CONICET | Buscador de Institutos y Recursos Humanos

Ca2+ acts as a ubiquitous second messenger to control a wide variety of physiological processes. Ca2+ signals trigger fertilization, control development and differentiation, coordinate cellular functions, and have an essential role in the platelet activation cascade. The increase in platelet intracellular calcium concentration regulates platelet integrin activation, granule secretion and lead to platelet activation and thrombus formation. A tight regulation of Ca+2 signaling is necessary to prevent inappropriate or excessive thrombus formation. In many cells, including platelets, the adequate Ca2+ homeostasis is regulated by various mechanisms: sarco/endoplasmic reticulum Ca2+ ATPases (SERCA) and plasma membrane Ca2+ ATPases (PMCA) that utilize ATP hydrolysis to pump Ca2+ into the endoplasmic reticulum or outside the cell, respectively, and the Na+/Ca2+ exchangers (NCX protein families) that extrude cytosolic Ca2+ in exchange for extracellular Na+. There are two different NCX protein families: one that is dependent on K+ (NCKX), and other K+-independent (NCX). Three different genes for Na+/ Ca2 exchanger (NCX1, NCX2, and NCX3) have been described. NCX1 exchanger is highly expressed in the heart and NCX2, and NCX3 are found in lower amounts in brain, kidney, smooth muscle, and skeletal muscle. They have a stoichiometry of 3 Na+: 1Ca2+. On the other hand, five different NCKX genes have been identified (NCKX1, NCKX2, NCKX3, NCKX4 and NCKX5). The tissue distribution of NCKX1 is more restricted, originally thought to be limited to the retinal photoreceptors. In the NCKX1 exchanger, K+ is co-transported with Ca2+ with a stoichiometry of 4 Na+/ (l Ca2+ + 1 K+). Na+/ Ca2+ exchange activity has been demonstrated in human platelet plasma membrane vesicles by Kimura et al 1993. Ca2+ influx is dependent on the presence of extracellular K+, where the NCKX was found to be identical to the human retinal rod isoform, however, its physiologic function is not well understood. Previous reports have shown that the retinal rod Na+/Ca+-K+ exchanger contains a long N-terminal extracellular hydrophilic segment that is heavily glycosylated and contains significant amounts of sialic acid residues, but neither the nature of the exchanger nor its role in platelet physiology has been fully characterized. Therefore, the aim of this report is to contribute to a better understanding and characterization of the NCX1 and NCKX1 exchangers in a human megakaryocytic cell line (DAMI cells) and human platelet, in order to elucidate the expression and glycosylation status in human platelets. Here, we demonstrate the expression of the Na+/ Ca2+ (NCX1) and the Na+/Ca+-K+ (NCKX1) proteins in human megakaryocytic cell line and human normal platelets. We observed different bands, when analyzed by Western blot on polyacrylamide electrophoresis gels (SDS- PAGE). NCX1 displays a band of an apparent size of 120 KDa, and an additional band of 70 KDa, corresponding to the mature protein and a proteolytic fragment, respectively. Parallel studies in NCKX1 exchanger show 3 major bands, one of an apparent size of 220 KDa, and the other band also present in the Na+/ Ca2 exchanger. These results are consistent with the NCX and NCKX protein electrophoresis patterns previously reported in the bibliography in cardiac muscle and retinal rod photoreceptors. In order to identify the possible glycosylation sites of the exchangers, the sequence proteins were analyzed by computational programs (NetGlyc). This analysis showed 2 possible N-glycosylation sites in NCX protein (Asp 44 and 136) and different N- and O-glycosylation sites for NCKX1 in the hydrophilic N-terminal segment (Asp 290, 552 and 664; and Thr 329, 340, 412 and 413). Moreover, microsomal fractions enriched in plasma membrane of megakaryocytes cell lines were analyzed by SDS-PAGE and Western blot, incubated with different lectins (Concanavalin A and Wheat Germ Agglutinin), showing the presence of mannose and N-acetyl glucosamine (GlcNAC) and sialic acid residues, respectively. To characterize the glycosylation status of both exchangers in human megakaryocytic cell lines (DAMI), cells where treated with the N-linked glycosylation inhibitor, tunicamicyn (10-100 ìg/ml) for different hours (0 to 48 hs). Furthermore, to establish the presence of O-linked or N-linked sialo-oligosaccharides, enzymatic deglycosilation studies were performed. Protein exchangers were first treated with endo-N-glycosidase F (PGNaseF). No changes in mobility of the exchangers were observed after tunicamicyn and endo-N-glycosidase F treatment. These results are consistent with the ones reported by Kim et al in 1998, where they postulate that either these sites might not be N-glycosylated or the oligosaccharide chains could be small and their removal does not affect the mobility of the exchanger on SDS-polyacrylamide gels. O-glycosylation studies and lectin affinity chromatography analyses are in progress. Further studies will be necessary to identify the glycosylation status of both exchangers in human platelets. Future studies are destined to