Search

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.

Integrins function as bi-directional signaling transducers that regulate cell-cell and cell-matrix signals across the membrane. A key modulator of integrin activation is talin, a large cytoskeletal protein that exists in an autoinhibited state in quiescent cells. Due to the fundamental importance of integrin activation in a myriad of critical cellular processes, including migration, adhesion, morphology, and proliferation, the mechanistic basis for molecular regulation of talin activation has been under intense investigation. A major mechanism of integrin activation in platelets is through heterotrimeric G protein signaling regulating hemostasis and thrombosis. Here we provide evidence that Switch Region 2 of the ubiquitously expressed G protein (Ga13) directly interacts with talin, relieves its state of autoinhibition, and triggers integrin activation. This Ga13-talin interaction represents a novel mechanism for the regulation of integrin signaling. The most abundant platelet integrin complex, aIIbb3, is activated through the cytoskeletal protein talin. Talin is a large 235 kDa protein comprised of an N-terminal 45 kDa FERM domain, also known as the talin head domain (THD), and a series of helical bundles known as the rod domain. The talin head domain consists of 4 distinct lobes designated as F0-F3. Integrin binding and activation is mediated through the F3 region; a critically regulated domain in talin. Regulation of the F3 lobe is accomplished through autoinhibition via anti-parallel dimerization. In the anti-parallel dimerization model, the rod domain region of one talin molecule binds to the F3 lobe on an adjacent talin molecule, thus achieving the state of autoinhibition. Because platelet functionality requires integrins for adherence and thrombus formation, regulation of talin presents a critical node where pharmacological intervention is possible. Our experiments demonstrate that Ga13SR2 peptide recognized a single binding site on THD that is critical for talin autoinhibition. Subsequent experiments show a GST-SR2 fusion protein can sterically interfere with talin's autoinhibitory rod domain, and similar results were obtained using the full length Ga13. Furthermore, talin activity was investigated by intramolecular FRET. We established a novel assay using a full length talin molecule containing an N terminal GFP tag, and a red fluorescent dye binding motif incorporated into the autoinhibitory rod domain. In an autoinhibited anti-parallel dimer state, GFP acts as a donor to the red fluorescent dye on an adjacent talin molecule. The FRET signal would decrease when talin is activated. Using this assay, we show that Ga13 reduced FRET signal as compared to a Ga13 SR2 mutant (R227A) thus demonstrating Ga13 can activate talin in a cellular milieu. To investigate the therapeutic potential of Ga13 SR2, we evaluated two critical aspects of anti-thrombotic therapy; bleeding time and in vivo thrombosis. Upon administration of Ga13SR2 peptide in mice, the bleeding time was significantly less than aspirin-treated mice. In vivo thrombosis was assessed using a laser injury model. A significant decrease in thrombosis was observed in mice treated with Ga13 SR2 when compared to the scramble peptide. Together, these studies suggest that the Ga13 SR2-talin activation pathway represents a novel integrin signaling process triggering rapid recruitment and activation of circulating platelets to the site of vascular injury. Because of the highly conserved nature of Ga13, talin, and integrins, the broad implications of this regulatory mechanism may extend to a multitude of cell types and pathologies. Figure Ga13 mediated activation of talin by relieving anti-parallel dimerization. Inactive Ga13 (GDP bound) is activated by exchanging GDP for GTP, via intracellular signaling processes. Active Ga13 (GTP bound) relieves talin autoinhibition by displacing the head/rod domain interaction. Activated talin is free to bind the cytoplasmic tail of b integrins and facilitate integrin activation. Figure. Ga13 mediated activation of talin by relieving anti-parallel dimerization. Inactive Ga13 (GDP bound) is activated by exchanging GDP for GTP, via intracellular signaling processes. Active Ga13 (GTP bound) relieves talin autoinhibition by displacing the head/rod domain interaction. Activated talin is free to bind the cytoplasmic tail of b integrins and facilitate integrin activation. Disclosures No relevant conflicts of interest to declare.

Dematin is a critical component of the membrane junctional complex in red blood cells. It tethers the spectrin cytoskeleton proteins to the membrane and its genetic deletion in mice causes dissociation of the spectrin, actin and β-adducin from the membrane resulting in the collapse of the red blood cells (RBCs). As dematin lacks a transmembrane domain, it is still unclear how this critical component of the junctional complex is anchored to the RBC membrane. Our previous studies have shown that the multi-transmembrane glucose transporter-1 (GLUT1) interacts with dematin and β-adducin in human RBCs, suggesting a potential role for GLUT1 in recruiting dematin to the membrane. However, as mouse RBCs do not express a GLUT1 homologue, an equivalent membrane receptor for dematin and/or adducin in mice remains to be determined. Using multiple in vitro and in vivo biochemical assays, here we demonstrate that the ubiquitously expressed plasma membrane Na+/H+ exchanger, NHE1 (Slc9a1), is one of the receptors for dematin and β-adducin in mature mouse red blood cells. NHE1 directly interacts with the core domain of dematin. Moreover, the dematin headpiece domain mutant S381E, which binds to the core domain with a higher affinity than the wild type, abolished the biochemical interaction between dematin and NHE1. This observation suggests that NHE1 and dematin headpiece domain compete for the same binding site(s) on the core domain. Furthermore, this finding highlights a molecular mechanism whereby an intermolecular switch of dematin regulates its interaction with NHE1 by phosphorylation. Dematin and β-adducin directly interact with NHE1 at its membrane-proximal cytoplasmic domain, which in turn regulates NHE1 activity in response to growth factor stimuli and intracellular pH alterations. Accordingly,we observed an increased cellular sodium content in erythrocytes of dematin headpiece and adducin double knockout mice (DAKO), suggesting a higher NHE1 activity in DAKO erythrocytes. Unlike GLUT1, NHE1 is expressed in both mouse and human RBCs. Thus, our results provide a novel mechanism for linking NHE1 to membrane skeleton and multiple cell signaling pathways through dematin and adducin (Figure 1). Since NHE1 is one of the major regulators of intracellular pH and hypertonic stress, our findings raise the possibility that the dematin-adducin-NHE1 complex may modulate these functions in RBCs as well as in other cell types with broad impact on the regulation of the actin cytoskeleton and cell migration. Figure 1 Dematin and adducin link erythrocyte junctional complex to membrane via multiple receptors. A, WT RBC cytoskeleton. B, Mutant (DAKO) RBC cytoskeleton. Images show a grossly deranged membrane skeleton in DAKO as compared to wild type. Red arrows show enlarged pores and yellow arrows indicate the presence of aggregates. Bars correspond to 0.2 µM. C, Schematic diagram of dematin, adducin, and NHE1 linking the complex to multiple signaling pathways. D, Proposed new model of the erythrocyte junctional complex. Figure 1. Dematin and adducin link erythrocyte junctional complex to membrane via multiple receptors. A, WT RBC cytoskeleton. B, Mutant (DAKO) RBC cytoskeleton. Images show a grossly deranged membrane skeleton in DAKO as compared to wild type. Red arrows show enlarged pores and yellow arrows indicate the presence of aggregates. Bars correspond to 0.2 µM. C, Schematic diagram of dematin, adducin, and NHE1 linking the complex to multiple signaling pathways. D, Proposed new model of the erythrocyte junctional complex. Disclosures No relevant conflicts of interest to declare.

