Glycophorin C Research Articles

Dantu Blood Group Erythrocytes Form Large Plasmodium falciparum Rosettes Less Commonly.

Dantu erythrocytes, which express a hybrid glycophorin B/A protein, are protective against severe malaria. Recent studies have shown that Dantu impairs Plasmodium falciparum invasion by increasing erythrocyte membrane tension, but its effects on pathological host-parasite adhesion interactions such as rosetting, the binding of uninfected erythrocytes to P. falciparum-infected erythrocytes, have not been investigated previously. The expression of several putative host rosetting receptors-including glycophorin A (GYPA), glycophorin C (GYPC), complement receptor 1 (CR1), and band 3, which complexes with GYPA to form the Wrightb blood group antigen-are altered on Dantu erythrocytes. Here, we compare receptor expression, and rosetting at both 1 hour and 48 hours after mixing with mature trophozoite-stage Kenyan laboratory-adapted P. falciparum strain 11019 parasites in Dantu and non-Dantu erythrocytes. Dantu erythrocytes showed lower staining for GYPA and CR1, and greater staining for band 3, as observed previously, whereas Wrightb and GYPC staining did not vary significantly. No significant between-genotype differences in rosetting were seen after 1 hour, but the percentage of large rosettes was significantly less in both Dantu heterozygous (mean, 16.4%; standard error of the mean [SEM], 3.2) and homozygous donors (mean, 15.4%; SEM, 1.4) compared with non-Dantu erythrocytes (mean, 32.9%; SEM, 7.1; one-way analysis of variance, P = 0.025) after 48 hours. We also found positive correlations between erythrocyte mean corpuscular volume (MCV), the percentage of large rosettes (Spearman's rs = 0.5970, P = 0.0043), and mean rosette size (rs = 0.5206, P = 0.0155). Impaired rosetting resulting from altered erythrocyte membrane receptor expression and reduced MCV might add to the protective effect of Dantu against severe malaria.

Whole blood transcriptome profiling identifies gene expression subnetworks and a key gene characteristic of the rare type of osteomyelitis

Chronic non-bacterial osteomyelitis (CNO) is a rare and severe inflammatory bone disorder that can occur in the jaw. It is often associated with systemic conditions including autoimmune deficiency. Medical management of patients and establishment of a correct diagnosis are difficult as the etiology of the disease remains unknown. Therefore, little is known about the disease characteristics at the gene expression level. Here, we explored aspects of CNO based on whole blood RNA sequencing (>6 Gb per sample) of 11 patients and 9 healthy controls in Japan and on a recently developed method that is applicable to small datasets, can estimate a directed gene network, and extract a subnetwork of genes underlying patient characteristics. We identified nine subnetworks, comprising 26 differentially regulated edges and 36 genes, with the gene encoding glycophorin C (GYPC) presenting the highest discrimination ability. The expression of the gene was mostly lower in patients with CNO than in the healthy controls, suggesting an abnormal status of red cells in patients with CNO. This study enhances our understanding of CNO at the transcriptome level and further provides a framework for whole blood RNA sequencing and analysis of data obtained for a better diagnosis of the disease.

Identification of Potential Biomarkers Associated with Dilated Cardiomyopathy by Weighted Gene Coexpression Network Analysis.

Dilated cardiomyopathy (DCM) is one of the main causes of systolic heart failure and frequently has a genetic component. The molecular mechanisms underlying the onset and progression of DCM remain unclear. This study aimed to identify novel diagnostic biomarkers to aid in the treatment and diagnosis of DCM. The Gene Expression Omnibus (GEO) database was explored to extract two microarray datasets, GSE120895 and GSE17800, which were subsequently merged into a single cohort. Differentially expressed genes were analyzed in the DCM and control groups, followed by weighted gene coexpression network analysis to determine the core modules. Core nodes were identified by gene significance (GS) and module membership (MM) values, and four hub genes were predicted by the Lasso regression model. The expression levels and diagnostic values of the four hub genes were further validated in the datasets GSE19303. Finally, potential therapeutic drugs and upstream molecules regulating genes were identified. The turquoise module is the core module of DCM. Four hub genes were identified: GYPC (glycophorin C), MLF2 (myeloid leukemia factor 2), COPS7A (COP9 signalosome subunit 7A) and ARL2 (ADP ribosylation factor like GTPase 2). Subsequently, Hub genes showed significant differences in expression in both the dataset and the validation model by real-time quantitative PCR (qPCR). Four potential modulators and seven chemicals were also identified. Finally, molecular docking simulations of the gene-encoded proteins with small-molecule drugs were successfully performed. The results suggested that ARL2, MLF2, GYPC and COPS7A could be potential gene biomarkers for DCM.

