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Narla Mohandas - One of the best experts on this subject based on the ideXlab platform.
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Similarities and differenCes in the struCture and funCtion of 4.1G and 4.1R135, two protein 4.1 paralogues expressed in erythroid Cells.
The Biochemical journal, 2010Co-Authors: Wataru Nunomura, Narla Mohandas, Marilyn Parra, Philippe Gascard, Kengo Kinoshita, Yuichi TakakuwaAbstract:Membrane skeletal protein 4.1R is the prototypiCal member of a family of four highly paralogous proteins that inClude 4.1G, 4.1N and 4.1B. Two isoforms of 4.1R (4.1R 135 and 4.1R 80 ), as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R 80 is present in mature erythroCytes. Although the funCtion of 4.1R isoforms in erythroid Cells has been well CharaCterized, there is little or no information on the funCtion of 4.1G in these Cells. In the present study, we performed detailed CharaCterization of the interaCtion of 4.1G with various erythroid membrane proteins and the regulation of these interaCtions by CalCium-saturated Calmodulin. Like both isoforms of 4.1R, 4.1G bound to band 3, GlyCophorin C, CD44, p55 and Calmodulin. While both 4.1G and 4.1R 135 interaCt with similar affinity with CD44 and p55, there are signifiCant differenCes in the affinity of their interaCtion with band 3 and GlyCophorin C. This differenCe in affinity is related to the non-Conserved N-terminal headpieCe region of the two proteins that is upstream of the 30 kDa membrane-binding domain that harbours the binding sites for the various membrane proteins. The headpieCe region of 4.1G also Contains a high-affinity CalCium-dependent Calmodulin-binding site that plays a key role in modulating its interaCtion with various membrane proteins. We suggest that expression of the two paralogues of protein 4.1 with different affinities for band 3 and GlyCophorin C is likely to play a role in assembly of these two membrane proteins during terminal erythroid differentiation.
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hereditary spheroCytosis and hereditary elliptoCytosis aberrant protein sorting during erythroblast enuCleation
Blood, 2010Co-Authors: Marcela Salomao, Narla Mohandas, Ke Chen, Jonathan Villalobos, Joel Anne ChasisAbstract:During erythroblast enuCleation, membrane proteins distribute between extruded nuClei and retiCuloCytes. In hereditary spheroCytosis (HS) and hereditary elliptoCytosis (HE), defiCienCies of membrane proteins, in addition to those enCoded by the mutant gene, oCCur. ElliptoCytes, resulting from protein 4.1R gene mutations, laCk not only 4.1R but also GlyCophorin C, whiCh links the Cytoskeleton and bilayer. In HS resulting from ankyrin-1 mutations, band 3, Rh-assoCiated antigen, and GlyCophorin A are defiCient. The Current study was undertaken to explore whether aberrant protein sorting, during enuCleation, Creates these membrane-spanning protein defiCienCies. We found that although GlyCophorin C sorts to retiCuloCytes normally, it distributes to nuClei in 4.1R-defiCient HE Cells. Further, GlyCophorin A and Rh-assoCiated antigen, whiCh normally partition predominantly to retiCuloCytes, distribute to both nuClei and retiCuloCytes in an ankyrin-1-defiCient murine model of HS. We ConClude that aberrant protein sorting is one meChanistiC basis for protein defiCienCies in HE and HS.
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adduCin forms a bridge between the erythroCyte membrane and its Cytoskeleton and regulates membrane Cohesion
Blood, 2009Co-Authors: William A. Anong, Narla Mohandas, David M. Bodine, Taina Franco, Haiyan Chu, Tahlia L Weis, Emily E Devlin, Philip S. LowAbstract:The erythroCyte membrane skeleton is the best understood Cytoskeleton. BeCause its protein Components have homologs in virtually all other Cells, the membrane serves as a fundamental model of biologiC membranes. Modern textbooks portray the membrane as a 2-dimensional speCtrin-based membrane skeleton attaChed to a lipid bilayer through 2 linkages: band 3–ankyrin–β-speCtrin and GlyCophorin C–protein 4.1–β-speCtrin.1–7 Although evidenCe supports an essential role for the first bridge in regulating membrane Cohesion, rupture of the GlyCophorin C–protein 4.1 interaCtion has little effeCt on membrane stability.8 We demonstrate the existenCe of a novel band 3–adduCin–speCtrin bridge that ConneCts the speCtrin/aCtin/protein 4.1 junCtional Complex to the bilayer. As rupture of this bridge leads to spontaneous membrane fragmentation, we ConClude that the band 3–adduCin–speCtrin bridge is important to membrane stability. The required reloCation of part of the band 3 population to the speCtrin/aCtin junCtional Complex and its formation of a new bridge with adduCin neCessitates a signifiCant revision of aCCepted models of the erythroCyte membrane.
