Proteomics Clin. Appl. 2016, 10, 403–414
403
DOI 10.1002/prca.201500080
REVIEW
Erythrocyte and platelet proteomics in hematological disorders Abhijit Chakrabarti1 , Suchismita Halder1 and Shilpita Karmakar2 1 2
Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India Biophysics and Structural Genomics Division, Saha institute of Nuclear Physics, Kolkata, India
Erythrocytes undergo ineffective erythropoesis, hemolysis, and premature eryptosis in sickle cell disease and thalassemia. Abnormal hemoglobin variants associated with hemoglobinopathy lead to vesiculation, membrane instability, and loss of membrane asymmetry with exposal of phosphatidylserine. This potentiates thrombin generation resulting in activation of the coagulation cascade responsible for subclinical phenotypes. Platelet activation also results in the release of microparticles, which express and transfer functional receptors from platelet membrane, playing key roles in vascular reactivity and activation of intracellular signaling pathways. Over the last decade, proteomics had proven to be an important field of research in studies of blood and blood diseases. Blood cells and its fluidic components have been proven to be easy systems for studying differential expressions of proteins in hematological diseases encompassing hemoglobinopathies, different types of anemias, myeloproliferative disorders, and coagulopathies. Proteomic studies of erythrocytes and platelets reported from several groups have highlighted various factors that intersect the signaling networks in these anucleate systems. In this review, we have elaborated on the current scenario of anucleate blood cell proteomes in normal and diseased individuals and the cross-talk between the two major constituent cell types of circulating blood.
Received: July 27, 2015 Revised: October 26, 2015 Accepted: November 19, 2015
Keywords: Erythrocytes / Hematological disorders / Interactome / Platelets / Proteomics
1
Introduction
Erythrocytes travel through our circulatory system reversibly transferring oxygen from lungs to tissues and carries carbondioxide back through hemoglobin. Erythrocytes originate from hematopoietic stem cells that give rise to nucleated proerythroblast that matures to form anucleated erythrocytes. It has a lifespan of 120 days with a normal count of 4.7–6.1 × 106 /L. It constantly changes shape in circulation from a biconcave disc of 6.2–8.2 m diameter to a cigar shape [1]. Its relative simplicity and easy availability, lack of internal organelles, and important physiologic function has made eryCorrespondence: Professor Abhijit Chakrabarti, Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700064, India E-mail: [email protected], [email protected] Fax: +91–3323374637 Abbreviations: ACMT, asymptomatic constitutional macrothrombocytopenia; CML, chronic myeloid leukemia; EMPs, erythrocyte-derived microparticles; GP, G-protein; HbE, hemoglobin E  thalassemia; PDI, protein disulphide isomerase; Prdx2, peroxiredoxin 2; PS, phosphatidylserine; SCD, sickle cell disease C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
throcytes a major focus of proteomic study [2]. The disc-like structure of platelets was first recognized and described by Osler in his papers written way back in the year 1873 and 1874. Platelets are anucleate discoid cells and measure approximately 2.0–4.0 × 0.5m with a mean volume of 7–11 fL. The normal platelet count is 150–450 ×103 /L and a mean lifespan of 10 days in circulation. Ultrastructural characterization of platelets with electron microscopy demonstrated two types of platelets, those that had morphologic characteristics of resting platelets and those that had the morphologic characteristics typical of activated platelets. With activation, platelets transform from a disk to a “spiny sphere” with long pseudopodia. Platelets are derived from megakaryocytes during a complex differentiation process that involves the packaging of cytoplasmic components into discrete areas of megakaryocyte dendritic projections, called proplatelets [3]. Over the last decade, proteomics has been an important field of research for study of blood and blood diseases. It has been proven to be as effective as genomics in determining the protein content of the extracellular vesicles released from erythrocytes and platelets. Erythrocytes and platelets have also been well studied using proteomics approach due to inability and/or limited ability of protein synthesis in erythrocytes and platelets. Differential expression of proteins in hematological www.clinical.proteomics-journal.com
404
A. Chakrabarti et al.
disorders encompassing hemoglobinopathies, different types of anemias, myeloproliferative disorders, and coagulopathies have been studied by many groups. In this review, we would discuss the current status of the proteomic understanding of anucleate blood cells both in normal and individuals with hematological diseases highlighting the proteomic changes and cross-talk between the two major constituent cell types of circulating blood.