P-10 Methylation-Based Expression Loss of Glycophorin C (GYPC) Gene and Clinical Importance in Oral Cancer

P-10 Methylation-Based Expression Loss of Glycophorin C (GYPC) Gene and Clinical Importance in Oral Cancer

The Gardos effect drives erythrocyte senescence and leads to Lu/BCAM and CD44 adhesion molecule activation

Senescence of erythrocytes is characterized by a series of changes that precede their removal from the circulation, including loss of red cell hydration, membrane shedding, loss of deformability, phosphatidyl serine exposure, reduced membrane sialic acid content, and adhesion molecule activation. Little is known about the mechanisms that initiate these changes nor is it known whether they are interrelated. In this study, we show that Ca2+-dependent K+ efflux (the Gardos effect) drives erythrocyte senescence. We found that increased intracellular Ca2+ activates the Gardos channel, leading to shedding of glycophorin-C (GPC)-containing vesicles. This results in a loss of erythrocyte deformability but also in a marked loss of membrane sialic acid content. We found that GPC-derived sialic acid residues suppress activity of both Lutheran/basal cell adhesion molecule (Lu/BCAM) and CD44 by the formation of a complex on the erythrocyte membrane, and Gardos channel-mediated shedding of GPC results in Lu/BCAM and CD44 activation. This phenomenon was observed as erythrocytes aged and on erythrocytes that were otherwise prone to clearance from the circulation, such as sickle erythrocytes, erythrocytes stored for transfusion, or artificially dehydrated erythrocytes. These novel findings provide a unifying concept on erythrocyte senescence in health and disease through initiation of the Gardos effect.

Identification and validation of smoking-related genes in lung adenocarcinoma using an in vitro carcinogenesis model and bioinformatics analysis

BackgroundLung cancer is one of the most common carcinomas in the world, and lung adenocarcinoma (LUAD) is the most lethal and most common subtype of lung cancer. Cigarette smoking is the most leading risk factor of lung cancer, but it is still unclear how normal lung cells become cancerous in cigarette smokers. This study aims to identify potential smoking-related biomarkers associated with the progression and prognosis of LUAD, as well as their regulation mechanism using an in vitro carcinogenesis model and bioinformatics analysis.ResultsBased on the integration analysis of four Gene Expression Omnibus (GEO) datasets and our mRNA sequencing analysis, 2 up-regulated and 11 down-regulated genes were identified in both S30 cells and LUAD. By analyzing the LUAD dataset in The Cancer Gene Analysis (TCGA) database, 3 of the 13 genes, viz., glycophorin C (GYPC), NME/NM23 nucleoside diphosphate kinase 1 (NME1) and slit guidance ligand 2 (SLIT2), were found to be significantly correlated with LUAD patients’ smoking history. The expression levels of GYPC, NME1 and SLIT2 in S30 cells and lung cancer cell lines were validated by quantitative PCR, immunofluorescence, and western blot assays. Besides, these three genes are associated with tumor invasion depth, and elevated expression of NME1 was correlated with lymph node metastasis. The enrichment analysis suggested that these genes were highly correlated to tumorigenesis and metastasis-related biological processes and pathways. Moreover, the increased expression levels of GYPC and SLIT2, as well as decreased expression of NME1 were associated with a favorable prognosis in LUAD patients. Furthermore, based on the multi-omics data in the TCGA database, these genes were found to be regulated by DNA methylation.ConclusionIn conclusion, our observations indicated that the differential expression of GYPC, NME1 and SLIT2 may be regulated by DNA methylation, and they are associated with cigarette smoke-induced LUAD, as well as serve as prognostic factors in LUAD patients.