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Protein 4.1R-dependent multiprotein Complex: New insights into the struCtural organization of the red blood Cell membrane
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Marcela Salomao, Joel Anne Chasis, Xihui Zhang, Yang Yang, Soohee Lee, John H. Hartwig, Narla MohandasAbstract:Protein 4.1R (4.1R) is a multifunCtional Component of the red Cell membrane. It forms a ternary Complex with aCtin and speCtrin, whiCh defines the nodal junCtions of the membrane-skeletal network, and its attaChment to the transmembrane protein GlyCophorin C Creates a bridge between the protein network and the membrane bilayer. We now show that deletion of 4.1R in mouse red Cells leads to a large diminution of aCtin aCCompanied by extensive loss of Cytoskeletal lattiCe struCture, with formation of bare areas of membrane. Whereas band 3, the preponderant transmembrane Constituent, and proteins known to be assoCiated with it are present in normal or inCreased amounts, GlyCophorin C is missing and XK, Duffy, and Rh are muCh reduCed in the 4.1R-defiCient Cells. The inferenCe that these are assoCiated with 4.1R was borne out by the results of in vitro pull-down assays. Furthermore, whereas Western blot analysis showed normal levels of band 3 and Kell, flow CytometriC analysis using an antibody against the extraCellular region of band 3 or Kell revealed reduCtion of these two proteins, suggesting a Conformational Change of band 3 and Kell epitopes. Taken together, we suggest that 4.1R organizes a maCromoleCular Complex of skeletal and transmembrane proteins at the junCtional node and that perturbation of this maCromoleCular Complex not only is responsible for the well CharaCterized membrane instability but may also remodel the red Cell surfaCe.
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AdduCin Forms a Bridge between the SpeCtrin-ACtin JunCtional Complex and Band 3.
Blood, 2005Co-Authors: William A. Anong, Narla Mohandas, David M. Bodine, Nicholas O. Markham, Patrick G. Gallagher, Philip S. LowAbstract:The ClassiCal model of the human erythroCyte membrane (RBCM) shows two bridges ConneCting the lipid bilayer to the membrane skeleton: 1) a bridge attaChing the CytoplasmiC domain of band 3 (CDB3) to ankyrin, whiCh in turn binds β-speCtrin, and 2) a bridge linking the CytoplasmiC domain of GlyCophorin C to protein 4.1, whiCh in turn binds the speCtrin-aCtin junCtional Complex. ReCent data, however, suggest while disruption of the band 3-ankyrin- β-speCtrin interaCtion profoundly alters membrane stability, disruption of the GlyCophorin C-protein 4.1R linkage has no effeCt on membrane meChaniCal properties. In a searCh for additional bridges between the junCtional Complex and the bilayer, we disCovered that adduCin, a Component of the speCtrin-aCtin junCtional Complex, binds to band 3. EvidenCe for this interaCtion derives from a number of different experimental strategies. We have been able to show that: i) photoaCtivation of sulfo-SBED-labeled adduCin reConstituted onto the RBCM leads to label transfer to band 3, ii) adduCin binds to KI-stripped IOVs with a Kd of 280 nM and this binding is inhibited by an antibody to CDB3 (as well as by unlabeled adduCin), iii) IOVs derived from normal erythroCytes retain adduCin, whereas similar IOVs prepared from erythroCytes defiCient in band 3 retain very little adduCin, iv) the tail domain (but not the headpieCe domain) of β-adduCin binds to KI-stripped IOVs and this binding is Competed by both anti-CDB3 and intaCt adduCin, v) GST-labeled β-adduCin tail domain Can pull down band 3 in Co-pelleting studies and this Co-preCipitation is bloCked by anti-CDB3, and vi) the tail domain of β-adduCin direCtly binds CDB3. BeCause adduCin is an important struCtural Component of the junCtional Complex, these data suggest that the junCtional Complex is linked to the RBCM via CDB3 and that part of the band 3 population must be loCated adjaCent to the junCtional Complex. This putative new bridge between the RBCM and the speCtrin-aCtin skeleton may also help explain why β-adduCin knoCkout miCe have unstable erythroCyte membranes.
