- Email: [email protected]
Overlap of epitopes recognized by anti-carbonic anhydrase I IgG in patients with malignancy-related aplastic anemia-like syndrome and in patients with aplastic anemia
Immunology Letters 153 (2013) 47–49
Contents lists available at ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Overlap of epitopes recognized by anti-carbonic anhydrase I IgG in patients with malignancy-related aplastic anemia-like syndrome and in patients with aplastic anemia Barbora Jankovicova a , Ludovit Skultety b,c,d , Maria Dubrovcakova e , Martin Stern f , Zuzana Bilkova a , Jan Lakota e,∗ a
Department of Biological and Biochemical Sciences, University of Pardubice, Pardubice, Czech Republic Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovakia c Centre of Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia d Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic e Laboratory of Molecular Oncology, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia f Immunotherapy Laboratory, Department of Biomedicine, University Hospital Basel, Basel, Switzerland b
a r t i c l e
i n f o
Article history: Received 11 June 2013 Accepted 17 July 2013 Available online 26 July 2013 Keywords: Carbonic anhydrase I Epitope extraction Anti-CA I autoantibodies Aplastic anemia LC–MS/MS
a b s t r a c t High titers of anti-carbonic anhydrase I (anti-CA I) autoantibodies were detected in the sera of patients with malignancies who developed an aplastic anemia-like (AA-like) syndrome after a high-dose therapy (HDT) and autologous stem cell transplantation (ASCT). It was found, that the presence of these anti-CA I autoantibodies is associated with spontaneous tumor regression. The main immunodominant epitopes of carbonic anhydrase isoform I (CA I) have previously been identified using epitope extraction technique in combination with mass spectrometric detection and bioinformatic verification. Similarly, the sera of patients with bona fide aplastic anemia (AA) who poorly responded to immunosuppressive treatment with anti-thymocyte globulin (ATG) demonstrated high titers of anti-CA I antibodies. In order to reveal differences between these antibodies, we applied the same methodology of epitope mapping procedure. Surprisingly, the anti-CA I antibodies from the both groups of patients compatibly recognized the same four candidate CA I epitopes – DGLAV, NVGHS, SLKPI, SSEQL. This finding may indicate common pathophysiological mechanisms in these two syndromes. However, at this moment it remains unresolved if anti-CA I antibodies are implicated in marrow or tumor suppression or are just an epi-phenomenon. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Aplastic anemia (AA) is a rare, potentially life-threatening failure of haemopoiesis characterized by pancytopenia and bone-marrow aplasia. Most cases are acquired and mediated by autoreactive lymphocytes causing destruction of haemopoietic stem cells. Development of bone marrow transplantation (BMT) and potent immunosuppressive therapy in the 1970s greatly improved the prognosis of an illness that had previously been fatal in most cases within a year of diagnosis [1]. First-line treatment approaches include a combination of anti-thymocyte globulin (ATG) and cyclosporine A for patients without a sibling donor, and HLA identical sibling transplantation for patients younger than age 40 with a donor [2].
∗ Corresponding author at: Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 833 91 Bratislava, Slovakia. Tel.: +421 2 5932 7411. E-mail address: [email protected] (J. Lakota). 0165-2478/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.imlet.2013.07.006
Interestingly, an aplastic anemia-like (AA-like) syndrome may develop after high dose therapy (HDT) and autologous stem cell transplantation (ASCT) in patients with Hodgkin’s disease, multiple myeloma and some other malignant diseases. This AA-like syndrome is associated with spontaneous tumor regression [3]. There is increasing evidence that autoimmunity can inhibit the growth of solid tumors, so the pancytopenia occurring in AA or in AAlike syndromes may reflect an ongoing immune reaction against underlying malignancy (anti-tumor autoimmunity) or infection [4]. High titers of serum autoantibodies specifically reacting with carbonic anhydrase isoform I (CA I) were observed in these patients, and occurrence of such antibodies was associated with significantly increased survival. Anti-CA I antibodies have previously been reported in autoimmune diseases, e.g. autoimmune/idiopathic chronic pancreatitis (ACP/ICP), Sjögren’s syndrome (SjS) [5–7], idiopathic recurrent pregnancy loss (RPL) [8], connective tissue diseases (CTD) [9], systemic lupus erythematosus (SLE), and other rheumatic diseases [10]. In some autoimmune diseases, anti-CA I antibodies can be a reliable diagnostic indicator. It has recently been hypothesized that these diseases may have a common pathogenetic