2
Erythrocyte proteomics
Erythrocytes have been the focus of protein-based studies from the early days of 2DE in 1979 [4]. The first major contribution came from Goodman’s group who have separated the membrane and cytosolic fractions by further subfractionation using SEC followed by online MS/MS in a triple quadrupole mass spectrometer [5]. The next major boost in the number came from laboratory of Matthias Mann, where they have prefractionated membrane and cytoplasmic fractions using SDSPAGE followed by both in-gel and in-solution digestions and MS by Qq-TOF and FT-ICR-MS [6]. This resulted in a sharp increase in the identified protein numbers to 314 membrane proteins and 252 cytosolic proteins. They have also classified each protein according to their possible function using bioinformatics tools namely, cellular transport, metabolism, and protein complex formations while most membrane proteins, in addition to their role in cytoskeletal networks, account for signal transduction. We have earlier presented a review on the red cell proteomic studies discussing the scenario of differential expression of red cell protein in hemoglobin disorders [7]. Since then the latest review by Goodman’s group using the latest proteomic technique, current erythrocyte protein count stands at 2289 [8]. But we believe with increased sensitivity of detection, in future we could detect even very low copy number proteins and this list would expand. 2.1 Erythrocyte membrane proteomics The first classical 2DE–MALDI-MS based proteomics study was carried out by Low and co-workers, where they have employed both 1D and 2D gel-based separation followed by MALDI-MS to identify a total of about 70 membrane proteins [9]. Rabilloud established more sensitive methods of gel staining leading to visualizing greater number of protein spots in 2DE [10]. Following this, few gel-based studies on erythrocyte membrane came up, describing protein deregulation in hereditary spherocytosis [11–15], sickle cell anemia [16–18], and hemoglobin E  (HbE) thalassemia [19]. Few novel proteins were identified in malaria parasite infected erythrocyte membrane [20]. Blue-native/SDS PAGE techniques coupled with CyDye labeling were used to identify proteins of erythrocyte membranes in hemolytic anemia patients [21]. It has also been previously observed that proteolytic and oxidative damage accounts for major changes in the membrane skeletal proteins such as band 4.1, band 4.2, and ␣-spectrin [22]. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics Clin. Appl. 2016, 10, 403–414
Pasini et al. could annotate 314 membrane proteins [6]. De Palma et al. performed proteomic analysis of intact erythrocytes, isolated membranes, and Triton X-100 treated soluble fraction and a skeletal pellet. Using multidimensional protein identification technology (MuDPIT) and linear ion trap LTQ MS they have identified 299 unique proteins of which 211 were identified as being from the membrane [23]. Recently, Speicher and colleagues have separated erythrocyte membrane proteins by 1D SDS PAGE and found that when they cut the gels into 30 uniform slices, and performed ingel digestion of each, they were able to identify 842 unique proteins utilizing an LTQ-Orbitrap XL MS [24]. Till date most of the studies on posttranslational modifications of erythrocyte proteins have focused on the membrane proteins. Membrane skeletal proteins such as spectrin [25, 26], ankyrin [27], band 3 [28], band 4.1 [29, 30], adducin [31], and dematin [32] are known to be regulated by phosphorylation that in turn regulate the erythrocyte membrane architecture. Phosphoproteome of erythrocyte membranes is altered in sickle cell disease (SCD) patients as well as upon storage purpose for blood transfusion [33–35]. However, the phosphoproteome of erythrocyte is understudied and even profiling of all the phosphosites is far from completion. Ubiquitin is quite abundant inside erythrocytes and proteomics studies also predicted many erythrocyte proteins to be ubiquitinated [6]. From previous biochemical studies membrane proteins such as ␣-spectrin, ankyrin, and band 3 have been reported to be ubiquitinated [36–39]. Ubiquitination was never studied on a global proteomics scale inside erythrocyte and their functional implications remain an enigma till date. Delobel and coworkers studied carbonylation of proteins, in different populations, the hemoglobin-depleted soluble fraction, integral membrane, and cytoskeleton membrane protein fractions, showing the effect of oxidation on those fractions due to storage [40]. A glycomics study differentiates proteins based on their characteristic sugars thus contributing to isoform identification [41, 42]. This technique helps in the characterization of the erythrocyte carbohydrate heterogeneity responsible for blood group antigen diversity and immunology and provides possible insights in carbohydrate transfer during erythrocyte interaction with other cell populations (e.g. blood and endothelial cells) [43].