Athar H Chishti - One of the best experts on this subject based on the ideXlab platform.
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ReCeptor-Based IdentifiCation of Novel Peptide Ligands as Inhibitors of Blood Stage Malaria.
Blood, 2007Co-Authors: Sonja B Lauterbach, Roberto Lanzillotti, Huiqing Chen, Ravi S. Kane, Theresa L. Coetzer, Athar H ChishtiAbstract:Malaria parasite Plasmodium falCiparum uses multiple reCeptors on the surfaCe of human red blood Cells to attaCh and invade host Cells during blood stage infeCtion. GlyCophorins, inCluding GlyCophorin A, B, and C have been impliCated as host reCeptors and play an important role during Plasmodium falCiparum invasion in human erythroCytes, partiCularly in the sialiC aCid-dependent parasite strains. To identify the parasite proteins that Could bind to human GlyCophorins, we sCreened a phage display CDNA library of P. falCiparum (FCR3 strain, a sialiC aCid-dependent strain) using human GlyCophorins and native intaCt human erythroCytes as bait. After four rounds of library sCreening and panning, 35 phage Clones were identified that bound to purified GlyCophorins immobilized on the plastiC surfaCe. DNA sequenCing of 12 phage Clones revealed that they enCode the same 7-amino aCid sequenCe, ETTLKSF. Using immobilized intaCt human erythroCytes, a similar sCreening strategy led to the isolation of additional 20 phage Clones, and the DNA sequenCing of 6 suCh Clones again revealed the same 7-amino aCid sequenCe, ETTLKSF. In vitro binding of synthetiC ETTLKSF peptide to purified GlyCophorins and intaCt erythroCytes was Confirmed by ELISA and indireCt immunofluoresCenCe assays. Pull-down experiments demonstrated that the ETTLKSF peptide speCifiCally interaCts with GlyCophorin C, but not with GlyCophorin A and B, on human erythroCytes. The synthetiC ETTLKSF peptide bloCked merozoite invasion of P. falCiparum in human erythroCytes in a dose-dependent manner, whereas the Control peptides were without any effeCt. We have named the ETTLKSF peptide as GBL-1, the GlyCophorin Binding Ligand-1. We propose that further CharaCterization of GBL-1, the first peptide ligand that speCifiCally binds to human GlyCophorin C, Could lead to the development of novel anti-malarial agents that prevent P. falCiparum invasion in human erythroCytes via the GlyCophorin C reCeptor.
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THE PDZ DOMAIN OF HUMAN ERYTHROCYTE P55 MEDIATES ITS BINDING TO THE CYTOPLASMIC CARBOXYL TERMINUS OF GlyCophorin C: ANALYSIS OF THE BINDING INTERFACE BY IN VITRO MUTAGENESIS
The Journal of biological chemistry, 1997Co-Authors: Shirin M Marfatia, João H. Morais-cabral, Anthony C. Kim, Olwyn Byron, Athar H ChishtiAbstract:The PDZ domain, also known as the GLGF repeat/DHR domain, is an ∼90-amino aCid motif disCovered in a reCently identified family of proteins termed MAGUKs (membrane-assoCiated guanylatekinase homologues). SequenCe Comparison analysis has sinCe identified PDZ domains in over 50 proteins. Like SH2 and SH3 domains, the PDZ domains mediate speCifiC protein-protein interaCtions, whose speCifiCities appear to be diCtated by the primary struCture of the PDZ domain as well as its binding target. Using reCombinant fusion proteins and a blot overlay assay, we show that a single Copy of the PDZ domain in human erythroCyte p55 binds to the Carboxyl terminus of the CytoplasmiC domain of human erythroid GlyCophorin C. Deletion mutagenesis of 21 amino aCids at the amino terminus of the p55 PDZ domain Completely abrogates its binding aCtivity for GlyCophorin C. Using an alanine sCan and surfaCe plasmon resonanCe teChnique, we identify residues in the CytoplasmiC domain of GlyCophorin C that are CritiCal for its interaCtion with the PDZ domain. The reCognition speCifiCity of the p55 PDZ domain appears to be unique, sinCe the three PDZ domains of hDlg (human lymphoCyte homologue of the DrosophiladisCs large tumor suppressor) do not bind the CytoplasmiC domain of GlyCophorin C. Taken together with our previous studies, these results Complete the identifiCation of interaCting domains in the ternary Complex between p55, GlyCophorin C, and protein 4.1. ImpliCations of these findings are disCussed in terms of binding speCifiCity and the regulation of Cytoskeleton-membrane interaCtions.