Prognostic and clinico-pathological significance of BIN1 in breast cancer

Abstract Bridging integrator 1 (BIN1) is a potential tumor suppressor that behaves as silent or misplaced in breast, prostate, skin, lung, and colon cancers. Previous studies noted decreased BIN1 expression in breast cancer (BC). Despite this, BIN1's role in breast cancer is not well understood and remains to be elucidated. Here, we computationally analyzed BIN1 expression and its prognostic role in BC using various databases. Specifically, we found a down-regulated expression profile for the BIN1 gene in different BC subtypes using Oncomine, TCGA, and bcGenExMiner v4.2 web. The expression of BIN1 was found to be associated with various clinico-pathological conditions including estrogen receptor (ER), progesterone receptor (PR) basal-like subtype, and triple-negative breast cancer (TNBC) based on analysis of the bcGenExMiner v4.2 and TCGA databases. In addition to mRNA expression, the correlation between mRNA expression and BIN1 promoter methylation was established using TCGA datasets from UCSC Xena and ULCAN web, and we subsequently examined the relationship of BIN1 promoter methylation with different clinico-pathological conditions in BC patients. Data mining via Kaplan-Meier plotter indicated a positive association between lower BIN1 expression and low BC patient survival and various clinico-pathological conditions in BC patients. Furthermore, we identified a list of genes co-expressed with BIN1 in BC using TCGA data from the ULCAN web. Among the co-expressed genes, glycophorin C (GYPC) had the most predominant positive correlation. This correlation was further confirmed in BC using UCSC Xena and bcGenExMiner v4.2 webs. Overall, this study establishes that attenuated BIN1 expression might induce progression and poor prognosis in BC patients with GYPC association. This could be useful for clinical BC therapy.

The Gerbich blood group system: old knowledge, new importance.

Antigens of the Gerbich blood group system are expressed on glycophorin C (GPC) and glycophorin D (GPD), minor sialoglycoproteins of human erythrocytes. GPC and GPD help maintain erythrocyte shape of and contributes to the stability of its membrane. There are six high-prevalence Gerbich antigens: Ge2, Ge3, Ge4, GEPL (GE10), GEAT (GE11), GETI (GE12) and five low-prevalence Gerbich antigens: Wb (GE5), Lsa (GE6), Ana (GE7), Dha (GE8), GEIS (GE9). Some Gerbich antigens (Ge4, Wb, Dha, GEAT) are expressed only on GPC, two (Ge2, Ana) are expressed only on GPD, while others (Ge3, Lsa, GEIS, GEPL, GETI) are expressed on both GPC and GPD. Antibodies recognizing GPC/GPD may arise naturally (so-called "naturally-occurring RBC antibodies") or as the result of alloimmunization, and some of them may be clinically relevant. Gerbich antibodies usually do not cause serious hemolytic transfusion reactions (HTR); autoantibodies of anti-Ge2- or anti-Ge3 specificity can cause autoimmune hemolytic anemia (AIHA).

Genetic Evidence for Erythrocyte Receptor Glycophorin B Expression Levels Defining a Dominant Plasmodium falciparum Invasion Pathway into Human Erythrocytes.

ABSTRACTPlasmodium falciparum, the parasite that causes the deadliest form of malaria, has evolved multiple proteins known as invasion ligands that bind to specific erythrocyte receptors to facilitate invasion of human erythrocytes. The EBA-175/glycophorin A (GPA) and Rh5/basigin ligand-receptor interactions, referred to as invasion pathways, have been the subject of intense study. In this study, we focused on the less-characterized sialic acid-containing receptors glycophorin B (GPB) and glycophorin C (GPC). Through bioinformatic analysis, we identified extensive variation in glycophorin B (GYPB) transcript levels in individuals from Benin, suggesting selection from malaria pressure. To elucidate the importance of the GPB and GPC receptors relative to the well-described EBA-175/GPA invasion pathway, we used an ex vivo erythrocyte culture system to decrease expression of GPA, GPB, or GPC via lentiviral short hairpin RNA transduction of erythroid progenitor cells, with global surface proteomic profiling. We assessed the efficiency of parasite invasion into knockdown cells using a panel of wild-type P. falciparum laboratory strains and invasion ligand knockout lines, as well as P. falciparum Senegalese clinical isolates and a short-term-culture-adapted strain. For this, we optimized an invasion assay suitable for use with small numbers of erythrocytes. We found that all laboratory strains and the majority of field strains tested were dependent on GPB expression level for invasion. The collective data suggest that the GPA and GPB receptors are of greater importance than the GPC receptor, supporting a hierarchy of erythrocyte receptor usage in P. falciparum.

A post-genome-wide association study validating the association of the glycophorin C gene with serum hemoglobin level in pig.