The composition of the erythrocyte plasma membrane is extensively modified during the intracellular growth of the malaria parasite Plasmodium falciparum. It has been previously shown that an 80-kD phosphoprotein is associated with the plasma membrane of human red blood cells (RBCs) infected with trophozoite/schizont stage malaria parasites. However, the identity of this 80-kD phosphoprotein is controversial. One line of evidence suggests that this protein is a phosphorylated form of RBC protein 4.1 and that it forms a tight complex with the mature parasite-infected erythrocyte surface antigen. In contrast, evidence from another group indicates that the 80-kD protein is derived from the intracellular malaria parasite. To resolve whether the 80-kD protein is indeed RBC protein 4.1, we made use of RBCs obtained from a patient with homozygous 4.1(-) negative hereditary elliptocytosis. RBCs from this patient are completely devoid of protein 4.1. We report here that this lack of protein 4.1 is correlated with the absence of phosphorylation of the 80-kD protein in parasite- infected RBCs, a finding that provides conclusive evidence that the 80- kD phosphoprotein is indeed protein 4.1. In addition, we also identify and partially characterize a casein kinase that phosphorylates protein 4.1 in P falciparum-infected human RBCs. Based on these results, we suggest that the maturation of malaria parasites in human RBCs is accompanied by the phosphorylation of protein 4.1. This phosphorylation of RBC protein 4.1 may provide a mechanism by which the intracellular malaria parasite alters the mechanical properties of the host plasma membrane and modulates parasite growth and survival in vivo.

The composition of the erythrocyte plasma membrane is extensively modified during the intracellular growth of the malaria parasite Plasmodium falciparum. It has been previously shown that an 80-kD phosphoprotein is associated with the plasma membrane of human red blood cells (RBCs) infected with trophozoite/schizont stage malaria parasites. However, the identity of this 80-kD phosphoprotein is controversial. One line of evidence suggests that this protein is a phosphorylated form of RBC protein 4.1 and that it forms a tight complex with the mature parasite-infected erythrocyte surface antigen. In contrast, evidence from another group indicates that the 80-kD protein is derived from the intracellular malaria parasite. To resolve whether the 80-kD protein is indeed RBC protein 4.1, we made use of RBCs obtained from a patient with homozygous 4.1(-) negative hereditary elliptocytosis. RBCs from this patient are completely devoid of protein 4.1. We report here that this lack of protein 4.1 is correlated with the absence of phosphorylation of the 80-kD protein in parasite- infected RBCs, a finding that provides conclusive evidence that the 80- kD phosphoprotein is indeed protein 4.1. In addition, we also identify and partially characterize a casein kinase that phosphorylates protein 4.1 in P falciparum-infected human RBCs. Based on these results, we suggest that the maturation of malaria parasites in human RBCs is accompanied by the phosphorylation of protein 4.1. This phosphorylation of RBC protein 4.1 may provide a mechanism by which the intracellular malaria parasite alters the mechanical properties of the host plasma membrane and modulates parasite growth and survival in vivo.

In this issue of Blood , Rieger et al show that malaria parasite infiltration in the human placenta requires a specific geometry and affinity of host receptors to facilitate strong adhesion.[1][1] ![Figure][2] Schematic representation of the adhesion of P falciparum -infected erythrocytes to

Human erythrocyte p55 is a peripheral membrane protein that contains three distinct domains in its primary structure: an N-terminal domain, an SH3 motif, and a C-terminal guanylate kinase domain. We used naturally mutated red blood cells (RBCs) with primary genetic defects resulting in the absence of protein 4.1 (4.1[-] hereditary elliptocytosis) or glycophorin C (Leach elliptocytosis). The absence of either protein was associated with the absence of p55. On a stoichiometric basis, the reduction in glycophorin C (about 80%) was concomitant to the lack of p55 in RBCs devoid of protein 4.1. Similarly, the reduction of protein 4.1 (about 20%) was equivalent to the absence of p55 in RBCs devoid of glycophorin C. These correlations suggest that p55 is associated, in precise proportions, with the protein 4.1-glycophorin-C complex, linking the skeleton and the membrane. The protein 4.1-glycophorin-C cross-bridge is known to be critically important for the stability and mechanical properties of human RBC plasma membrane. Because isoforms of protein 4.1, glycophorin C, and p55 exist in many tissues, these results provide evidence of a linkage between the skeleton and the membrane that may have implications in many nonerythroid cells.