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reduCed invasion and growth of plasmodium falCiparum into elliptoCytiC red blood Cells with a Combined defiCienCy of protein 4 1 GlyCophorin C and p55
Blood, 1996Co-Authors: Athar H Chishti, J Palek, Derek R Fisher, G J MaaloufAbstract:In this investigation, we have measured the invasion and growth of the malaria parasite Plasmodium falCiparum into elliptoCytiC red blood Cells (RBCs) obtained from subjeCts with homozygous hereditary elliptoCytosis. These elliptoCytiC RBCs have been previously CharaCterized to possess moleCular defeCts in protein 4.1 and GlyCophorin C. Our results show that the invasion of Plasmodium falCiparum into these protein 4.1 (-) RBCs is signifiCantly reduCed. GlyCophorin C (-) LeaCh RBCs were similarly resistant to parasite invasion in vitro. The intraCellular development of parasites that invaded protein 4.1 (-) RBCs was also dramatiCally reduCed. In Contrast, no suCh reduCtion of intraCellular parasite growth was observed in the GlyCophorin C (-) LeaCh RBCs. In ConjunCtion with our reCent finding that a third protein termed p55 is also defiCient in protein 4.1 (-) and GlyCophorin C (-) RBCs, the present data undersCore the importanCe of the membrane-assoCiated ternary Complex between protein 4.1, GlyCophorin C, and p55 during the invasion and growth of malaria parasites into human RBCs.
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identifiCation of the protein 4 1 binding interfaCe on GlyCophorin C and p55 a homologue of the drosophila disCs large tumor suppressor protein
Journal of Biological Chemistry, 1995Co-Authors: Shirin M Marfatia, Daniel Branton, Athar H ChishtiAbstract:AbstraCt Protein 4.1 is the prototype of a family of proteins that inClude ezrin, talin, brain tumor suppressor merlin, and tyrosine phosphatases. All members of the protein 4.1 superfamily share a highly Conserved N-terminal 30-kDa domain whose biologiCal funCtion is poorly understood. It is believed that the attaChment of the Cytoskeleton to the membrane may be mediated via this 30-kDa domain, a funCtion that requires formation of multiprotein Complexes at the plasma membrane. In this investigation, synthetiCally tagged peptides and baCterially expressed proteins were used to map the protein 4.1 binding site on human erythroid GlyCophorin C, a transmembrane glyCoprotein, and on human erythroid p55, a palmitoylated peripheral membrane phosphoprotein. The results show that the 30-kDa domain of protein 4.1 binds to a 12-amino aCid segment within the CytoplasmiC domain of GlyCophorin C and to a positively Charged, 39-amino aCid motif in p55. SequenCes similar to this Charged motif are Conserved in other members of the p55 superfamily, inCluding the Drosophila disCs-large tumor suppressor protein. Our data provide new insights into how protein 4.1, GlyCophorin C, p55, and their non-erythroid homologues, interaCt with the Cytoskeleton to exert their physiologiCal effeCts.
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in vitro binding studies suggest a membrane assoCiated Complex between erythroid p55 protein 4 1 and GlyCophorin C
Journal of Biological Chemistry, 1994Co-Authors: Shirin M Marfatia, Daniel Branton, Athar H ChishtiAbstract:AbstraCt p55 is a palmitoylated peripheral membrane phosphoprotein of human erythroCytes. Primary struCture of p55 inCludes a single Copy of the SH3 motif, a COOH-terminal guanylate kinase domain, and an NH2-terminal domain of unknown funCtion. Although the funCtion of p55 is not known, interest in this Component has been heightened by its similarity to the Drosophila tumor suppressor disCs-large (dlg). In this report we provide evidenCe for the direCt assoCiation of p55 with the NH2-terminal 30-kDa domain of protein 4.1, a key Component of the erythroid membrane skeleton. In addition, p55 also binds to the CytoplasmiC domain of GlyCophorin C, a transmembrane protein of red blood Cells. We also provide evidenCe demonstrating the direCt assoCiation of the 30-kDa domain of protein 4.1 with the CytoplasmiC domain of GlyCophorin C. Taken together, these results suggest the existenCe of a novel ternary Complex at the erythroid plasma membrane involving protein 4.1, p55, and GlyCophorin C. SinCe isoforms of protein 4.1, p55, and GlyCophorin C are present in many non-erythroid Cells, the binding interaCtions may be prototypiCal of similar assoCiations that modulate Cytoskeletal-membrane linkage of broad signifiCanCe.
