide sequence

2256.0428 1929.0076 1580.7914 1186.6864

58-76 40-57 214-227 138-149

0 0 0 0

EIINVGHSFHVNFEDNDNR HDTSLKPISVSYNPATAK ESISVSSEQLAQFR ADGLAVIGVLMK

a

MC ) missed cleavages.

fragments of BSA (data not shown). Fragments belonging to CA I were repeatedly isolated from this mixture and no fragments of BSA were observed in elution fractions. The zero nonspecific sorption has thus been confirmed. To further characterize the epitopes and verify the results from trypsin digestion experiments, we have performed a similar analysis with another proteolytic enzyme, R-chymotrypsin, possessing a different specifity, digesting on the Ctermini of peptides after Phe, Tyr, and Trp residues. A carrier with immobilized R-chymotrypsin was prepared by the same procedure as trypsin carrier (see Section 2.4). The final activity of immobilized R-chymotrypsin (1.95 IU/mg of carrier) was determined with the low-molecular weight substrate SUPHEPA. The optimized cleavage time was established to be 90 min, when 9 out of 12 theoretical peptides (i.e., 69.2% coverage) in the range of MW 600-5000 were observed (Figure 3B). The following steps, such as epitope extraction and MS/MS analysis, were performed similarly as described in the previous text. In the case of R-chymotrypsin, the MS/MS spectra of the elution fractions repeatedly demonstrated the presence of 3 following fragments: SSLAEAASKADGLAVIGVLMKVGEANPKLQKVLDALQAIKTKGKRAPF, IICKESISVSSEQLAQF, and NPATAKEIINVGHSF (Table 2). The autoantibodies in the serum of patient with multiple myeloma after HDT and ASCT treatment, used in this work, exhibited cross-reactivity with CA I, II, IX, and XII. To assess

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CA I Epitopes Recognized by Specific Autoantibodies Table 2. R-Chymotryptic Fragments of CA I Repeatedly Identified in Elution Fractions by LC-MS/MS mass

position

MCa

modifications

4932.8069 1881.9626 1597.8332

129-176 210-226 52-66

0 0 0

Cys_CM: 212 (1939.9681)

a

peptide sequence

SSLAEAASKADGLAVIGVLMKVGEANPKLQKVLDALQAIKTKGKRAPF IICKESISVSSEQLAQF NPATAKEIINVGHSF

MC ) missed cleavages.

Figure 5. Multiple sequence alignment of human CA I, CA II, CA IX, and CA XII performed using Clustal 2.0.1. The figure was prepared using program CHROMA.31 Highlighted regions correspond to experimentally detected peptides: magenta to SLKPI, red to NVGHS, cyan to DGLAV, and yellow to SSEQL. Red and green arrows above the sequence correspond to tryptic and chymotryptic fragments identified in elution fractions by LC-MS/MS, respectively.

Figure 6. Analysis of solvent accessible surface area of individual residues of conserved peptides within the CA I structure (PDB code 1AZM). For each residue of detected peptides, the graph (top) shows the percentage of total accessible surface area (ASA) which is exposed to the solvent. The location of each peptide in the CA I structure (PDB code 1AZM) is represented in a corresponding color (SLKPI magenta, NVGHS red, DGLAV cyan, and SSEQL yellow) in the secondary structure representation (middle) and by the solvent accessible surface (bottom). Zinc ion and inhibitor molecules located in the enzyme active site are depicted in green.

which regions of the peptides detected by epitope mapping on CA I could represent a common epitope, we analyzed conservation of peptide sequences in CA I, II, IX, and XII. This analysis detected four short continuous sequences which shared more than 75% homology among isoforms: SLKPI, NVGHS, DGLAV, and SSEQL (Figure 5). Three of these sequences were detected in epitope mapping experiments upon CA I fragmentation of both typsin and R-chymotrypsin pro-

teases. Peptide bearing sequence SLKPI was only detected in the elution of fragments generated by tryptic digest. Possible reason for this could be the nonaccessibility of epitope within a long R-chymotrypsin fragment. We analyzed the position of potential conserved epitopes on CA I three-dimensional structure and their accessibility for antibody recognition in the context of the native CA I molecule (Figure 6). Peptide SLKPI (residues 43-47) is a part of a loop Journal of Proteome Research • Vol. 9, No. 10, 2010 5177

research articles located on the surface of the CA I molecule, which lacks any particular secondary structure. With the exception of the hydrophobic side chains of L44 and I47, the peptide is rather exposed to the solvent, and thus accessible for potential autoantibody binding. Peptide NVGHS, residues 61-65, forms a loop connecting two β-strands of the central antiparallel β-sheet, which supports the enzyme active site. The loop is also fairly exposed on the surface of the molecule, with the exception of the terminal residues of this sequence, which are more buried in the protein core. Peptide DGLAV, residues 139-143, is the least exposed of the discussed peptides. Residues L141, A142, and V143 are part of the antiparallel β-sheet structure and are located at the bottom of the deep cavity of the enzyme catalytic site. Accessibility of this peptide is rather limited. Peptide SSEQL, residues 219-223, is in the CA I structure part of a short R-helix. The residues are fairly well exposed on the surface. The only exception is residue L223, which packs against the hydrophobic protein core. Sequence homology and structural analysis corroborated by the experimental epitope mapping resulted in detecting of potential conserved epitopes on CA surface available for antibody recognition in the patient’s serum.

4. Conclusion A combination of the epitope extraction technique, a traditional approach of epitope mapping, together with LC-MS/MS and bioinformatic analyses has been used for identification of epitopes recognized by anti-CA autoantibodies. Using anti-CA I autoantibodies isolated from the serum of a patient, who spontaneously regressed after HDT with ASCT, and who developed aplastic anemia type syndrome, we identified 4 epitopes: NVGHS, DGLAV, SSEQL, and SLKPI. According to compatible results of LC-MS/MS and bioinformatic analyses, we consider the discussed epitopes as relevant potential therapeutic targets for peptide-based vaccination. We believe that the presented results can be useful for disclosure of a mechanism of tumor growth inhibition in relation to the appearance and dynamics of autoantibody formation and to the disease prognosis of monitored patients and might be helpful in clinical practices for development of new therapeutic or diagnostic approaches.

Acknowledgment. The authors wish to acknowledge The Ministry of Education of Czech Republic (project No. MSMT 0021627502), The State program of research and development BITCET (SPVV 337/2003), The Norway and EEA grants (SK 0095), Structural funds of EU - research and development program TRANSMED (No. 2624012008) for financial support of the research. In part, this work was supported by Projects AV0Z50520514 and AV0Z40550506 awarded by the Academy of Sciences of the Czech Republic, grant No. 1M0505 awarded by the Ministry of Education of the Czech Republic, and grant No. GA203/09/0820 awarded by Czech Grant Foundation. We thank Devon Maloy for a critical proofreading of the manuscript. References (1) Supuran, C. T. Carbonic anhydrases: Catalytic and inhibition mechanism, distribution and physiological roles. In Carbonic Anhydrase: Its Inhibitors and Activators; Scozzafava, A. , Supuran, C. T. , Conway, J. , Eds.; CRC Press: Boca Raton, FL, 2004; pp 124. (2) Scozzafava, A.; Supuran, C. T. Carbonic anhydrase inhibitors. Curr. Med. Chem.: Immunol. Endocr. Metab. Agents 2001, 1, 61–97.

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