48
B. Jankovicova et al. / Immunology Letters 153 (2013) 47–49
mechanism based on autoimmune reactions against a common antigen [10,11]. Interestingly, serum antibodies to CAs I and II found in patients with ICP and SjS are not cross-reactive with each other, suggesting that these antibodies might be detected as a consequence of the cross-reactivity of an antibody against another antigen that mimics CAs I or II [5,12,13]. The epitope extraction technique with tandem mass spectrometry (MS/MS) detection and bioinformatic analyses has already been applied to identify the main immunogenic epitopes of CA I in patients with AA-like syndrome [14]. Four candidate epitopes of CA I (NVGHS, DGLAV, SSEQL, and SLKPI) were recognized by anti-CA I autoantibodies isolated from the serum of a patient with multiple myeloma, who spontaneously regressed after HDT with ASCT, and who developed AA-like syndrome. The positivity of the anti-CA I autoantibodies in serum of 38% patients with AA (20 from a total of 53) [15] was associated with older age at diagnosis of AA, poor response to immunosuppressive treatment with ATG, and inferior long-term survival. This work aims to identify and compare immunodominant epitopes of CA I which are recognized by anti-CA I antibodies isolated from anti-CA I autoantibodies highly positive patients’ sera of both, the group of patients with malignancy regression, and the bona fide AA patients who poorly responded to immunosuppressive treatment with ATG. 2. Materials and methods 2.1. Patients Anti-CA I antibody positive sera of 3 patients with AA were collected at University Hospital Basel as described previously [16]. The sera of 2 patients with malignancy who developed AA-like syndrome after HDT and ASCT treatment (performed according to standard European Bone Marrow Transplantation group protocols) were obtained from the National Cancer Institute Bratislava [3,14]. Serum samples were stored at −80 ◦ C until analysis. Patients gave written informed consent to the analysis, and the study was approved by the local institutional review board. 2.2. Specific anti-CA I IgG purification In a two-step procedure, the IgG fraction was isolated from patients’ sera by affinity chromatography with protein A, and subsequently underwent affinity chromatography with immobilized CA I, which is described in detail in [14]. 2.3. Epitope extraction The procedure consisting of enzymatic fragmentation of CA I and specific immunocapturing of generated peptides by affinity carrier with covalently immobilized anti-CA I IgG molecules has been applied as described previously [14]. Eluted peptide mixtures were analyzed by automated nanoflow RP-UPLC system coupled to a Q-Tof Premier (Waters, Milford, MA, USA) tandem mass spectrometer (LC–MS/MS). For protein identification, a novel multiplex approach called MSE was used [17]. The obtained MS/MS data were processed using the ProteinLynx Global Server v. 2.4 (Waters, UK). The resulting data were searched against human CA I database (http://www.uniprot.org/uniprot/P00915). 3. Results and discussion This work is a follows up of our previous publication [14], where we identified the epitopes of CA I that react with anti-CA I autoantibodies isolated from serum of patient with multiple myeloma, who developed AA-like syndrome and spontaneously regressed after HDT and ASCT [3]. We mapped four immunodominant epitopes
(NVGHS, DGLAV, SSEQL, and SLKPI) which shared more than 75% homology among cross-reactive isoforms CA I, II, IX, and XII. Analysis of their position in 3D structure of the native CA I revealed partial (NVGHS and DGLAV) or total (SLKPI and SSEQL) surface exposure related to accessibility of potential autoantibodies [14]. It was observed that high titers of these anti-CA I autoantibodies are associated with a survival benefit for patients suffering from various malignant diseases (e.g. Hodgkin’s disease, non-Hodgkin’s lymphoma, multiple myeloma, Ewing’s sarcoma) [3]. Since, the presence of the anti-CA I specific antibodies was detected in the sera of patients who developed AA-like syndrome we wondered if they are present also in the sera of patients with bona fide AA of different severity. Surprisingly, the anti-CA I antibodies were detected in 38% of analyzed sera (20 from a total of 53) [15]. Thus, we decided to analyze if there is any sequence similarity or cross-reactivity with respective antigen between these antibodies of the both groups of patients (AA and AA-like syndrome). We applied the epitope mapping procedure [14] based on algorithm of epitope extraction composed of effective