Ewa Jaskiewicz - One of the best experts on this subject based on the ideXlab platform.
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Plasmodium reiChenowi EBA-140 merozoite ligand binds to GlyCophorin D on Chimpanzee red blood Cells, shedding new light on origins of Plasmodium falCiparum.
Parasites & vectors, 2017Co-Authors: Agata Zerka, Radoslaw Kaczmarek, Marcin Czerwinski, Ewa JaskiewiczAbstract:All symptoms of malaria are Caused by the intraerythroCytiC proliferation of Plasmodium merozoites. Merozoites invade erythroCytes using multiple binding ligands that reCognise speCifiC surfaCe reCeptors. It has been suggested that adaptation of Plasmodium parasites to infeCt speCifiC hosts is driven by Changes in genes enCoding Plasmodium erythroCyte-binding ligands (EBL) and retiCuloCyte-binding ligands (RBL). Homologs of both EBL and RBL, inCluding the EBA-140 merozoite ligand, have been identified in P. falCiparum and P. reiChenowi, whiCh infeCt humans and Chimpanzees, respeCtively. The P. falCiparum EBA-140 was shown to bind human GlyCophorin C, a minor erythroCyte sialoglyCoprotein. Until now, the erythroCyte reCeptor for the P. reiChenowi EBA-140 remained unknown. The baCulovirus expression veCtor system was used to obtain the reCombinant EBA-140 Region II, and flow Cytometry and immunoblotting methods were applied to CharaCterise its speCifiCity. We showed that the Chimpanzee GlyCophorin D is the reCeptor for the P. reiChenowi EBA-140 ligand on Chimpanzee red blood Cells. We propose that the development of GlyCophorin C speCifiCity is spurred by the P. falCiparum lineage. We speCulate that the P. falCiparum EBA-140 evolved to hijaCk GPC on human erythroCytes during divergenCe from its ape anCestor.
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Plasmodium reiChenowi EBA-140 merozoite ligand binds to GlyCophorin D on Chimpanzee red blood Cells, shedding new light on origins of Plasmodium falCiparum
BMC, 2017Co-Authors: Agata Zerka, Radoslaw Kaczmarek, Marcin Czerwinski, Ewa JaskiewiczAbstract:AbstraCt BaCkground All symptoms of malaria are Caused by the intraerythroCytiC proliferation of Plasmodium merozoites. Merozoites invade erythroCytes using multiple binding ligands that reCognise speCifiC surfaCe reCeptors. It has been suggested that adaptation of Plasmodium parasites to infeCt speCifiC hosts is driven by Changes in genes enCoding Plasmodium erythroCyte-binding ligands (EBL) and retiCuloCyte-binding ligands (RBL). Homologs of both EBL and RBL, inCluding the EBA-140 merozoite ligand, have been identified in P. falCiparum and P. reiChenowi, whiCh infeCt humans and Chimpanzees, respeCtively. The P. falCiparum EBA-140 was shown to bind human GlyCophorin C, a minor erythroCyte sialoglyCoprotein. Until now, the erythroCyte reCeptor for the P. reiChenowi EBA-140 remained unknown. Methods The baCulovirus expression veCtor system was used to obtain the reCombinant EBA-140 Region II, and flow Cytometry and immunoblotting methods were applied to CharaCterise its speCifiCity. Results We showed that the Chimpanzee GlyCophorin D is the reCeptor for the P. reiChenowi EBA-140 ligand on Chimpanzee red blood Cells. ConClusions We propose that the development of GlyCophorin C speCifiCity is spurred by the P. falCiparum lineage. We speCulate that the P. falCiparum EBA-140 evolved to hijaCk GPC on human erythroCytes during divergenCe from its ape anCestor
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The baCulovirus-expressed binding region of Plasmodium falCiparum EBA-140 ligand and its GlyCophorin C binding speCifiCity.