elucidate the role of the glycosylation in the targeting and activity of the NCX and NCKX exchangers in human megakaryocytic cell line and in human platelets and to correlate the possible role of these exchangers with the thrombus-hemorrhagic events shown in Congenital Disorders of Glycosylation diseases. There are two different NCX protein families: one that is dependent on K+ (NCKX), and other K+-independent (NCX). Three different genes for Na+/ Ca2 exchanger (NCX1, NCX2, and NCX3) have been described. NCX1 exchanger is highly expressed in the heart and NCX2, and NCX3 are found in lower amounts in brain, kidney, smooth muscle, and skeletal muscle. They have a stoichiometry of 3 Na+: 1Ca2+. On the other hand, five different NCKX genes have been identified (NCKX1, NCKX2, NCKX3, NCKX4 and NCKX5). The tissue distribution of NCKX1 is more restricted, originally thought to be limited to the retinal photoreceptors. In the NCKX1 exchanger, K+ is co-transported with Ca2+ with a stoichiometry of 4 Na+/ (l Ca2+ + 1 K+). Na+/ Ca2+ exchange activity has been demonstrated in human platelet plasma membrane vesicles by Kimura et al 1993. Ca2+ influx is dependent on the presence of extracellular K+, where the NCKX was found to be identical to the human retinal rod isoform, however, its physiologic function is not well understood. Previous reports have shown that the retinal rod Na+/Ca+-K+ exchanger contains a long N-terminal extracellular hydrophilic segment that is heavily glycosylated and contains significant amounts of sialic acid residues, but neither the nature of the exchanger nor its role in platelet physiology has been fully characterized. Therefore, the aim of this report is to contribute to a better understanding and characterization of the NCX1 and NCKX1 exchangers in a human megakaryocytic cell line (DAMI cells) and human platelet, in order to elucidate the expression and glycosylation status in human platelets. Here, we demonstrate the expression of the Na+/ Ca2+ (NCX1) and the Na+/Ca+-K+ (NCKX1) proteins in human megakaryocytic cell line and human normal platelets. We observed different bands, when analyzed by Western blot on polyacrylamide electrophoresis gels (SDS- PAGE). NCX1 displays a band of an apparent size of 120 KDa, and an additional band of 70 KDa, corresponding to the mature protein and a proteolytic fragment, respectively. Parallel studies in NCKX1 exchanger show 3 major bands, one of an apparent size of 220 KDa, and the other band also present in the Na+/ Ca2 exchanger. These results are consistent with the NCX and NCKX protein electrophoresis patterns previously reported in the bibliography in cardiac muscle and retinal rod photoreceptors. In order to identify the possible glycosylation sites of the exchangers, the sequence proteins were analyzed by computational programs (NetGlyc). This analysis showed 2 possible N-glycosylation sites in NCX protein (Asp 44 and 136) and different N- and O-glycosylation sites for NCKX1 in the hydrophilic N-terminal segment (Asp 290, 552 and 664; and Thr 329, 340, 412 and 413). Moreover, microsomal fractions enriched in plasma membrane of megakaryocytes cell lines were analyzed by SDS-PAGE and Western blot, incubated with different lectins (Concanavalin A and Wheat Germ Agglutinin), showing the presence of mannose and N-acetyl glucosamine (GlcNAC) and sialic acid residues, respectively. To characterize the glycosylation status of both exchangers in human megakaryocytic cell lines (DAMI), cells where treated with the N-linked glycosylation inhibitor, tunicamicyn (10-100 ìg/ml) for different hours (0 to 48 hs). Furthermore, to establish the presence of O-linked or N-linked sialo-oligosaccharides, enzymatic deglycosilation studies were performed. Protein exchangers were first treated with endo-N-glycosidase F (PGNaseF). No changes in mobility of the exchangers were observed after tunicamicyn and endo-N-glycosidase F treatment. These results are consistent with the ones reported by Kim et al in 1998, where they postulate that either these sites might not be N-glycosylated or the oligosaccharide chains could be small and their removal does not affect the mobility of the exchanger on SDS-polyacrylamide gels. O-glycosylation studies and lectin affinity chromatography analyses are in progress. Further studies will be necessary to identify the glycosylation status of both exchangers in human platelets. Future studies are destined to elucidate the role of the glycosylation in the targeting and activity of the NCX and NCKX exchangers in human megakaryocytic cell line and in human platelets and to correlate the possible role of these exchangers with the thrombus-hemorrhagic events shown in Congenital Disorders of Glycosylation diseases. Na+/ Ca2+ exchange activity has been demonstrated in human platelet plasma membrane vesicles by Kimura et al 1993. Ca2+ influx is dependent on the presence of extracellular K+, where the NCKX was found to be identical to the human retinal rod isoform, however, its physiologic function is not well understood. Previous reports have shown that the retinal rod Na+/Ca+-K+ exchanger contains a long N-terminal