2.2 Erythrocyte cytosolic proteomics The numbers of gel-based proteomics studies on erythrocyte cytosol have been surprisingly low. The specific reason for this low number of studies was the presence of high abundance of hemoglobin, which masks the presence of all other proteins in the cytosol [7]. D’Amici et al. detected 838 protein spots in total by analyzing fractions using 2D IEFSDS-PAGE [44]. There are limited number of commercially available kits for hemoglobin depletion such as Proteominer [45–47] and Hemovoid (thalassemia major, TM) depletion www.clinical.proteomics-journal.com
405
Proteomics Clin. Appl. 2016, 10, 403–414
[48] in addition to Ni(II)-Sepharose affinity chromatography followed by ion-exchange chromatography [49] and SPSephadex (Sulphopropyl-Sephadex) cation exchanger matrix to deplete hemoglobin in cyano-met form [50]. This resulted in a 20-fold increase of visual detection of protein spots in a Coomassie-stained 17 cm 2D gel (418 spots vs.
403
DOI 10.1002/prca.201500080
REVIEW
Erythrocyte and platelet proteomics in hematological disorders Abhijit Chakrabarti1 , Suchismita Halder1 and Shilpita Karmakar2 1 2
Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India Biophysics and Structural Genomics Division, Saha institute of Nuclear Physics, Kolkata, India
Erythrocytes undergo ineffective erythropoesis, hemolysis, and premature eryptosis in sickle cell disease and thalassemia. Abnormal hemoglobin variants associated with hemoglobinopathy lead to vesiculation, membrane instability, and loss of membrane asymmetry with exposal of phosphatidylserine. This potentiates thrombin generation resulting in activation of the coagulation cascade responsible for subclinical phenotypes. Platelet activation also results in the release of microparticles, which express and transfer functional receptors from platelet membrane, playing key roles in vascular reactivity and activation of intracellular signaling pathways. Over the last decade, proteomics had proven to be an important field of research in studies of blood and blood diseases. Blood cells and its fluidic components have been proven to be easy systems for studying differential expressions of proteins in hematological diseases encompassing hemoglobinopathies, different types of anemias, myeloproliferative disorders, and coagulopathies. Proteomic studies of erythrocytes and platelets reported from several groups have highlighted various factors that intersect the signaling networks in these anucleate systems. In this review, we have elaborated on the current scenario of anucleate blood cell proteomes in normal and diseased individuals and the cross-talk between the two major constituent cell types of circulating blood.