PloS one, 2015Co-Authors: Joanna Rydzak, Radoslaw Kaczmarek, Marcin Czerwinski, Jolanta Lukasiewicz, Jolanta Tyborowska, Boguslaw Szewczyk, Ewa JaskiewiczAbstract:The erythroCyte binding ligand 140 (EBA-140) is a member of the Plasmodium falCiparum DBL family of erythroCyte binding proteins, whiCh are Considered as prospeCtive Candidates for malaria vaCCine development. The EBA-140 ligand is a paralogue of the well-CharaCterized P. falCiparum EBA-175 protein. They share homology of domain struCture, inCluding Region II, whiCh Consists of two homologous F1 and F2 domains and is responsible for ligand-erythroCyte reCeptor interaCtion during invasion. In this report we desCribe, for the first time, the GlyCophorin C speCifiCity of the reCombinant, baCulovirus-expressed binding region (Region II) of P. falCiparum EBA-140 ligand. It was found that the reCombinant EBA-140 Region II binds to the endogenous and reCombinant GlyCophorin C, but does not bind to GerbiCh-type GlyCophorin C, neither normal nor reCombinant, whiCh laCks amino aCid residues 36–63 of its polypeptide Chain. Our results emphasize the CruCial role of this GlyCophorin C region in EBA-140 ligand binding. Moreover, the EBA-140 Region II did not bind either to GlyCophorin D, the trunCated form of GlyCophorin C laCking the N-glyCan or to desialylated GPC. These results draw attention to the role of GlyCophorin C glyCans in EBA-140 binding. The full identifiCation of the EBA-140 binding site on GlyCophorin C moleCule, Consisting most likely of its glyCans and peptide baCkbone, may help to design therapeutiCs or vaCCines that target the erythroCyte binding merozoite ligands.
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ReCombinant forms of GlyCophorin C as a tool for CharaCterization of epitopes for new murine monoClonal antibodies with anti-GlyCophorin C speCifiCity.
Transfusion medicine (Oxford England), 2002Co-Authors: Ewa Jaskiewicz, Marcin Czerwinski, S. Murata, M. Uchikawa, T. Miyazaki, H. Ikeda, Elwira LisowskaAbstract:Summary GlyCophorin C (GPC) and GlyCophorin D (GPD) are minor but important Components of human RBC membranes. They Carry the high-frequenCy antigens Ge2, Ge3 and Ge4 of the GerbiCh blood group system. The epitopes for five new monoClonal antibodies (MoAbs) with anti-GPC speCifiCity were CharaCterized. Two antibodies (4G11 and 5B11) reaCted with glyCosylated N-terminal epitopes, and three reaCted with internal epitopes of GPC. PepsCan analysis showed that the MoAb RB11 required for binding the EPDP sequenCe, oCCurring twiCe in GPC polypeptide Chain. The MoAb 7F11 reCognized the sequenCe 13PLSLEPDP20, and the MoAb RB8 did not reaCt with synthetiC peptides. Further CharaCterization of the internal epitopes was performed in fluoresCenCe-aCtivated Cell sorter (FACS) with the use of reCombinant GPC and its variant forms transiently expressed on COS-7 Cells. The results indiCated that the MoAb RB11 reCognized distinCtly its target sequenCe EPDP only in a normal GPC moleCule. The reaCtivity of the MoAb 7F11 with the PLSLEPDP sequenCe was Confirmed and found to be enhanCed by the O-glyCan at the Ser15 residue. The MoAb RB8 reCognized the glyCopeptidiC epitope in proximity to the Ser15 residue, requiring the presenCe of O-glyCan. The Combination of immunoChemiCal teChniques with the use of the reCombinant forms of GPC has made it possible to define the role of sugar Chains in the reCognition of peptidiC epitopes in glyCosylated antigen and sheds new light on the GerbiCh system antigens.
Shirin M Marfatia - One of the best experts on this subject based on the ideXlab platform.