extracellular hydrophilic segment that is heavily glycosylated and contains significant amounts of sialic acid residues, but neither the nature of the exchanger nor its role in platelet physiology has been fully characterized. Therefore, the aim of this report is to contribute to a better understanding and characterization of the NCX1 and NCKX1 exchangers in a human megakaryocytic cell line (DAMI cells) and human platelet, in order to elucidate the expression and glycosylation status in human platelets. Here, we demonstrate the expression of the Na+/ Ca2+ (NCX1) and the Na+/Ca+-K+ (NCKX1) proteins in human megakaryocytic cell line and human normal platelets. We observed different bands, when analyzed by Western blot on polyacrylamide electrophoresis gels (SDS- PAGE). NCX1 displays a band of an apparent size of 120 KDa, and an additional band of 70 KDa, corresponding to the mature protein and a proteolytic fragment, respectively. Parallel studies in NCKX1 exchanger show 3 major bands, one of an apparent size of 220 KDa, and the other band also present in the Na+/ Ca2 exchanger. These results are consistent with the NCX and NCKX protein electrophoresis patterns previously reported in the bibliography in cardiac muscle and retinal rod photoreceptors. In order to identify the possible glycosylation sites of the exchangers, the sequence proteins were analyzed by computational programs (NetGlyc). This analysis showed 2 possible N-glycosylation sites in NCX protein (Asp 44 and 136) and different N- and O-glycosylation sites for NCKX1 in the hydrophilic N-terminal segment (Asp 290, 552 and 664; and Thr 329, 340, 412 and 413). Moreover, microsomal fractions enriched in plasma membrane of megakaryocytes cell lines were analyzed by SDS-PAGE and Western blot, incubated with different lectins (Concanavalin A and Wheat Germ Agglutinin), showing the presence of mannose and N-acetyl glucosamine (GlcNAC) and sialic acid residues, respectively. To characterize the glycosylation status of both exchangers in human megakaryocytic cell lines (DAMI), cells where treated with the N-linked glycosylation inhibitor, tunicamicyn (10-100 ìg/ml) for different hours (0 to 48 hs). Furthermore, to establish the presence of O-linked or N-linked sialo-oligosaccharides, enzymatic deglycosilation studies were performed. Protein exchangers were first treated with endo-N-glycosidase F (PGNaseF). No changes in mobility of the exchangers were observed after tunicamicyn and endo-N-glycosidase F treatment. These results are consistent with the ones reported by Kim et al in 1998, where they postulate that either these sites might not be N-glycosylated or the oligosaccharide chains could be small and their removal does not affect the mobility of the exchanger on SDS-polyacrylamide gels. O-glycosylation studies and lectin affinity chromatography analyses are in progress. Further studies will be necessary to identify the glycosylation status of both exchangers in human platelets. Future studies are destined to elucidate the role of the glycosylation in the targeting and activity of the NCX and NCKX exchangers in human megakaryocytic cell line and in human platelets and to correlate the possible role of these exchangers with the thrombus-hemorrhagic events shown in Congenital Disorders of Glycosylation diseases. significant amounts of sialic acid residues, but neither the nature of the exchanger nor its role in platelet physiology has been fully characterized. Therefore, the aim of this report is to contribute to a better understanding and characterization of the NCX1 and NCKX1 exchangers in a human megakaryocytic cell line (DAMI cells) and human platelet, in order to elucidate the expression and glycosylation status in human platelets. Here, we demonstrate the expression of the Na+/ Ca2+ (NCX1) and the Na+/Ca+-K+ (NCKX1) proteins in human megakaryocytic cell line and human normal platelets. We observed different bands, when analyzed by Western blot on polyacrylamide electrophoresis gels (SDS- PAGE). NCX1 displays a band of an apparent size of 120 KDa, and an additional band of 70 KDa, corresponding to the mature protein and a proteolytic fragment, respectively. Parallel studies in NCKX1 exchanger show 3 major bands, one of an apparent size of 220 KDa, and the other band also present in the Na+/ Ca2 exchanger. These results are consistent with the NCX and NCKX protein electrophoresis patterns previously reported in the bibliography in cardiac muscle and retinal rod photoreceptors. In order to identify the possible glycosylation sites of the exchangers, the sequence proteins were analyzed by computational programs (NetGlyc). This analysis showed 2 possible N-glycosylation sites in NCX protein (Asp 44 and 136) and different N- and O-glycosylation sites for NCKX1 in the hydrophilic N-terminal segment (Asp 290, 552 and 664; and Thr 329, 340, 412 and 413). Moreover, microsomal fractions enriched in plasma membrane of megakaryocytes cell lines were analyzed by SDS-PAGE and Western blot, incubated with different lectins (Concanavalin A and Wheat Germ Agglutinin), showing the presence of mannose and N-acetyl glucosamine (GlcNAC) and