ObjectiveThis study aimed to validate the statistical evidence from the genome-wide association study (GWAS) as true-positive and to better understand the effects of the glycophorin C (GYPC) gene on serum hemoglobin traits.MethodsOur initial GWAS revealed the presence of two single nucleotide polymorphisms (SNPs) (ASGA0069038 and ALGA0084612) for the hemoglobin concentration trait (HGB) in the 2.48 Mb region of SSC15. From this target region, GYPC was selected as a promising gene that associated with serum HGB traits in pigs. SNPs within the GYPC gene were detected by sequencing. Thereafter, we performed association analysis of the variant with the serum hemoglobin level in three pig populations.ResultsWe identified one SNP (g.29625094 T>C) in exon 3 of the GYPC gene. Statistical analysis showed a significant association of the SNP with the serum hemoglobin level on day 20 (p<0.05). By quantitative real-time polymerase chain reaction, the GYPC gene was expressed in eight different tissues.ConclusionThese results might improve our understanding of GYPC function and provide evidence for its association with serum hemoglobin traits in the pig. These results also indicate that the GYPC gene might serve as a useful marker in pig breeding programs.

Baculovirus-expressed Plasmodium reichenowi EBA-140 merozoite ligand is host specific

Plasmodium reichenowi, an ape malaria parasite is morphologically identical and genetically similar to Plasmodium falciparum, infects chimpanzees but not humans. Genomic studies revealed that all primate malaria parasites belong to Laverania subgenus. Laverania parasites exhibit strict host specificity, but the molecular mechanisms underlying these host restrictions remain unexplained.Plasmodium merozoites express multiple binding ligands that recognize specific receptors on erythrocytes, including micronemal proteins belonging to P. falciparum EBL family. It was shown that erythrocyte binding antigen-175 (EBA-175), erythrocyte binding ligand-1 (EBL-1), erythrocyte binding antigen-140 (EBA-140) recognize erythrocyte surface sialoglycoproteins - glycophorins A, B, C, respectively. EBA-140 merozoite ligand hijacks glycophorin C (GPC), a minor erythrocyte sialoglycoprotein, to invade the erythrocyte through an alternative invasion pathway.A homolog of P. falciparum EBA-140 protein was identified in P. reichenowi. The amino acid sequences of both EBA-140 ligands are very similar, especially in the conservative erythrocyte binding region (Region II). It has been suggested that evolutionary changes in the sequence of EBL proteins may be associated with Plasmodium host restriction.In this study we obtained, for the first time, the recombinant P. reichenowi EBA-140 ligand Region II using baculovirus expression vector system. We show that the ape EBA-140 Region II is host specific and binds to chimpanzee erythrocytes in the dose and sialic acid dependent manner. Further identification of the erythrocyte receptor for this ape ligand is of great interests, since it may reveal the molecular basis of host restriction of both P. reichenowi and its deadliest human counterpart, P. falciparum.

A Stem Cell Strategy Identifies Glycophorin C as a Major Erythrocyte Receptor for the Rodent Malaria Parasite Plasmodium berghei.

The clinical complications of malaria are caused by the parasite expansion in the blood. Invasion of erythrocytes is a complex process that depends on multiple receptor-ligand interactions. Identification of host receptors is paramount for fighting the disease as it could reveal new intervention targets, but the enucleated nature of erythrocytes makes genetic approaches impossible and many receptors remain unknown. Host-parasite interactions evolve rapidly and are therefore likely to be species-specific. As a results, understanding of invasion receptors outside the major human pathogen Plasmodium falciparum is very limited. Here we use mouse embryonic stem cells (mESCs) that can be genetically engineered and differentiated into erythrocytes to identify receptors for the rodent malaria parasite Plasmodium berghei. Two proteins previously implicated in human malaria infection: glycophorin C (GYPC) and Band-3 (Slc4a1) were deleted in mESCs to generate stable cell lines, which were differentiated towards erythropoiesis. In vitro infection assays revealed that while deletion of Band-3 has no effect, absence of GYPC results in a dramatic decrease in invasion, demonstrating the crucial role of this protein for P. berghei infection. This stem cell approach offers the possibility of targeting genes that may be essential and therefore difficult to disrupt in whole organisms and has the potential to be applied to a variety of parasites in diverse host cell types.