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THE PDZ DOMAIN OF HUMAN ERYTHROCYTE P55 MEDIATES ITS BINDING TO THE CYTOPLASMIC CARBOXYL TERMINUS OF GlyCophorin C: ANALYSIS OF THE BINDING INTERFACE BY IN VITRO MUTAGENESIS
The Journal of biological chemistry, 1997Co-Authors: Shirin M Marfatia, João H. Morais-cabral, Anthony C. Kim, Olwyn Byron, Athar H ChishtiAbstract:The PDZ domain, also known as the GLGF repeat/DHR domain, is an ∼90-amino aCid motif disCovered in a reCently identified family of proteins termed MAGUKs (membrane-assoCiated guanylatekinase homologues). SequenCe Comparison analysis has sinCe identified PDZ domains in over 50 proteins. Like SH2 and SH3 domains, the PDZ domains mediate speCifiC protein-protein interaCtions, whose speCifiCities appear to be diCtated by the primary struCture of the PDZ domain as well as its binding target. Using reCombinant fusion proteins and a blot overlay assay, we show that a single Copy of the PDZ domain in human erythroCyte p55 binds to the Carboxyl terminus of the CytoplasmiC domain of human erythroid GlyCophorin C. Deletion mutagenesis of 21 amino aCids at the amino terminus of the p55 PDZ domain Completely abrogates its binding aCtivity for GlyCophorin C. Using an alanine sCan and surfaCe plasmon resonanCe teChnique, we identify residues in the CytoplasmiC domain of GlyCophorin C that are CritiCal for its interaCtion with the PDZ domain. The reCognition speCifiCity of the p55 PDZ domain appears to be unique, sinCe the three PDZ domains of hDlg (human lymphoCyte homologue of the DrosophiladisCs large tumor suppressor) do not bind the CytoplasmiC domain of GlyCophorin C. Taken together with our previous studies, these results Complete the identifiCation of interaCting domains in the ternary Complex between p55, GlyCophorin C, and protein 4.1. ImpliCations of these findings are disCussed in terms of binding speCifiCity and the regulation of Cytoskeleton-membrane interaCtions.
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identifiCation of the protein 4 1 binding interfaCe on GlyCophorin C and p55 a homologue of the drosophila disCs large tumor suppressor protein
Journal of Biological Chemistry, 1995Co-Authors: Shirin M Marfatia, Daniel Branton, Athar H ChishtiAbstract:AbstraCt Protein 4.1 is the prototype of a family of proteins that inClude ezrin, talin, brain tumor suppressor merlin, and tyrosine phosphatases. All members of the protein 4.1 superfamily share a highly Conserved N-terminal 30-kDa domain whose biologiCal funCtion is poorly understood. It is believed that the attaChment of the Cytoskeleton to the membrane may be mediated via this 30-kDa domain, a funCtion that requires formation of multiprotein Complexes at the plasma membrane. In this investigation, synthetiCally tagged peptides and baCterially expressed proteins were used to map the protein 4.1 binding site on human erythroid GlyCophorin C, a transmembrane glyCoprotein, and on human erythroid p55, a palmitoylated peripheral membrane phosphoprotein. The results show that the 30-kDa domain of protein 4.1 binds to a 12-amino aCid segment within the CytoplasmiC domain of GlyCophorin C and to a positively Charged, 39-amino aCid motif in p55. SequenCes similar to this Charged motif are Conserved in other members of the p55 superfamily, inCluding the Drosophila disCs-large tumor suppressor protein. Our data provide new insights into how protein 4.1, GlyCophorin C, p55, and their non-erythroid homologues, interaCt with the Cytoskeleton to exert their physiologiCal effeCts.
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in vitro binding studies suggest a membrane assoCiated Complex between erythroid p55 protein 4 1 and GlyCophorin C
Journal of Biological Chemistry, 1994Co-Authors: Shirin M Marfatia, Daniel Branton, Athar H ChishtiAbstract:AbstraCt p55 is a palmitoylated peripheral membrane phosphoprotein of human erythroCytes. Primary struCture of p55 inCludes a single Copy of the SH3 motif, a COOH-terminal guanylate kinase domain, and an NH2-terminal domain of unknown funCtion. Although the funCtion of p55 is not known, interest in this Component has been heightened by its similarity to the Drosophila tumor suppressor disCs-large (dlg). In this report we provide evidenCe for the direCt assoCiation of p55 with the NH2-terminal 30-kDa domain of protein 4.1, a key Component of the erythroid membrane skeleton. In addition, p55 also binds to the CytoplasmiC domain of GlyCophorin C, a transmembrane protein of red blood Cells. We also provide evidenCe demonstrating the direCt assoCiation of the 30-kDa domain of protein 4.1 with the CytoplasmiC domain of GlyCophorin C. Taken together, these results suggest the existenCe of a novel ternary Complex at the erythroid plasma membrane involving protein 4.1, p55, and GlyCophorin C. SinCe isoforms of protein 4.1, p55, and GlyCophorin C are present in many non-erythroid Cells, the binding interaCtions may be prototypiCal of similar assoCiations that modulate Cytoskeletal-membrane linkage of broad signifiCanCe.