Introduction: With an estimated 15 million patients and no drug that addresses its etiology, sickle cell disease (SCD) remains an area of unmet need. Vaso-occlusive pain crisis (VOPC), the hallmark of SCD, is initiated by sickle RBCs (sRBCs) recruiting leukocytes and platelets to potentiate vessel occlusion. ADP released by sRBCs is a potent activator of platelets, and sickle cell patients are known to have activated platelets in circulation both at steady state and during VOPC. However, the mechanism underlying platelet dysfunction in SCD is not fully understood. Platelet activation mediated by the protease activated receptors (PAR1 and PAR4 in humans, PAR3 and PAR4 in mice), triggers PLC-β activation resulting in calcium mobilization. The increased calcium flux leads to activation of GPIIbIIIa/aIIbb3, GP1b, and P-selectin involved in platelet aggregation, adhesion, and rolling. Prior evidence has established a role of the calcium-activated cysteine protease, calpain-1 in platelet activation. Washed platelets from calpain-1 knockout C57BL/6 mice demonstrated impaired platelet aggregation. However, due to the critical contribution of sRBCs to platelet dysfunction in SCD, whole blood (impedance) aggregometry represents a physiological assessment of platelet aggregation. Methods: Townes SCD mice (SS) were backcrossed with calpain-1 knockout (CKO) mice to generate SCD mice lacking calpain-1 (SSCKO). Humanized mice (AA) were used as controls. Using flow cytometry, we evaluated in vivo platelet activation following stimulation with ADP and Thrombin GPRP. Platelet counts were obtained via ADVIA 120 and flow cytometry. For platelet aggregation, 500 μL of blood was harvested from the vena cava of AA, SS, SSCKO, and CKO mice. Whole blood aggregation in response to PAR4 stimulation was assessed using the Roche Multiplate Analyzer. A separate group of mice were challenged with hypoxia/reoxygenation (H/R) treatment (3 hours of 10.5% O2, followed by 4 hours of 21% O2) prior to platelet aggregation testing. SCD mice are characterized by tissue infarcts suggestive of thrombus formation. To examine whether H/R treatment induces formation of fibrin thrombi, we harvested brain, lungs, heart, kidneys, liver, and spleen following blood collection, and performed histology. Results: Compared to AA, SS and SSCKO mice are thrombocytopenic. Similar to Berkeley, Townes mouse platelets are activated in vivo, demonstrated by activated GPIIbIIIa on circulating platelets. At steady state, PAR4 agonist-induced platelet aggregation is similar in AA and SS mice (64 U v. 53 U, n = 6-10/group, p = 0.3). As depicted in Fig 1., SSCKO mice show significantly reduced platelet aggregation compared to SS mice (13 U v 53 U, p Figure 1. Impaired steady state whole blood platelet aggregation in SSCKO mice (panel 3). Figure 1. Impaired steady state whole blood platelet aggregation in SSCKO mice (panel 3). Figure 2. H/R relieves calpain-1-mediated inhibition of platelet aggregation in SS-CKO mice (panel 3). Figure 2. H/R relieves calpain-1-mediated inhibition of platelet aggregation in SS-CKO mice (panel 3). Disclosures Jakubowski: Eli Lilly and Company: Employment, Equity Ownership. Frelinger:GL Synthesis: Research Funding; Celerion: Research Funding; Megakaryon: Research Funding; Bristol-Myers Squibb: Research Funding; Sysmex: Research Funding; Eisai: Research Funding; Baxalta: Research Funding; Pfizer: Research Funding; GE Global Research: Research Funding; NIH: Research Funding.