Received: July 27, 2015 Revised: October 26, 2015 Accepted: November 19, 2015
Keywords: Erythrocytes / Hematological disorders / Interactome / Platelets / Proteomics
1
Introduction
Erythrocytes travel through our circulatory system reversibly transferring oxygen from lungs to tissues and carries carbondioxide back through hemoglobin. Erythrocytes originate from hematopoietic stem cells that give rise to nucleated proerythroblast that matures to form anucleated erythrocytes. It has a lifespan of 120 days with a normal count of 4.7–6.1 × 106 /L. It constantly changes shape in circulation from a biconcave disc of 6.2–8.2 m diameter to a cigar shape [1]. Its relative simplicity and easy availability, lack of internal organelles, and important physiologic function has made eryCorrespondence: Professor Abhijit Chakrabarti, Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700064, India E-mail: [email protected], [email protected] Fax: +91–3323374637 Abbreviations: ACMT, asymptomatic constitutional macrothrombocytopenia; CML, chronic myeloid leukemia; EMPs, erythrocyte-derived microparticles; GP, G-protein; HbE, hemoglobin E  thalassemia; PDI, protein disulphide isomerase; Prdx2, peroxiredoxin 2; PS, phosphatidylserine; SCD, sickle cell disease C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
throcytes a major focus of proteomic study [2]. The disc-like structure of platelets was first recognized and described by Osler in his papers written way back in the year 1873 and 1874. Platelets are anucleate discoid cells and measure approximately 2.0–4.0 × 0.5m with a mean volume of 7–11 fL. The normal platelet count is 150–450 ×103 /L and a mean lifespan of 10 days in circulation. Ultrastructural characterization of platelets with electron microscopy demonstrated two types of platelets, those that had morphologic characteristics of resting platelets and those that had the morphologic characteristics typical of activated platelets. With activation, platelets transform from a disk to a “spiny sphere” with long pseudopodia. Platelets are derived from megakaryocytes during a complex differentiation process that involves the packaging of cytoplasmic components into discrete areas of megakaryocyte dendritic projections, called proplatelets [3]. Over the last decade, proteomics has been an important field of research for study of blood and blood diseases. It has been proven to be as effective as genomics in determining the protein content of the extracellular vesicles released from erythrocytes and platelets. Erythrocytes and platelets have also been well studied using proteomics approach due to inability and/or limited ability of protein synthesis in erythrocytes and platelets. Differential expression of proteins in hematological www.clinical.proteomics-journal.com
404
A. Chakrabarti et al.
disorders encompassing hemoglobinopathies, different types of anemias, myeloproliferative disorders, and coagulopathies have been studied by many groups. In this review, we would discuss the current status of the proteomic understanding of anucleate blood cells both in normal and individuals with hematological diseases highlighting the proteomic changes and cross-talk between the two major constituent cell types of circulating blood.
2
Erythrocyte proteomics
Erythrocytes have been the focus of protein-based studies from the early days of 2DE in 1979 [4]. The first major contribution came from Goodman’s group who have separated the membrane and cytosolic fractions by further subfractionation using SEC followed by online MS/MS in a triple quadrupole mass spectrometer [5]. The next major boost in the number came from laboratory of Matthias Mann, where they have prefractionated membrane and cytoplasmic fractions using SDSPAGE followed by both in-gel and in-solution digestions and MS by Qq-TOF and FT-ICR-MS [6]. This resulted in a sharp increase in the identified protein numbers to 314 membrane proteins and 252 cytosolic proteins. They have also classified each protein according to their possible function using bioinformatics tools namely, cellular transport, metabolism, and protein complex formations while most membrane proteins, in addition to their role in cytoskeletal networks, account for signal transduction. We have earlier presented a review on the red cell proteomic studies discussing the scenario of differential expression of red cell protein in hemoglobin disorders [7]. Since then the latest review by Goodman’s group using the latest proteomic technique, current erythrocyte protein count stands at 2289 [8]. But we believe with increased sensitivity of detection, in future we could detect even very low copy number proteins and this list would expand. 