fragmentation of CA I and subsequent bioaffinity capturing of signature peptides that carry the prospective immunodominant epitope(s). Superparamagnetic microparticles were used as a solid phase in all steps including covalent immobilization of proteolytic enzymes (trypsin and ␣-chymotrypsin), CA I antigen and the specific antiCA I antibodies. The immunospecifically captured and subsequently released peptide fragments of CA I carrying the immunodominant epitopes were detected and identified by ultrasensitive MS/MS analyses (Fig. 1). In the first step, specific proteolytic fragmentation of CA I was performed using immobilized trypsin or ␣-chymotrypsin. The digestion performed by immobilized proteases benefits from their high proteolytic activity, the absence of autolytic fragments in the peptide mixture, and low frequency of missed cleavages [18]. In the next step, the IgGs from anti-CA I autoantibodies highly positive sera of the both groups of patients were isolated using PureProteome magnetic beads coated with Protein A (Fig. 1). To obtain the specific anti-CA I antibodies, the IgG fractions of patients’ sera were applied to the carrier with CA I as an affinity ligand (binding capacity: 3.1 nmol/0.125 ml). The capture efficiency of IgGs was monitored by SDS-PAGE, the total amount of isolated IgG was evaluated spectrophotometrically. Purified anti-CA I IgG molecules were then immobilized orientedly to the SiMAG-Hydrazide magnetic microparticles. The mixture of proteolytically fragmented CA I was loaded on the anti-CA I IgG immunosorbent. Unbound peptides were washed out by an equilibration buffer, and the high salt buffer containing 0.2 M and 1 M NaCl was applied to remove non-specifically sorbed molecules. The captured peptides were eluted with trifluoroacetic acid (0.05%). All fractions were subsequently analyzed by LCMS/MS technique. The novel MSE 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 was employed to identify the peptides [17]. The analysis of the elution fractions of both sample sets repeatedly revealed the presence of the following 4 (EIINVGHSFHVNFEDNDNR, HDTSLKPISVSYNPATAK, ESISVSSEQLAQFR, and ADGLAVIGVLMK) 3 (IICKESISVSSEQLAQF, NPATAKEIINVGHSF, and and SSLAEAASKADGLAVIGVLMKVGEANPKLQKVLDALQAIKTKGKRAPF) peptides obtained by digestion with trypsin and ␣-chymotrypsin, respectively. These overlapping peptides were recognized not only by anti-CA I IgG antibodies of patients with malignancies who developed an AA-like syndrome after HDT and ASCT but also with the antibodies of patients with bona fide AA who poorly responded to immunosuppressive treatment with ATG. Thus, the result suggests a match with recognized immunodominant epitopes DGLAV, NVGHS, SLKPI, SSEQL of CA I and it indicates that
B. Jankovicova et al. / Immunology Letters 153 (2013) 47–49
49
Fig. 1. The workflow of epitope mapping procedure.
the specific anti-CA I IgGs from both groups of patients are fully compatible. This finding suggests a common pathophysiological mechanism in these two syndromes, however, at this moment we do not know yet if these antibodies are really implicated in marrow or tumor suppression or are just an epi-phenomenon. Thus, further experiments are needed to confirm this hypothesis. 4. Conclusions Anti-CA I autoantibodies isolated from sera of patients with aplastic anemia who poorly responded to immunosuppressive treatment with ATG recognize the same immunodominant CA I epitopes (NVGHS, DGLAV, SSEQL, and SLKPI) as those from the sera of a patients, who developed AA-like syndrome and spontaneously regressed after HDT and ASCT. The epitope extraction technique based on application of superparamagnetic beads followed by LC–MS/MS detection revealed their potential to identify these compatible epitopes, which we consider as relevant therapeutic targets for peptide-based vaccination. We also believe that the presented results can be useful for disclosure of a mechanism of marrow or tumor suppression and they might be helpful for monitoring the disease in clinical practice. Acknowledgments Part of the cost of this study was supported by the Ministry of Education, Youth and Sports of the Czech Republic, Project CZ.1.07/2.3.00/30.0021 “Enhancement of R&D Tools of Excellence at the University of Pardubice” as well as by the grants: 2/0156/11 of the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and the Slovak Academy of Sciences, the 26240120030 (TRANSMED2), and 26240220062 (Center for innovative research of anticancer and antiviral strategies) supported by the Research & Development Operational Programme funded by the ERDF and the IMIC institutional research concept RVO61388971. The work of JL has been supported by Foundation for Transplantation. References [1] Brodsky RA, 1647–56.