Dematin is an actin binding and bundling protein originally identified as a component of the erythrocyte membrane junctional complex. A widely expressed member of the villin-family of adaptor proteins, dematin regulates RhoA activity and cell shape in fibroblasts. Actin binding and bundling activity of dematin is regulated by phosphorylation of its headpiece domain by the cAMP-dependent protein kinase. Despite its extensive biochemical characterization, the physiological function of dematin in mature erythrocytes remains unknown. We used a conditional gene disruption strategy by generating a targeting construct that has the potential for full body gene knockout as well as tissue-specific deletion of dematin gene using the Cre-lox gene deletion system. Wild type, heterozygous, and homozygous progeny were obtained in a typical Mendelian ratio of 1:2:1. Dramatic splenomegaly in 7-week old full length dematin knockout (FLKO) mice was observed with the average spleen weight 10-fold higher than those of the wild type littermates. Flow cytometry showed a ~16-fold increase in reticulocytes (Fig.1A), which was also seen in the blood smear (Fig.1B,C). Severe hemolytic anemia is most likely the cause of relative pallor observed in FLKO mice at day 1 after birth. The adult FLKO mice continue to show relatively smaller body size as compared to wild type and heterozygous mice. These findings are consistent with severe anemia and compensatory erythropoiesis. FLKO mice exhibit typical signs of anisocytosis, microcytosis, macrocytosis, and polychromasia, which are indicative of tremendous variation in RBC cell size and the premature release of reticulocytes from the bone marrow. Moreover, additional RBC abnormalities, including poikilocytosis, acanthocytosis, fragmented RBC, and spherocytes, are consistent with severe hemolytic disease. By scanning EM, the FLKO erythrocytes showed dramatic variation in shape and size. The spherocytes, microcytic vesiculation, and the protruding structures are observed in FKLO mice, as well as extensive intravascular hemolysis (Fig. 1D,E). RBC half-life measurements in vivo by NHS-biotin labeling and flow cytometry showed mutant cells almost immediately cleared from the circulation in FLKO mice. A seven-week chase experiment showed that the half-life of RBCs was reduced from 22 days in wild type and heterozygous mice to less than 3 days in FLKO mice. The hematological phenotype of FLKO mice indicated reduced RBC count, hemoglobin, and hematocrit with increase in the RBC distribution width. Collectively, these findings indicate that the mechanical strength of RBC membrane strictly relies on the presence of full length dematin. We employed membrane fractionation, in vitro protein domain mapping, transmission/scanning electron microscopy, and dynamic deformability measurements to investigate the underlying mechanisms of extreme membrane fragility in FLKO erythrocytes. We also examined the protein profile of RBC ghosts. Surprisingly, the major cytoskeletal proteins remained unchanged in the FLKO ghosts; however, a marked reduction of spectrin, adducin, and actin was observed. When normalized against band 3, these proteins were reduced by 60%, 90%, and 90%, respectively. Since these membrane proteins are essential for RBC stability, our findings suggest a specific role of dematin in recruiting or maintaining a stable association of essential cytoskeletal proteins in the plasma membrane. These results raise the possibility that dematin may directly interact with adducin, and together anchor the spectrin molecules to the plasma membrane. Our findings provide the first in vivo evidence that dematin is essential for the maintenance of erythrocyte shape and membrane mechanical properties by regulating the integrity of the spectrin-actin junctions. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: Sickle cell disease (SCD) is caused by the substitution of a valine residue for glutamic acid in the β-globin chain of hemoglobin. The resulting sickle hemoglobin rapidly polymerizes upon deoxygenation, forming sickle RBCs (sRBCs) with reduced deformability. Less deformable sRBCs have an impaired ability to navigate the microvasculature, thereby potentiating vaso-occlusive crises, the hallmark of SCD. Deoxygenation-mediated sickling also induces influx of calcium leading to calpain-1 mediated RBC dehydration via the Gardos channel. Calpains are calcium-activated cysteine proteases involved in numerous cellular processes. In mammals, calpain-1 and calpain-2 are the dominantly expressed isoforms, along with the endogenous inhibitor, calpastatin. Previously, we have shown that genetic deletion of calpain-1 improves RBC deformability in C57BL/6 mice (Wieschhaus et al, Biochem J 2012). Similarly, pharmacological inhibition of calpain-1 prevents dehydration of sRBCs in the SAD model of mild SCD (De Franceschi, et al, FASEB J 2013). Here, our objective is to determine whether genetic deletion of calpain-1 in the HbSS-Townes mice – a severe SCD mouse model, improves RBC deformability in both normoxic and hypoxic conditions. Methods: Transgenic SCD mice (HbSS-Townes) expressing >99% human HbS were backcrossed with calpain-1 knockout (CKO) mice to obtain sickle mice lacking calpain-1 (HbSS-Townes-CKO). Casein zymography was performed to confirm the complete loss of calpain-1 activity in the RBCs of the HbSS-Townes-CKO mice. Thiazole orange-based FACS revealed that HbSS-Townes and HbSS-Townes-CKO mice express ~60% reticulocytes. The elevated reticulocyte count prompted us to evaluate the effect of calpain-1 deficiency on reticulocyte deformability. We utilized a microfluidic-based flow cytometer to assess single reticulocyte deformability following thiazole orange staining upon treatment with calcium ionophore (A23187) and CaCl2. We further probed the molecular mechanisms of RBC deformability by assessing calcium-induced proteolysis of RBC cytoskeletal proteins. RBCs were harvested from HbSS-Townes and HbSS-Townes-CKO mice, treated with A23187 and CaCl2 to activate calpain-1, and subjected to RBC ghosts preparation and gel electrophoresis. Cytoskeletal proteins were visualized and quantified. Guided by our previous findings that calpain-1 knockout (CKO) mice exhibit a clot retraction defect (Kuchay, et al, MCB 2007), we harvested un-coagulated blood from HbSS-Townes and HbSS-Townes-CKO mice, and assessed their clot formation phenotype in calcium-free RPMI medium at different time intervals. Results: Casein zymography revealed a complete loss of calpain-1 enzyme activity (indicated by lack of casein cleavage) in the HbSS-Townes-CKO mice. Furthermore, reticulocytes from HbSS-Townes-CKO mice are more deformable than their HbSS-Townes counterparts under steady state as well as upon treatment with calcium ionophore and CaCl2. An improvement in sickle reticulocyte deformability has significant potential to prevent vaso-occlusive crisis under situations of hypoxia-induced ischemia reperfusion injury – a hypothesis that is currently being tested. In addition, our preliminary results show that RBC ghosts from HbSS-Townes mice exhibit a transglutaminase-mediated cross-linking defect in the cytoskeleton that is restored in the HbSS-Townes-CKO mice. This finding has significant implications for an increased in vivo RBC life span of HbSS-Townes-CKO mice. Finally, we found that HbSS-Townes-CKO mice display a whole blood clot retraction defect, similar to our previous findings on platelets harvested from calpain-1 knockout (CKO) mice. Our findings showing improved sickle cell deformability and clot retraction phenotype are consistent with recently published findings demonstrating an interaction between RBCs and platelets in clot formation and in vivo thrombosis. To test this intriguing hypothesis, we are currently evaluating whether HbSS-Townes-CKO mice exhibit reduced thrombus formation upon laser-induced in vivo thrombosis. Together, our results report the first mouse model of severe sickle cell disease lacking calpain-1 activity, and unveil a new role for calpain-1 in the multi-step process regulating SCD pathogenesis. These findings might serve as a basis for exploring calpain-1 inhibition as a therapeutic target for SCD. Disclosures No relevant conflicts of interest to declare.

Only 5% to 10% of band 3 null mice survive the neonatal period. To determine the cause of death, 3 adult and 11 newborn band 3 null mice were submitted for histopathologic examination. All but 1 pup showed evidence of thrombosis including: (1) large thrombotic lesions in the heart, which were partially organized, calcified in some fields, and endothelialized, indicating a process that developed premortem (3 of 3 adults and 6 of 11 pups). (2) Subcapsular necrotic areas in the liver suggestive of premortem ischemic events caused by arteriolar occlusions (8 of 11 pups). (3) Large vein thrombi (4 of 11 pups). To investigate the etiology of this hypercoagulable state, we have used the Russell’s viper venom test (RVV) to show that red blood cells (RBCs) from band 3 null mice significantly shorten the RVV clotting time of normal plasma in a dose-dependent fashion, whereas RBCs from normal mice have no effect, suggesting that the membrane of band 3 null RBCs provides a suitable surface for activation of the prothrombinase complex. Using flow cytometry, we have examined the phosphatidylserine (PS)-specific binding of fluorescein isothiocyanate (FITC)-annexin V to normal and band 3 null RBCs. A subpopulation of cells (3% to 5% of RBCs) with increased FITC-annexin V binding was detected in band 3 null RBCs as compared with normal RBCs. Furthermore, the entire cell population of band 3 null RBCs shows a measurable increase in the mean fluorescence intensity, suggesting that band 3 null RBCs may have increased PS exposure on the outer membrane leaflet. These findings are further supported by direct fluorescence microscopy of normal and band 3 null RBCs labeled with FITC-annexin V. Based on these observations, we postulate that the high mortality of band 3 null mice may be related to a hypercoagulable state, which appears to originate from changes in the phospholipid composition of the membrane leading to PS exposure on the outer leaflet.© 1998 by The American Society of Hematology.