2.1 Erythrocyte membrane proteomics The first classical 2DE–MALDI-MS based proteomics study was carried out by Low and co-workers, where they have employed both 1D and 2D gel-based separation followed by MALDI-MS to identify a total of about 70 membrane proteins [9]. Rabilloud established more sensitive methods of gel staining leading to visualizing greater number of protein spots in 2DE [10]. Following this, few gel-based studies on erythrocyte membrane came up, describing protein deregulation in hereditary spherocytosis [11–15], sickle cell anemia [16–18], and hemoglobin E  (HbE) thalassemia [19]. Few novel proteins were identified in malaria parasite infected erythrocyte membrane [20]. Blue-native/SDS PAGE techniques coupled with CyDye labeling were used to identify proteins of erythrocyte membranes in hemolytic anemia patients [21]. It has also been previously observed that proteolytic and oxidative damage accounts for major changes in the membrane skeletal proteins such as band 4.1, band 4.2, and ␣-spectrin [22]. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics Clin. Appl. 2016, 10, 403–414
Pasini et al. could annotate 314 membrane proteins [6]. De Palma et al. performed proteomic analysis of intact erythrocytes, isolated membranes, and Triton X-100 treated soluble fraction and a skeletal pellet. Using multidimensional protein identification technology (MuDPIT) and linear ion trap LTQ MS they have identified 299 unique proteins of which 211 were identified as being from the membrane [23]. Recently, Speicher and colleagues have separated erythrocyte membrane proteins by 1D SDS PAGE and found that when they cut the gels into 30 uniform slices, and performed ingel digestion of each, they were able to identify 842 unique proteins utilizing an LTQ-Orbitrap XL MS [24]. Till date most of the studies on posttranslational modifications of erythrocyte proteins have focused on the membrane proteins. Membrane skeletal proteins such as spectrin [25, 26], ankyrin [27], band 3 [28], band 4.1 [29, 30], adducin [31], and dematin [32] are known to be regulated by phosphorylation that in turn regulate the erythrocyte membrane architecture. Phosphoproteome of erythrocyte membranes is altered in sickle cell disease (SCD) patients as well as upon storage purpose for blood transfusion [33–35]. However, the phosphoproteome of erythrocyte is understudied and even profiling of all the phosphosites is far from completion. Ubiquitin is quite abundant inside erythrocytes and proteomics studies also predicted many erythrocyte proteins to be ubiquitinated [6]. From previous biochemical studies membrane proteins such as ␣-spectrin, ankyrin, and band 3 have been reported to be ubiquitinated [36–39]. Ubiquitination was never studied on a global proteomics scale inside erythrocyte and their functional implications remain an enigma till date. Delobel and coworkers studied carbonylation of proteins, in different populations, the hemoglobin-depleted soluble fraction, integral membrane, and cytoskeleton membrane protein fractions, showing the effect of oxidation on those fractions due to storage [40]. A glycomics study differentiates proteins based on their characteristic sugars thus contributing to isoform identification [41, 42]. This technique helps in the characterization of the erythrocyte carbohydrate heterogeneity responsible for blood group antigen diversity and immunology and provides possible insights in carbohydrate transfer during erythrocyte interaction with other cell populations (e.g. blood and endothelial cells) [43].
2.2 Erythrocyte cytosolic proteomics The numbers of gel-based proteomics studies on erythrocyte cytosol have been surprisingly low. The specific reason for this low number of studies was the presence of high abundance of hemoglobin, which masks the presence of all other proteins in the cytosol [7]. D’Amici et al. detected 838 protein spots in total by analyzing fractions using 2D IEFSDS-PAGE [44]. There are limited number of commercially available kits for hemoglobin depletion such as Proteominer [45–47] and Hemovoid (thalassemia major, TM) depletion www.clinical.proteomics-journal.com
405
Proteomics Clin. Appl. 2016, 10, 403–414
[48] in addition to Ni(II)-Sepharose affinity chromatography followed by ion-exchange chromatography [49] and SPSephadex (Sulphopropyl-Sephadex) cation exchanger matrix to deplete hemoglobin in cyano-met form [50]. This resulted in a 20-fold increase of visual detection of protein spots in a Coomassie-stained 17 cm 2D gel (418 spots vs.