STEVOR Is a Plasmodium falciparum Erythrocyte Binding Protein that Mediates Merozoite Invasion and Rosetting

Variant surface antigens play an important role in Plasmodium falciparum malaria pathogenesis and in immune evasion by the parasite. Although most work to date has focused on P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1), two other multigene families encoding STEVOR and RIFIN are expressed in invasive merozoites and on the infected erythrocyte surface. However, their role during parasite infection remains to be clarified. Here we report that STEVOR functions as an erythrocyte-binding protein that recognizes Glycophorin C (GPC) on the red blood cell (RBC) surface and that its binding correlates with the level of GPC on the RBC surface. STEVOR expression on the RBC leads to PfEMP1-independent binding of infected RBCs to uninfected RBCs (rosette formation), while antibodies targeting STEVOR in the merozoite can effectively inhibit invasion. Our results suggest a PfEMP1-independent role for STEVOR in enabling infected erythrocytes at the schizont stage to form rosettes and in promoting merozoite invasion.

Phosphatidylinositol-4,5 bisphosphate (PIP2) inhibits apo-calmodulin binding to protein 4.1

Membrane skeletal protein 4.1R80 plays a key role in regulation of erythrocyte plasticity. Protein 4.1R80 interactions with transmembrane proteins, such as glycophorin C (GPC), are regulated by Ca2+-saturated calmodulin (Ca2+/CaM) through simultaneous binding to a short peptide (pep11; A264KKLWKVCVEHHTFFRL) and a serine residue (Ser185), both located in the N-terminal 30kDa FERM domain of 4.1R80 (H·R30). We have previously demonstrated that CaM binding to H·R30 is Ca2+-independent and that CaM binding to H·R30 is responsible for the maintenance of H·R30 β-sheet structure. However, the mechanisms responsible for the regulation of CaM binding to H·R30 are still unknown. To investigate this, we took advantage of similarities and differences in the structure of Coracle, the Drosophila sp. homologue of human 4.1R80, i.e. conservation of the pep11 sequence but substitution of the Ser185 residue with an alanine residue. We show that the H·R30 homologue domain of Coracle, Cor30, also binds to CaM in a Ca2+-independent manner and that the Ca2+/CaM complex does not affect Cor30 binding to the transmembrane protein GPC. We also document that both H·R30 and Cor30 bind to phosphatidylinositol-4,5 bisphosphate (PIP2) and other phospholipid species and that that PIP2 inhibits Ca2+-free CaM but not Ca2+-saturated CaM binding to Cor30. We conclude that PIP2 may play an important role as a modulator of apo-CaM binding to 4.1R80 throughout evolution.

Antibody‐mediated glycophorin C coligation on K562 cells induces phosphatidylserine exposure and cell death in an atypical apoptotic process

Glycophorin C (GPC) is necessary in the maintenance of red blood cell structure. Severe autoimmune hemolytic anemia and hemolytic disease of the fetus and newborn (HDFN) have been associated with Gerbich (Ge) blood group system antigens expressed on GPC. Previous in vitro studies with cord blood progenitor cells have shown that anti-Ge suppresses erythropoiesis. Here, we evaluated the K562 erythroleukemic cell line to study the cellular effects of a murine anti-GPC. Cell proliferation was evaluated after treatment with anti-GPC. Flow cytometry was used to evaluate exofacial phosphatidylserine (PS) expression and cell viability (propidium iodide binding). Cell morphology was evaluated under light microscopy with cytospin preparations stained with May-Grünwald Giemsa. Anti-GPC dramatically inhibited K562 proliferation and increased PS expression, consistent with cytoplasmic blebbing, suggesting evidence of apoptosis. Z-VAD-FMK, an inhibitor of classical apoptosis, was unable to reverse the suppressive effect of anti-GPC. However, hemin was able to attenuate growth suppression. Together, the data suggest that anti-GPC suppresses erythroid proliferation through the induction of nonclassical apoptosis.

Reduction of Erythropoiesis in MDS with 5q Deletion Is Associated with Aberrant SPARC and TNF-Alpha Gene Expression and Predicts the Efficacy of Lenalidomide Therapy.