Glycophorin A is the major transmembrane sialoglycoprotein of red blood cells. It has been shown to contribute to the expression of the MN and Wright blood group antigens, to act as a receptor for the malaria parasite Plasmodium falciparum and Sendai virus, and along with the anion transporter, band 3, may contribute to the mechanical properties of the red blood cell membrane. Several lines of evidence suggest a close interaction between glycophorin A and band 3 during their biosynthesis. Recently, we have generated mice where the band 3 expression was completely eliminated by selective inactivation of the AE1 anion exchanger gene, thus allowing us to study the effect of band 3 on the expression of red blood cell membrane proteins. In this report, we show that the band 3 −/− red blood cells contain protein 4.1, adducin, dematin, p55, and glycophorin C. In contrast, the band 3 −/− red blood cells are completely devoid of glycophorin A (GPA), as assessed by Western blot and immunocytochemistry techniques, whereas the polymerase chain reaction (PCR) confirmed the presence of GPA mRNA. Pulse-label and pulse-chase experiments show that GPA is not incorporated in the membrane and is rapidly degraded in the cytoplasm. Based on these findings and other published evidence, we propose that band 3 plays a chaperone-like role, which is necessary for the recruitment of GPA to the red blood cell plasma membrane.

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.

Abstract 5124 Background & Objectives: Acute lymphoblastic leukemia (ALL) is a complex genetic disease involving many fusion oncogenes (FGs) (Xu et al., 2012). The frequency of various FO can vary in different ethnic groups & these FGs have important implications for prognosis & treatment outcome (van-Dongen et al, 1999). Methods: We studied FGs in 101 pediatric ALL patients using Interphase FISH & RT-PCR (van-Dongen et al, 1999), & their association with clinical features & treatment outcome. Results: Five most common FGs i. e. BCR-ABL [t(9;22)], TCF3-PBX1 [t(1;19)], ETV6-RUNX1 [t(12;21)], MLL-AF4 [t(4;11)] & SIL-TAL1 (del 1p32) were found in 89/101 (88. 1%) patients. Frequency of BCR-ABL was 44. 5% (45/101) (Table 1). BCR-ABL positive patients had a significantly lower survival (43. 73 ± 4. 24 weeks) (Figure 1)& higher white cell count as compared to others except patients with MLL-AF4. The highest relapse-free survival (RFS) was documented in ETV6-RUNX1 (14. 167 months) followed closely by those cases in which no gene was detected (13/100). RFS in BCR-ABL, MLL-ASF4, TCF-PBX4 & SIL-TAL1 was less than 10 months (7. 994, 3. 559, 5. 500 & 8. 080 months respectively) (Figure 2 & 3). BCR-ABL: Frequency of occurrence was directly proportional to age (3 less than 2 year age group, 16 in the 2–7 year age group & 26 in the older than 7 group. Total leukocyte count (TLC) was higher when compared to patients with other oncogenes. Organomegaly was not common. BCR-ABL positivity was associated with low remission rates & shortened survival. ETV6-RUNX1: The median age of the patients was 1. 85 years. The gene frequency was highest in patients younger than 2 years. TLC was not very high & patients had a good prognosis. MLL-AF4: 17 patients had MLL-AF4 gene rearrangement with a median age of 9 years. Five patients were younger than 2 years, two between 2 & 7 years, & ten patients were in the 7–15 age group. Majority of our patients were older unlike the usual occurrence where most of the patients are infants. TCF3-PBX1: This FG occurs in around 2% of patients. Only two female patients were diagnosed with this translocation. Both the patients were over 2 years of age. It was associated with an inferior outcome in the context of response to chemotherapy & a higher risk of CNS relapse although small numbers preclude any firm conclusions. SIL-TAL1: Patients were older than 2 years, with the majority falling in the age range 7 to 15 years. The immunophenotype data were available in all SIL-TAL1 patients showing this fusion gene was associated with T-ALL with organomegaly being observed frequently. Discussion & Conclusion: This is the first study from Pakistan correlating molecular markers with disease biology & treatment outcome in pediatric ALL. Our study revealed the highest reported frequency of BCR-ABL FO in pediatric ALL, in consistent with various other reports from Pakistan & rest of the world ((Iqbal & Akhtar, 2007; Faiz et al., 2011; (Gaynon et al., 1997; Iacobucci et al., 2012).) which, consequently, was associated with poor overall survival. The data indicates an immediate need for incorporation of tyrosine kinase inhibitors in the treatment of BCR-ABL+ pediatric ALL in this population & the development of facilities for stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract 371 Terminal differentiation of mammalian erythroid precursors involves enucleation, a process required for the production of reticulocytes. The signaling pathways and the molecular components mediating the final enucleation step are not well understood, although the resemblance of enucleation to asymmetric cell division has been suggested. The enucleation of erythroblasts can be replicated in vitro; however, the enucleation efficiency is not optimal under in vitro culture conditions. A functional role of both microfilaments and microtubules has been suggested in the regulation of erythroblast enucleation. We hypothesized that molecular motors known to regulate asymmetric cell division might also play a functional role in erythroblast enucleation. GAKIN (also called KIF13B) is a kinesin-3 motor implicated in the regulation of cell polarity pathways. Its Drosophila homologue Khc-73 regulates polarity formation during the asymmetric cell division of neuroblasts. We generated a mutant mouse line of GAKIN and examined the efficiency of erythroblast enucleation using the fetal liver in vitro erythroid precursor culture system originally developed by the Lodish group. The GAKIN mutant mouse line was produced from the embryonic stem (ES) cells containing a genomic insertion of the beta-galactosidase-neomycin gene. The beta-geo insertion was mapped within intron 38 of the GAKIN gene, thus removing the CAP-Gly domain located at the C-terminus of GAKIN. This allowed us to visualize specific tissue expression of GAKIN, in addition to the subcellular localization of a GAKIN-lacZ fusion protein by lacZ activity staining. The lacZ activity revealed that the GAKIN-lacZ fusion protein is expressed in bone marrow macrophages and erythroblasts, while also highly concentrated at contact sites between macrophages and erythroblasts. GAKIN mutant mice are viable and fertile, and complete blood analysis did not reveal any discernible phenotype. To test for the enucleation efficiency, erythroid precursors were isolated from fetal liver stage at day 14.5 embryos and enucleation was quantified by flow cytometry after 2 days of in vitro culture. The GAKIN-lacZ fusion protein appeared as a single dot representing the microtubule organizing center (MTOC) in the erythroid precursors isolated from fetal liver (Fig. 1). However, GAKIN mutant erythroblasts enucleated at the same efficiency as wild type erythroblasts under steady state conditions in vitro. Since the mammalian genome contains a close homologue of GAKIN/KIF13B, termed KIF13A, a possibility exists that KIF13A functionally compensates for GAKIN mutation in critical pathways. To address this issue, we generated KIF13A null mice using a similar gene disruption strategy. Again, the KIF13A mutant erythroblasts did not show any measurable change in enucleation efficiency under similar conditions. However, fetal liver erythroblasts isolated from GAKIN and KIF13A double mutant mice exhibited a significant enhancement of enucleation efficiency. This finding suggests that together, GAKIN and KIF13A negatively regulate the erythroblast enucleation by modulating the microtubule-based cytoskeleton. We propose that efficient erythroid enucleation in vivo involves signaling mechanisms inhibiting both GAKIN and KIF13A motors. Since erythrocytes from adult double mutant mice appear to be hematologically normal, our findings raise the possibility of enhancing the in vitro production of functional erythrocytes by inhibiting GAKIN and KIF13A activity in CD34 positive stem cells. Disclosures: No relevant conflicts of interest to declare.