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IntraCellular interaCtions between protein 4.1 and GlyCophorin C on transport vesiCles, as determined by fluoresCenCe Correlation speCtrosCopy
FEBS Letters, 2012Co-Authors: Shotaro Tanaka, Yuichi TakakuwaAbstract:InteraCtion of protein 4.1 (4.1R) with the transmembrane protein GlyCophorin C (GPC) regulates the funCtions of erythroCyte membrane. FluoresCenCe Correlation speCtrosCopy (FCS) was used to define the interaCtion of EGFP-4.1R with DsRed-GPC on transport vesiCles (TVs) by measuring their fluCtuation in living Cells. DsRed-GPC expressed in HeLa Cells was delivered to the plasma membrane through slow vesiCle transport. EGFP-4.1R, whiCh freely diffused in the Cytosol when expressed alone, diffused slowly when Co-expressed with DsRed-GPC, indiCating assoCiation of EGFP-4.1R with TVs. FluoresCenCe Cross-Correlation speCtrosCopy (FCCS) showed direCt interaCtion of EGFP-4.1R with DsRed-GPC on TVs. The present study demonstrates that 4.1R binds to GPC on TVs in living Cells.
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Similarities and differenCes in the struCture and funCtion of 4.1G and 4.1R135, two protein 4.1 paralogues expressed in erythroid Cells.
The Biochemical journal, 2010Co-Authors: Wataru Nunomura, Narla Mohandas, Marilyn Parra, Philippe Gascard, Kengo Kinoshita, Yuichi TakakuwaAbstract:Membrane skeletal protein 4.1R is the prototypiCal member of a family of four highly paralogous proteins that inClude 4.1G, 4.1N and 4.1B. Two isoforms of 4.1R (4.1R 135 and 4.1R 80 ), as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R 80 is present in mature erythroCytes. Although the funCtion of 4.1R isoforms in erythroid Cells has been well CharaCterized, there is little or no information on the funCtion of 4.1G in these Cells. In the present study, we performed detailed CharaCterization of the interaCtion of 4.1G with various erythroid membrane proteins and the regulation of these interaCtions by CalCium-saturated Calmodulin. Like both isoforms of 4.1R, 4.1G bound to band 3, GlyCophorin C, CD44, p55 and Calmodulin. While both 4.1G and 4.1R 135 interaCt with similar affinity with CD44 and p55, there are signifiCant differenCes in the affinity of their interaCtion with band 3 and GlyCophorin C. This differenCe in affinity is related to the non-Conserved N-terminal headpieCe region of the two proteins that is upstream of the 30 kDa membrane-binding domain that harbours the binding sites for the various membrane proteins. The headpieCe region of 4.1G also Contains a high-affinity CalCium-dependent Calmodulin-binding site that plays a key role in modulating its interaCtion with various membrane proteins. We suggest that expression of the two paralogues of protein 4.1 with different affinities for band 3 and GlyCophorin C is likely to play a role in assembly of these two membrane proteins during terminal erythroid differentiation.
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regulation of protein 4 1r p55 and GlyCophorin C ternary Complex in human erythroCyte membrane
Journal of Biological Chemistry, 2000Co-Authors: Wataru Nunomura, Yuichi Takakuwa, Marilyn Parra, John G Conboy, Narla MohandasAbstract:AbstraCt Three binary protein-protein interaCtions, GlyCophorin C (GPC)-4.1R, GPC-p55, and p55–4.1R, Constitute the GPC-4.1R-p55 ternary Complex in the erythroCyte membrane. Little is known regarding the moleCular basis for the interaCtion of 4.1R with either GPC or p55 and regarding the role of 4.1R in regulating the various protein-protein interaCtions that Constitute the GPC-4.1R-p55 ternary Complex. In the present study, we present evidenCe that sequenCes in the 30-kDa domain enCoded by exon 8 and exon 10 of 4.1R Constitute the binding interfaCes for GPC and p55, respeCtively. We further show that 4.1R inCreases the affinity of p55 binding to GPC by an order of magnitude, implying that 4.1R modulates the interaCtion between p55 and GPC. Finally, we doCument that binding of Calmodulin to 4.1R deCreases the affinity of 4.1R interaCtions with both p55 and GPC in a Ca2+-dependent manner, implying that the GPC-4.1R-p55 ternary protein Complex Can undergo dynamiC regulation in the erythroCyte membrane. Taken together, these findings have enabled us to identify an important role for 4.1R in regulating the GPC-4.1R-p55 ternary Complex in the erythroCyte membrane.