Jones
RJ.
Aplastic
anaemia.
Lancet
2005;365(9471):
[2] Passweg JR, Marsh JC. Aplastic anemia: first-line treatment by immunosuppression and sibling marrow transplantation. American Society of Hematology Education Program 2010;2010:36–42. [3] Lakota J, Skultety L, Dubrovcakova M, Altaner C. Presence of serum carbonic anhydrase autoantibodies in patients relapsed after autologous stem cell transplantation indicates an improved prognosis. Neoplasma 2008;55(6):488–92. [4] Nissen C, Stern M. Acquired immune mediated aplastic anemia: is it antineoplastic? Autoimmunity Reviews 2009;9(1):11–6. [5] Kino-Ohsaki J, Nishimori I, Morita M, Okazaki K, Yamamoto Y, Onishi S, et al. Serum antibodies to carbonic anhydrase I and II in patients with idiopathic chronic pancreatitis and Sjögren’s syndrome. Gastroenterology 1996;110(5):1579–86. [6] Bartolomé MJ, de las Heras G, López-Hoyos M. Low-avidity antibodies to carbonic anhydrase-I and -II in autoimmune chronic pancreatitis. ScientificWorld Journal 2002;2:1560–8. [7] Frulloni L, Bovo P, Brunelli S, Vaona B, Di Francesco V, Nishimori I, et al. Elevated serum levels of antibodies to carbonic anhydrase I and II in patients with chronic pancreatitis. Pancreas 2000;20(4):382–8. [8] Karahan SC, Guven S, Mentese A, Bacak A, Kopuz M, Ozeren M. Serum anticarbonic anhydrase I and II antibodies and idiopathic recurrent pregnancy loss. Reproductive BioMedicine Online 2009;19(6):859–63. [9] Caccavo D, Afeltra A, Rigon A, Vadacca M, Zobel BB, Zennaro D, et al. Antibodies to carbonic anhydrase in patients with connective tissue diseases: relationship with lung involvement. International Journal of Immunopathology and Pharmacology 2008;21(3):659–67. [10] Itoh Y, Reichlin M. Antibodies to carbonic anhydrase in systemic lupus erythematosus and other rheumatic diseases. Arthritis & Rheumatism 1992;35(1):73–82. [11] Botrè F, Botrè C, Podestà E, Podda M, Invernizzi P. Effect of anti-carbonic anhydrase antibodies on carbonic anhydrases I and II. Clinical Chemistry 2003;49(7):1221–3. [12] Nishimori I, Miyaji E, Morimoto K, Nagao K, Kamada M, Onishi S. Serum antibodies to carbonic anhydrase IV in patients with autoimmune pancreatitis. Gut 2005;54(2):274–81. [13] Nishimori I, Onishi S. Carbonic anhydrase isozymes in the human pancreas. Digestive and Liver Disease 2001;33(1):68–74. [14] Skultety L, Jankovicova B, Svobodova Z, Mader P, Rezacova P, Dubrovcakova M, et al. 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. Journal of Proteome Research 2010;9(10):5171–9. [15] Lakota J, Lanz A, Dubrovcakova M, Jankovicova B, Gonzalez A, Stern M. Antibodies against carbonic anhydrase in patients with aplastic anemia. Acta Haematologica 2012;128(3):190–4. [16] Speck B, Nissen C, Tichelli A, Gratwohl A. Treatment of aplastic anemia. Oncologist 1996;1(6):367–70. [17] Plumb RS, Johnson KA, Rainville P, Smith BW, Wilson ID, Castro-Perez JM, et al. UPLC/MS(E); a new approach for generating molecular fragment information for biomarker structure elucidation. Rapid Communications in Mass Spectrometry 2006;20(13):1989–94. [18] Bilkova Z, Slovakova M, Minc N, Futterer C, Cecal R, Horak D, et al. Functionalized magnetic microand nanoparticles: optimization and application to -chip tryptic digestion. Electrophoresis 2006;27(9):1811–24.