5115 Introduction: Acute lymphoblastic leukemia (ALL) is a heterogeneous lymphoid disorder with many genetic abnormalities of which fusion oncogenes (FGs) are very common with a known role in leukemogenesis ( Harrison & Foroni , 2002). Although prognostic significance of FGs is well characterized in pediatric ALL, the role of FGs in adult ALL is not well established (Moorman et al., 2007). Methods: We studied the frequency and association of five most common FGs namely BCR-ABL, MLL-AF4, ETV6-RUNX11, E2A-PBX1 and SIL-TAL1 with disease biology and treatment outcome in adult ALL. FGs were studied at diagnosis in 104 adult ALL patients using RT-PCR (Van-Dongen et al, 1999) and Interphase FISH. Results: FGs were found in 78. 8% (82/104) subjects ([Table 1][1]). Overall survival (OS) and relapse free survival (RFS) were 26. 17 and 11. 147 months, respectively ([Figures 1][2]–[2][3]). Patients with MLL-AF4 (12. 19%) showed an elevated total leukocyte count (TLC), prominent organomegaly, frequent central nervous system (CNS) involvement, and a poor clinical outcome (OS=8. 8 months). SIL-TAL1 (35. 36%) was associated with lymphadenopathy, frequent organomegaly, low platelets count and poor survival. Patients with BCR-ABL (20. 3%) had high TLC (p-value 30,000 | 10 (48) | 1 (20) | 3 (70) | 13 (45) | 7 (41.1) | 1 | | Hepatomegaly | | | | | | | | No | 11 (52) | 2 (40) | 5 (50) | 24 (83) | 15 (88) | 22 | | yes | 10 (48) | 3 (60) | 5 (50) | 5 (17) | 2 (12) | | | Splenomegaly | | | | | | | | No | 5 (24) | 5 (100) | 5 (50) | 21 (72) | 14 (82) | 20 | | Yes | 16 (76) | | 5 (50) | 8 (28) | 3 (18) | 2 | | Lymphadenopathy | | | | | | | | No | 3 (14) | 4 (80) | 5 (50) | 21 (21) | 2 (12) | 8 | | yes | 18 (86) | 1 (20) | 5 (50) | 8 (28) | 15 (88) | 14 | | Platelets | | | | | | | | 50,000 | 1 (5) | 4 (80) | 1 (10) | 23 (79) | 12 (71) | 20 | | Immunophenotyping | | | | | | | | T-ALL | | | | 24 (83) | | | | B-ALL | 3 (14) | 2 (40) | 2 (20) | | 9 (53) | 2 | | Unknown | 19 (90) | 3 (60) | 8 (80) | 5 (17) | 8 (47) | 20 | | CR | | | | | | | | 4 weeks | 14 (66) | 1 (20) | 2 (20) | 1 (3) | 3 (18) | 3 | | No remission | 3 (14) | | 2 (20) | 8 (28) | 3 (18) | 4 | Table 1: Comparison of the clinical characteristic of different FGs in ALL adult patients ![Figure][4] ![Figure][4] Disclosures: No relevant conflicts of interest to declare. [1]: #T1 [2]: #F1 [3]: #F2 [4]: pending:yes

The composition of the erythrocyte plasma membrane is extensively modified during the intracellular growth of the malaria parasite Plasmodium falciparum. It has been previously shown that an 80-kD phosphoprotein is associated with the plasma membrane of human red blood cells (RBCs) infected with trophozoite/schizont stage malaria parasites. However, the identity of this 80-kD phosphoprotein is controversial. One line of evidence suggests that this protein is a phosphorylated form of RBC protein 4.1 and that it forms a tight complex with the mature parasite-infected erythrocyte surface antigen. In contrast, evidence from another group indicates that the 80-kD protein is derived from the intracellular malaria parasite. To resolve whether the 80-kD protein is indeed RBC protein 4.1, we made use of RBCs obtained from a patient with homozygous 4.1(-) negative hereditary elliptocytosis. RBCs from this patient are completely devoid of protein 4.1. We report here that this lack of protein 4.1 is correlated with the absence of phosphorylation of the 80-kD protein in parasite-infected RBCs, a finding that provides conclusive evidence that the 80-kD phosphoprotein is indeed protein 4.1. In addition, we also identify and partially characterize a casein kinase that phosphorylates protein 4.1 in P falciparum-infected human RBCs. Based on these results, we suggest that the maturation of malaria parasites in human RBCs is accompanied by the phosphorylation of protein 4.1. This phosphorylation of RBC protein 4.1 may provide a mechanism by which the intracellular malaria parasite alters the mechanical properties of the host plasma membrane and modulates parasite growth and survival in vivo.