AbstractAbstract 2826Background of anemia in myelodysplastic syndromes (MDS) with 5q deletion (del(5q)) is a haplo-insufficiency of the RPS14 gene. However, degree of anemia and quantity of erythropoiesis vary markedly between MDS patients with del(5q), even with a similar degree of RPS14 insufficiency, so that the question arises whether other influences may play a further relevant role in erythroid failure in this disease.From 35 patients with low or intermediate-1 risk MDS with del(5q) who developed a transfusion-dependent anemia and were recruited and treated with lenalidomide (10 mg / d) within the MDS-003 study 38 +/− 33 months after diagnosis of MDS, bone marrow biopsies taken at 6 to 12-month time intervals after diagnosis of disease were evaluated for changes of erythropoiesis having occurred prior to lenalidomide therapy by morphometric, immunohistochemical, and molecular methods. A second group of patients with MDS treated with best supportive care (n = 307) served as a control. From each bone marrow biopsy, 20 000 nucleated cells were evaluated marking (1) nucleated erythroid cells (NEC) immunohistochemically using anti-HbA, glycophorin-C (GPC), CD71, and CD34 antibodies and (2) marrow macrophages by anti-CD68 antibody. By analogy to burst and colony forming units from in-vitro models, the numbers of erythroid precursor cells (EPC) were estimated by the numbers of erythroblastic islands (IslE) within marrow. Morphometrically assessed erythropoiesis was related to the mRNA expression of RPS14, SPARC, and TNF-alpha genes, nuclear p53 protein expression of NEC and their relevance for transfusion dependence and AML-free survival time of patients.5q deletion predicted erythroid hypoplasia independently of the type of MDS, evidence of marrow fibrosis and AML transformation (P < 0.0005). During early-phase MDS with del(5q), anemia correlated with p53 overexpression of NEC and decreased numbers of HbA+ NEC (P < 0.00005). After the first year of disease, two alterations of erythropoiesis became more relevant which did not correlate to p53 overexpression of NEC: (1) a decline in the numbers of GPC+HbA- NEC indicating impaired differentiation of EPC to proerythroblasts which correlated with RPS14 insufficiency, and (2) a progressive decrease in the number of IslE indicating a block of differentiation at an earlier stage, the IslE forming EPC, which did not correlate to RPS14 gene expression, but to two other independent factors, a reduced SPARC and an increased TNF-alpha gene expression (P < 0.00005). The degree of TNF-alpha expression correlated to the number of a central component of the IslE, the (iron-presenting) macrophages, the potential source of TNF-alpha overexpression. Both alterations contributed significantly to erythroid hypoplasia and progressive transfusion dependence (P < 0.00005). Response to lenalidomide therapy continued for > 4 years (100% AML-free survival) when started during a phase with high numbers of p53 overexpressing NEC, but was short or poor with < 20 % AML-free survival when started during a phase when differentiation of erythropoiesis was impaired at a less mature stage (P = 0.0001). On the basis of these observations, a score predicting the efficacy of lenalidomide in individual patients could be constructed. Conclusions:In MDS with del(5q), erythropoiesis significantly changes over time. In the early stage of disease, maturation defect of erythropoiesis mainly affects HbA+ NEC and correlates to p53 overexpression of NEC. In the course of disease, erythropoiesis becomes hypoplastic due to differentiation defects at the level of more immature erythroid (precursor) cells relating to a p53-independent mechanism of RPS14 insufficiency and altered SPARC as well as TNF-alpha gene expression. Efficacy of lenalidomide therapy is significantly impaired in the case of vanishing IslE, reduced SPARC and increased TNF-alpha gene expression. Disclosures:Giagounidis:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Knight:Celgene Corp.: Employment.

Intracellular interactions between protein 4.1 and glycophorin C on transport vesicles, as determined by fluorescence correlation spectroscopy

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. Structured summary of protein interactionsGPC and 4.1R30physically interact by fluorescence correlation spectroscopy (View interaction)

【Original Contribution】 Characterization of Mouse Monoclonal Antibodies to Human Protein 4.1R FERM Domain: Epitope Mapping and Application to FERM Domain Binding to Red Blood Cell Inside-out Vesicles