Contents lists available at ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Overlap of epitopes recognized by anti-carbonic anhydrase I IgG in patients with malignancy-related aplastic anemia-like syndrome and in patients with aplastic anemia Barbora Jankovicova a , Ludovit Skultety b,c,d , Maria Dubrovcakova e , Martin Stern f , Zuzana Bilkova a , Jan Lakota e,∗ a
Department of Biological and Biochemical Sciences, University of Pardubice, Pardubice, Czech Republic Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovakia c Centre of Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia d Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic e Laboratory of Molecular Oncology, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia f Immunotherapy Laboratory, Department of Biomedicine, University Hospital Basel, Basel, Switzerland b
a r t i c l e
i n f o
Article history: Received 11 June 2013 Accepted 17 July 2013 Available online 26 July 2013 Keywords: Carbonic anhydrase I Epitope extraction Anti-CA I autoantibodies Aplastic anemia LC–MS/MS
a b s t r a c t High titers of anti-carbonic anhydrase I (anti-CA I) autoantibodies were detected in the sera of patients with malignancies who developed an aplastic anemia-like (AA-like) syndrome after a high-dose therapy (HDT) and autologous stem cell transplantation (ASCT). It was found, that the presence of these anti-CA I autoantibodies is associated with spontaneous tumor regression. The main immunodominant epitopes of carbonic anhydrase isoform I (CA I) have previously been identified using epitope extraction technique in combination with mass spectrometric detection and bioinformatic verification. Similarly, the sera of patients with bona fide aplastic anemia (AA) who poorly responded to immunosuppressive treatment with anti-thymocyte globulin (ATG) demonstrated high titers of anti-CA I antibodies. In order to reveal differences between these antibodies, we applied the same methodology of epitope mapping procedure. Surprisingly, the anti-CA I antibodies from the both groups of patients compatibly recognized the same four candidate CA I epitopes – DGLAV, NVGHS, SLKPI, SSEQL. This finding may indicate common pathophysiological mechanisms in these two syndromes. However, at this moment it remains unresolved if anti-CA I antibodies are implicated in marrow or tumor suppression or are just an epi-phenomenon. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Aplastic anemia (AA) is a rare, potentially life-threatening failure of haemopoiesis characterized by pancytopenia and bone-marrow aplasia. Most cases are acquired and mediated by autoreactive lymphocytes causing destruction of haemopoietic stem cells. Development of bone marrow transplantation (BMT) and potent immunosuppressive therapy in the 1970s greatly improved the prognosis of an illness that had previously been fatal in most cases within a year of diagnosis [1]. First-line treatment approaches include a combination of anti-thymocyte globulin (ATG) and cyclosporine A for patients without a sibling donor, and HLA identical sibling transplantation for patients younger than age 40 with a donor [2].
∗ Corresponding author at: Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 833 91 Bratislava, Slovakia. Tel.: +421 2 5932 7411. E-mail address: [email protected] (J. Lakota). 0165-2478/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.imlet.2013.07.006
Interestingly, an aplastic anemia-like (AA-like) syndrome may develop after high dose therapy (HDT) and autologous stem cell transplantation (ASCT) in patients with Hodgkin’s disease, multiple myeloma and some other malignant diseases. This AA-like syndrome is associated with spontaneous tumor regression [3]. There is increasing evidence that autoimmunity can inhibit the growth of solid tumors, so the pancytopenia occurring in AA or in AAlike syndromes may reflect an ongoing immune reaction against underlying malignancy (anti-tumor autoimmunity) or infection [4]. High titers of serum autoantibodies specifically reacting with carbonic anhydrase isoform I (CA I) were observed in these patients, and occurrence of such antibodies was associated with significantly increased survival. Anti-CA I antibodies have previously been reported in autoimmune diseases, e.g. autoimmune/idiopathic chronic pancreatitis (ACP/ICP), Sjögren’s syndrome (SjS) [5–7], idiopathic recurrent pregnancy loss (RPL) [8], connective tissue diseases (CTD) [9], systemic lupus erythematosus (SLE), and other rheumatic diseases [10]. In some autoimmune diseases, anti-CA I antibodies can be a reliable diagnostic indicator. It has recently been hypothesized that these diseases may have a common pathogenetic
48