Abstract 365 Dematin is an actin binding protein that was originally identified in the human erythrocyte membranes; however, dematin polypeptides are detectable in many non-erythroid cells. In erythrocytes, dematin is located at the spectrin-actin junctions thus linking the skeleton to the plasma membrane. This junctional complex serves to maintain both erythrocyte shape and membrane stability. Dematin is phosphorylated by multiple protein kinases, including the cAMP-dependent protein kinase and protein kinase C, and its actin bundling activity is regulated by phosphorylation. Dematin is also an excellent substrate of calpain-1, a calcium-dependent cysteine protease. Because of the functional similarities between the core components of the membrane skeleton in erythrocytes and platelets, we sought to investigate the physiological function of dematin in platelets. The remodeling of the actin cytoskeleton is known to regulate platelet activation and secretion of platelet granule contents. Western blotting demonstrated abundant expression of the ∼52 kDa polypeptide of dematin in both human and mouse platelets. To evaluate the functional role of dematin in platelets, we utilized the dematin headpiece knockout (HPKO) mouse model previously generated in our laboratory. Headpiece domain of dematin is an actin binding module sharing sequence similarity with the villin-family of cytoskeletal proteins. Importantly, the in vivo deletion of the headpiece domain of dematin resulted in substantial diminution of calcium mobilization in response to multiple agonists of platelet activation (Fig. 1). The reduced calcium mobilization in HPKO platelets was associated with significant inhibition of the platelet aggregation and granule secretion pathways. The HPKO platelets exhibit decreased activation of both the integrin αIIbβ3 receptor and RhoA upon platelet stimulation. Moreover, the HPKO platelets display aberrant morphology upon spreading on the fibrinogen and vWF-coated surfaces. Consistent with these findings, the HPKO mice show a significant clot retraction defect associated with a general tail bleeding phenotype. Mechanistically, the basal level of cAMP remained unaltered in the HPKO platelets suggesting the independence of the observed phenotype upon changes in the cAMP concentration. Immunofluorescence analysis indicated that dematin is associated with two platelet membrane compartments known to be involved in calcium fluxes, i.e., the dense tubular system (DTS) and the plasma membrane. Studies are currently in progress to identify the dematin-associated complex in platelets by Western blotting and proteomics approaches. Together, these results unveil dematin as a novel regulator of calcium homeostasis in platelets with functional implications for the development of new anti-platelet therapies. Disclosures: No relevant conflicts of interest to declare.

Human erythrocyte p55 is a peripheral membrane protein that contains three distinct domains in its primary structure: an N-terminal domain, an SH3 motif, and a C-terminal guanylate kinase domain. We used naturally mutated red blood cells (RBCs) with primary genetic defects resulting in the absence of protein 4.1 (4.1[-] hereditary elliptocytosis) or glycophorin C (Leach elliptocytosis). The absence of either protein was associated with the absence of p55. On a stoichiometric basis, the reduction in glycophorin C (about 80%) was concomitant to the lack of p55 in RBCs devoid of protein 4.1. Similarly, the reduction of protein 4.1 (about 20%) was equivalent to the absence of p55 in RBCs devoid of glycophorin C. These correlations suggest that p55 is associated, in precise proportions, with the protein 4.1-glycophorin-C complex, linking the skeleton and the membrane. The protein 4.1-glycophorin-C cross-bridge is known to be critically important for the stability and mechanical properties of human RBC plasma membrane. Because isoforms of protein 4.1, glycophorin C, and p55 exist in many tissues, these results provide evidence of a linkage between the skeleton and the membrane that may have implications in many nonerythroid cells.

Abstract 1990 Poster Board I-1012 Erythrocyte dematin is a widely expressed actin-binding and bundling protein, and functions as a suppressor of RhoA signaling in fibroblasts (Mohseni and Chishti, Molecular Cell Biology 28: 4712-4718, 2008). Dematin is a substrate of multiple protein kinases, and its actin bundling activity is regulated by cAMP dependent protein kinase. Recently, we identified a novel interaction between dematin and glucose transporter-1 (GLUT1) that is critically important for erythrocyte shape and membrane mechanical properties (Khan et al., Journal of Biological Chemistry 283:14600-14609, 2008). Since homologues of dematin and GLUT1 exist in many non-erythroid cells, we proposed that a conserved mechanism might couple related sugar transporters, such as the insulin-responsive glucose transporter-4 (GLUT4), to the actin cytoskeleton via dematin. Immunocytochemistry established the presence of dematin in 3T3-L1 adipocytes, and a small pool of dematin and GLUT4-containing vesicles co-localized in 3T3-L1 cells under both basal and insulin-stimulated conditions. Plasma membrane sheet assays indicate that upon insulin stimulation, dematin translocates to the plasma membrane along with GLUT4, resulting in partial co-localization at the plasma membrane. Furthermore, dematin RNAi treated 3T3-L1 cells show reduced GLUT4 protein expression, suggesting that dematin may regulate a sub-population of GLUT4 via the lysosomal degradation pathway in adipocytes. Importantly, glucose transport was reduced by ∼28% in 3T3-L1 adipocytes depleted of dematin, and by ∼15% in the dematin headpiece knockout (HPKO) mouse primary adipocytes. Since a significant amount of dematin did not co-localize with GLUT4 in the cytosol and plasma membrane, biochemical interaction between dematin and GLUT4 could not be verified using immunoprecipitation and transfection assays. Although dematin does not bind directly to GLUT4 under these conditions, a possibility existed that this interaction may be transient and mediated through an adaptor protein. Interestingly, dematin contains seven 14-3-3 binding sites, and 14-3-3 adaptor has been shown to be functionally involved in GLUT4 trafficking. We demonstrate that phosphorylated dematin binds to 14-3-3 in 3T3-L1 adipocytes under both basal and insulin stimulated conditions. Mutagenesis studies identify serine-85 on dematin as the primary phospho-binding site for 14-3-3zeta. Furthermore, using pharmacological inhibitors, Akt is identified as the likely protein kinase that phosphorylates dematin to mediate the biochemical interactions between dematin and 14-3-3zeta. Together, our results identify erythrocyte dematin as a potential regulator of glucose transporter trafficking and degradation pathways in adipocytes with functional implications for glucose homeostasis, diabetes, and obesity. Disclosures: No relevant conflicts of interest to declare.