sialic acid residues, respectively. To characterize the glycosylation status of both exchangers in human megakaryocytic cell lines (DAMI), cells where treated with the N-linked glycosylation inhibitor, tunicamicyn (10-100 ìg/ml) for different hours (0 to 48 hs). Furthermore, to establish the presence of O-linked or N-linked sialo-oligosaccharides, enzymatic deglycosilation studies were performed. Protein exchangers were first treated with endo-N-glycosidase F (PGNaseF). No changes in mobility of the exchangers were observed after tunicamicyn and endo-N-glycosidase F treatment. These results are consistent with the ones reported by Kim et al in 1998, where they postulate that either these sites might not be N-glycosylated or the oligosaccharide chains could be small and their removal does not affect the mobility of the exchanger on SDS-polyacrylamide gels. O-glycosylation studies and lectin affinity chromatography analyses are in progress. Further studies will be necessary to identify the glycosylation status of both exchangers in human platelets. Future studies are destined to elucidate the role of the glycosylation in the targeting and activity of the NCX and NCKX exchangers in human megakaryocytic cell line and in human platelets and to correlate the possible role of these exchangers with the thrombus-hemorrhagic events shown in Congenital Disorders of Glycosylation diseases. In order to identify the possible glycosylation sites of the exchangers, the sequence proteins were analyzed by computational programs (NetGlyc). This analysis showed 2 possible N-glycosylation sites in NCX protein (Asp 44 and 136) and different N- and O-glycosylation sites for NCKX1 in the hydrophilic N-terminal segment (Asp 290, 552 and 664; and Thr 329, 340, 412 and 413). Moreover, microsomal fractions enriched in plasma membrane of megakaryocytes cell lines were analyzed by SDS-PAGE and Western blot, incubated with different lectins (Concanavalin A and Wheat Germ Agglutinin), showing the presence of mannose and N-acetyl glucosamine (GlcNAC) and sialic acid residues, respectively. To characterize the glycosylation status of both exchangers in human megakaryocytic cell lines (DAMI), cells where treated with the N-linked glycosylation inhibitor, tunicamicyn (10-100 ìg/ml) for different hours (0 to 48 hs). Furthermore, to establish the presence of O-linked or N-linked sialo-oligosaccharides, enzymatic deglycosilation studies were performed. Protein exchangers were first treated with endo-N-glycosidase F (PGNaseF). No changes in mobility of the exchangers were observed after tunicamicyn and endo-N-glycosidase F treatment. These results are consistent with the ones reported by Kim et al in 1998, where they postulate that either these sites might not be N-glycosylated or the oligosaccharide chains could be small and their removal does not affect the mobility of the exchanger on SDS-polyacrylamide gels. O-glycosylation studies and lectin affinity chromatography analyses are in progress. Further studies will be necessary to identify the glycosylation status of both exchangers in human platelets. Future studies are destined to elucidate the role of the glycosylation in the targeting and activity of the NCX and NCKX exchangers in human megakaryocytic cell line and in human platelets and to correlate the possible role of these exchangers with the thrombus-hemorrhagic events shown in Congenital Disorders of Glycosylation diseases. To characterize the glycosylation status of both exchangers in human megakaryocytic cell lines (DAMI), cells where treated with the N-linked glycosylation inhibitor, tunicamicyn (10-100 ìg/ml) for different hours (0 to 48 hs). Furthermore, to establish the presence of O-linked or N-linked sialo-oligosaccharides, enzymatic deglycosilation studies were performed. Protein exchangers were first treated with endo-N-glycosidase F (PGNaseF). No changes in mobility of the exchangers were observed after tunicamicyn and endo-N-glycosidase F treatment. These results are consistent with the ones reported by Kim et al in 1998, where they postulate that either these sites might not be N-glycosylated or the oligosaccharide chains could be small and their removal does not affect the mobility of the exchanger on SDS-polyacrylamide gels. O-glycosylation studies and lectin affinity chromatography analyses are in progress. Further studies will be necessary to identify the glycosylation status of both exchangers in human platelets. Future studies are destined to elucidate the role of the glycosylation in the targeting and activity of the NCX and NCKX exchangers in human megakaryocytic cell line and in human platelets and to correlate the possible role of these exchangers with the thrombus-hemorrhagic events shown in Congenital Disorders of Glycosylation diseases. Future studies are destined to elucidate the role of the glycosylation in the targeting and activity of the NCX and NCKX exchangers in human megakaryocytic cell line and in human platelets and to correlate the possible role of these exchangers with the thrombus-hemorrhagic events shown in Congenital Disorders of Glycosylation diseases.