B. Jankovicova et al. / Immunology Letters 153 (2013) 47–49
mechanism based on autoimmune reactions against a common antigen [10,11]. Interestingly, serum antibodies to CAs I and II found in patients with ICP and SjS are not cross-reactive with each other, suggesting that these antibodies might be detected as a consequence of the cross-reactivity of an antibody against another antigen that mimics CAs I or II [5,12,13]. The epitope extraction technique with tandem mass spectrometry (MS/MS) detection and bioinformatic analyses has already been applied to identify the main immunogenic epitopes of CA I in patients with AA-like syndrome [14]. Four candidate epitopes of CA I (NVGHS, DGLAV, SSEQL, and SLKPI) were recognized by anti-CA I autoantibodies isolated from the serum of a patient with multiple myeloma, who spontaneously regressed after HDT with ASCT, and who developed AA-like syndrome. The positivity of the anti-CA I autoantibodies in serum of 38% patients with AA (20 from a total of 53) [15] was associated with older age at diagnosis of AA, poor response to immunosuppressive treatment with ATG, and inferior long-term survival. This work aims to identify and compare immunodominant epitopes of CA I which are recognized by anti-CA I antibodies isolated from anti-CA I autoantibodies highly positive patients’ sera of both, the group of patients with malignancy regression, and the bona fide AA patients who poorly responded to immunosuppressive treatment with ATG. 2. Materials and methods 2.1. Patients Anti-CA I antibody positive sera of 3 patients with AA were collected at University Hospital Basel as described previously [16]. The sera of 2 patients with malignancy who developed AA-like syndrome after HDT and ASCT treatment (performed according to standard European Bone Marrow Transplantation group protocols) were obtained from the National Cancer Institute Bratislava [3,14]. Serum samples were stored at −80 ◦ C until analysis. Patients gave written informed consent to the analysis, and the study was approved by the local institutional review board. 2.2. Specific anti-CA I IgG purification In a two-step procedure, the IgG fraction was isolated from patients’ sera by affinity chromatography with protein A, and subsequently underwent affinity chromatography with immobilized CA I, which is described in detail in [14]. 2.3. Epitope extraction The procedure consisting of enzymatic fragmentation of CA I and specific immunocapturing of generated peptides by affinity carrier with covalently immobilized anti-CA I IgG molecules has been applied as described previously [14]. Eluted peptide mixtures were analyzed by automated nanoflow RP-UPLC system coupled to a Q-Tof Premier (Waters, Milford, MA, USA) tandem mass spectrometer (LC–MS/MS). For protein identification, a novel multiplex approach called MSE was used [17]. The obtained MS/MS data were processed using the ProteinLynx Global Server v. 2.4 (Waters, UK). The resulting data were searched against human CA I database (http://www.uniprot.org/uniprot/P00915). 3. Results and discussion This work is a follows up of our previous publication [14], where we identified the epitopes of CA I that react with anti-CA I autoantibodies isolated from serum of patient with multiple myeloma, who developed AA-like syndrome and spontaneously regressed after HDT and ASCT [3]. We mapped four immunodominant epitopes
(NVGHS, DGLAV, SSEQL, and SLKPI) which shared more than 75% homology among cross-reactive isoforms CA I, II, IX, and XII. Analysis of their position in 3D structure of the native CA I revealed partial (NVGHS and DGLAV) or total (SLKPI and SSEQL) surface exposure related to accessibility of potential autoantibodies [14]. It was observed that high titers of these anti-CA I autoantibodies are associated with a survival benefit for patients suffering from various malignant diseases (e.g. Hodgkin’s disease, non-Hodgkin’s lymphoma, multiple myeloma, Ewing’s sarcoma) [3]. Since, the presence of the anti-CA I specific antibodies was detected in the sera of patients who developed AA-like syndrome we wondered if they are present also in the sera of patients with bona fide AA of different severity. Surprisingly, the anti-CA I antibodies were detected in 38% of analyzed sera (20 from a total of 53) [15]. Thus, we decided to analyze if there is any sequence similarity or cross-reactivity with respective antigen between these antibodies of the both groups of patients (AA and AA-like syndrome). We applied the epitope mapping procedure [14] based on algorithm of epitope extraction composed of effective fragmentation of CA I and subsequent bioaffinity capturing of signature peptides that carry the prospective immunodominant epitope(s). Superparamagnetic