In human erythrocytes, the 80-kDa isoform of protein 4.1R, 4.1R80, maintains mechanical membrane stability and deformability as a result of multiple protein-protein interactions. 4.1R80 binds to transmembrane proteins glycophorin C (GPC) and band 3 via its 30-kDa N-terminal FERM (Four one-Ezrin-Radixin-Moesin) domain (referred to as “R30” in the present study) and to spectrin and actin via a 10-kDa domain. Although the sites in R30 responsible for interaction with transmembrane proteins have been extensively studied, the identity of these sites has been challenged recently. Antibodies, in particular monoclonal antibodies (mAbs), are powerful tools not only for immunochemical studies but also for functional analyses such as the monitoring of the dynamic interactions of R30 with its binding partners. In the present study, we have generated mouse mAbs against recombinant R30 protein, and characterized their respective recognition epitopes in R30 using various recombinant proteins. Four representative clones, #5, #7, #9, #13 recognized the Y131DPELHGVDYVSDFKLAPN (pep8.1), Q150 TKELEEKVMELHKSYR (pep8.2), M1HCKVSLLDDTVYECVVE (pep4) and Q247EQYESTIGFKLPSYRA (pep13) epitopes, respectively. These sequences are located in the N-,α- and C-lobe structure of R30, respectively. IAsys-based in vitro binding analyses enabled us to demonstrate that the binding of R30 to pH 11-treated inside-out-vesicles (IOVs) was reduced by two combinations of mAbs (#5 and #9 or #7 and #9) but not by any of the mAbs alone and to confirm that the GPC binding site of R30 was located at or near pep8.1 and pep8.2 in the α- lobe of R30. Our study validates that this novel panel of mAbs constitutes a powerful tool for various types of analyses of 4.1R.

Structural stabilization of protein 4.1R FERM domain upon binding to apo-calmodulin: novel insights into the biological significance of the calcium-independent binding of calmodulin to protein 4.1R

In erythrocytes, 4.1R80 (80 kDa isoform of protein 4.1R) binds to the cytoplasmic tail of the transmembrane proteins band 3 and GPC (glycophorin C), and to the membrane-associated protein p55 through the N- (N-terminal), α- (α-helix-rich) and C- (C-terminal) lobes of R30 [N-terminal 30 kDa FERM (4.1/ezrin/radixin/moesin) domain of protein 4.1R] respectively. We have shown previously that R30 binds to CaM (calmodulin) in a Ca2+-independent manner, the equilibrium dissociation constant (Kd) for R30-CaM binding being very similar (in the submicromolar range) in the presence or absence of Ca2+. In the present study, we investigated the consequences of CaM binding on R30's structural stability using resonant mirror detection and FTIR (Fourier-transform IR) spectroscopy. After a 30 min incubation above 40° C, R30 could no longer bind to band 3 or to GPC. In contrast, R30 binding to p55, which could be detected at a temperature as low as 34° C, was maintained up to 44° C in the presence of apo-CaM. Dynamic light scattering measurements indicated that R30, either alone or complexed with apo-CaM, did not aggregate up to 40° C. FTIR spectroscopy revealed that the dramatic variations in the structure of the β-sheet structure of R30 observed at various temperatures were minimized in the presence of apo-CaM. On the basis of Kd values calculated at various temperatures, ΔCp and ΔG° for R30 binding to apo-CaM were determined as -10 kJ · K(-1) · mol-1 and ~ -38 kJ · mol(-1) at 37° C (310.15 K) respectively. These data support the notion that apo-CaM stabilizes R30 through interaction with its β-strand-rich C-lobe and provide a novel function for CaM, i.e. structural stabilization of 4.1R80.

Membrane compartmentalization in Southeast Asian ovalocytosis red blood cells

Red blood cells (RBCs) from individuals with Southeast Asian ovalocytosis (SAO) contain a mutant band 3 protein that causes the formation of unique linear oligomers in the RBC membrane. We used single-particle tracking to measure the lateral diffusion of individual glycophorin C (GPC), band 3, and CD58 proteins in membranes of intact SAO RBCs and normal RBCs (nRBCs). GPC, an integral protein that binds with high affinity to the RBC membrane skeleton, showed oscillatory motion within confinement areas that were smaller in SAO RBCs than in nRBCs. The additional confinement in SAO RBCs could be due to membrane stiffening associated with the SAO phenotype. Band 3 in both SAO RBCs and nRBCs also showed confined motion over short times (ms) and distances (nm), and the area of confinement was smaller in SAO RBCs than in nRBCs. These data presumably reflect the constraints imposed by band 3 oligomerization. Similarly, the glycosylphosphatidylinositol-linked protein CD58 showed loosely confined diffusion in nRBCs and a substantially higher degree of confinement in SAO RBCs. Restricted protein mobility could contribute to the altered adherence of parasite-infected RBCs to vascular endothelium that is thought to protect individuals with SAO from severe manifestations of malaria.