Abstract 779 The Feline Leukemia Virus Subgroup CReceptor, FLVCR, a member of the Major Facilitator Superfamily (MFS) of secondary permeases, plays a critical role in erythropoiesis through regulation of cellular heme export. In addition, the receptor is highly expressed at sites of heme trafficking, such as the small intestine, liver, and macrophages. Accordingly, neonatal conditional knockdown of FLVCR function in a mouse model results in arrested erythroid differentiation and the rapid development of systemic iron overload (Keel SB, et al. 2008 Science 319:825–28). As there are wide disparities between FLVCR protein and gene expression, we hypothesized that, similar to other MFS transporters such as the iron exporter ferroportin, FLVCR expression is likely regulated in a post-translational manner through control of its intracellular trafficking. Thus, we performed yeast-two-hybrid screens of a human liver cDNA library using the intracytosolic C-terminal potential PDZ domain–binding motif of FLVCR. This screen identified a candidate binding protein, PTPN3, a tyrosine phosphatase containing both PDZ and FERM (cytoskeletal attachment) domains. Using in vitro and in vivo co-immunoprecipitation assays, we demonstrate that PTPN3 interacts with and regulates FLVCR expression. Notably, in co-transfection assays, we demonstrate marked positive regulation of FLVCR expression by PTPN3 (∼4-fold). Furthermore, a pool of PTPN3 that associates with the cell membrane specifically immunoprecipitates FLVCR, suggesting their existence in a protein complex at the cell surface. As predicted, deletion of the PDZ domain–binding motif of FLVCR or the PTPN3 PDZ domain, or both, abrogates their interaction in vivo. In addition, a single amino acid mutation of PTPN3 that results in a catalytically inactive phosphatase significantly diminishes its interaction with the receptor, indicating the potential importance of phosphorylation/dephosphorylation of FLVCR for trafficking. By analogy to the ferroportin trafficking pathway (De Domenico I, et al. 2009 Proc. Natl. Acad. Sci. 106:3800–805), we surmise that tyrosine residues located within the intracellular loop of FLVCR (Y319, Y321) are the likely regulatory targets of phosphorylation/dephosphorylation reactions. Thus, both the scaffolding and the phosphatase functions of PTPN3 are important for the regulation of FLVCR expression. Finally, our preliminary analysis of mutation of the dileucine motif located within the intracytosolic N-terminus of FLVCR suggests the motif also contributes to correct cell membrane targeting in transfected cells. In addition to analyzing endogenous interactions between FLVCR and PTPN3, we are currently investigating the importance of both FLVCR trafficking in polarized and erythroid cell lines, utilizing defined deletion constructs, and FLVCR function using heme transport assays. Notwithstanding its importance in erythropoiesis and systemic iron homeostasis, functional insights into FLVCR regulation will increase our understanding of its role in cellular protection from heme toxicity, an issue of considerable relevance for hemolytic anemias, thalassemia, and malaria. Disclosures: No relevant conflicts of interest to declare.

Erythrocyte p55 is a prototypical member of a family of scaffolding proteins known as Membrane Associated Guanylate Kinase Homologues (MAGUKs). MAGUKs are multi-domain proteins that couple signals from specialized sites at the plasma membrane to intracellular signal transduction pathways and the cytoskeleton. P55 was originally identified in the erythrocytes as part of a ternary complex with protein 4.1R and glycophorin C, providing a critical linkage between the actin cytoskeleton and the plasma membrane. Although p55 is expressed in a variety of tissues, especially hematopoietic cells, its biological function is unclear. Here, using a p55 knockout mouse model, we show that p55 plays a prominent role in the regulation of neutrophil polarization. Neutrophils are the first respondents during infection and injury, adopting a highly polarized morphology when stimulated with chemotactic factors. G proteincoupled surface receptors recognize the external chemotactic gradient and translate it into an internal gradient of signaling molecules. At the front of the cell, accumulation of the lipid product phosphatidylinositol-3,4,5-trisphosphate (PIP3), activation of the small GTPase Rac, and polymerization of F-actin stimulates a positive feedback loop promoting pseudopod formation. Here, we show that neutrophils lacking p55 form multiple transient pseudopods at the sides and back of the cell upon stimulation. P55 is required for limiting the pseudopod in the direction of chemoattractant. As a result, these neutrophils do not migrate efficiently up a chemotactic gradient in vitro. Biochemical analysis indicates that total F-actin polymerization and total Rac activation is similar between wild type and p55 knockout neutrophils. However, we found that phosphorylation of AKT, the major kinase downstream of the phosphatidylinositol 3-kinase (PI3K)-PIP3 pathway, is almost completely blocked in p55 knockout neutrophils. This finding suggests that p55 exerts its functional effect by regulating PIP3 accumulation or its localization at the membrane, which is responsible for amplification of the frontness signal and stability of the leading edge pseudopod. Consistent with this finding, the p55 null mice are significantly more susceptible to spontaneous and induced infections. Taken together, we have identified p55 as a novel mediator of the frontness signal in neutrophils that promotes polarization and efficient chemotaxis.

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.

There is considerable interest in the elucidation of the mechanism that governs the linkage of elongated spectrin molecules to the erythrocyte plasma membrane. The mechanism by which the “head” region of the spectrin dimer, which participates in tetramer formation, binds to the membrane via ankyrin and band 3 has been reasonably well characterized. However, the mechanism by which the tail end of the spectrin dimer is anchored to the plasma membrane is not completely understood. Dematin and adducin are actin binding proteins located at the spectrin-actin junctions or “junctional complex” in the erythrocyte membrane. Individual suppression of their function in mice by the gene deletion exerts a modest effect on erythrocyte shape and membrane stability. In contrast, the combined deletion of dematin and adducin genes results in severe defects of erythrocyte shape, membrane instability, and hemolysis. Based on these findings, we proposed a model whereby dematin and adducin could function as a molecular bridge linking the junctional complex to the plasma membrane. Using a combination of cell surface labeling, immunoprecipitation, and vesicle proteomics, we have identified glucose transporter-1 as the receptor for dematin and adducin in the human erythrocyte membrane. This finding is the first description of a transmembrane protein that binds to dematin and adducin, thus providing a rationale for the attachment of the cytoskeletal junctional complex to the lipid bilayer via glucose transporter-1. Since homologues of dematin, adducin, and glucose transporter-1 exist in many non-erythroid cells, we propose that a conserved mechanism may exist that couples sugar and other related transporters to the actin cytoskeleton.