microparticles were used as a solid phase in all steps including covalent immobilization of proteolytic enzymes (trypsin and ␣-chymotrypsin), CA I antigen and the specific antiCA I antibodies. The immunospecifically captured and subsequently released peptide fragments of CA I carrying the immunodominant epitopes were detected and identified by ultrasensitive MS/MS analyses (Fig. 1). In the first step, specific proteolytic fragmentation of CA I was performed using immobilized trypsin or ␣-chymotrypsin. The digestion performed by immobilized proteases benefits from their high proteolytic activity, the absence of autolytic fragments in the peptide mixture, and low frequency of missed cleavages [18]. In the next step, the IgGs from anti-CA I autoantibodies highly positive sera of the both groups of patients were isolated using PureProteome magnetic beads coated with Protein A (Fig. 1). To obtain the specific anti-CA I antibodies, the IgG fractions of patients’ sera were applied to the carrier with CA I as an affinity ligand (binding capacity: 3.1 nmol/0.125 ml). The capture efficiency of IgGs was monitored by SDS-PAGE, the total amount of isolated IgG was evaluated spectrophotometrically. Purified anti-CA I IgG molecules were then immobilized orientedly to the SiMAG-Hydrazide magnetic microparticles. The mixture of proteolytically fragmented CA I was loaded on the anti-CA I IgG immunosorbent. Unbound peptides were washed out by an equilibration buffer, and the high salt buffer containing 0.2 M and 1 M NaCl was applied to remove non-specifically sorbed molecules. The captured peptides were eluted with trifluoroacetic acid (0.05%). All fractions were subsequently analyzed by LCMS/MS technique. The novel MSE 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 was employed to identify the peptides [17]. The analysis of the elution fractions of both sample sets repeatedly revealed the presence of the following 4 (EIINVGHSFHVNFEDNDNR, HDTSLKPISVSYNPATAK, ESISVSSEQLAQFR, and ADGLAVIGVLMK) 3 (IICKESISVSSEQLAQF, NPATAKEIINVGHSF, and and SSLAEAASKADGLAVIGVLMKVGEANPKLQKVLDALQAIKTKGKRAPF) peptides obtained by digestion with trypsin and ␣-chymotrypsin, respectively. These overlapping peptides were recognized not only by anti-CA I IgG antibodies of patients with malignancies who developed an AA-like syndrome after HDT and ASCT but also with the antibodies of patients with bona fide AA who poorly responded to immunosuppressive treatment with ATG. Thus, the result suggests a match with recognized immunodominant epitopes DGLAV, NVGHS, SLKPI, SSEQL of CA I and it indicates that
B. Jankovicova et al. / Immunology Letters 153 (2013) 47–49
49
Fig. 1. The workflow of epitope mapping procedure.
the specific anti-CA I IgGs from both groups of patients are fully compatible. This finding suggests a common pathophysiological mechanism in these two syndromes, however, at this moment we do not know yet if these antibodies are really implicated in marrow or tumor suppression or are just an epi-phenomenon. Thus, further experiments are needed to confirm this hypothesis. 4. Conclusions Anti-CA I autoantibodies isolated from sera of patients with aplastic anemia who poorly responded to immunosuppressive treatment with ATG recognize the same immunodominant CA I epitopes (NVGHS, DGLAV, SSEQL, and SLKPI) as those from the sera of a patients, who developed AA-like syndrome and spontaneously regressed after HDT and ASCT. The epitope extraction technique based on application of superparamagnetic beads followed by LC–MS/MS detection revealed their potential to identify these compatible epitopes, which we consider as relevant therapeutic targets for peptide-based vaccination. We also believe that the presented results can be useful for disclosure of a mechanism of marrow or tumor suppression and they might be helpful for monitoring the disease in clinical practice. Acknowledgments Part of the cost of this study was supported by the Ministry of Education, Youth and Sports of the Czech Republic, Project CZ.1.07/2.3.00/30.0021 “Enhancement of R&D Tools of Excellence at the University of Pardubice” as well as by the grants: 2/0156/11 of the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and the Slovak Academy of Sciences, the 26240120030 (TRANSMED2), and 26240220062 (Center for innovative research of anticancer and antiviral strategies) supported by the Research & Development Operational Programme funded by the ERDF and the IMIC institutional research concept RVO61388971. The work of JL has been supported by Foundation for Transplantation. References [1] Brodsky RA, 1647–56.
Jones
RJ.
Aplastic
anaemia.
Lancet
2005;365(9471):
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