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1.
Haematologica ; 107(1): 167-177, 2022 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-33406813

RESUMEN

Erythroblast maturation in mammals is dependent on organelle clearance throughout terminal erythropoiesis. We studied the role of the outer mitochondrial membrane protein voltage-dependent anion channel-1 (VDAC1) in human terminal erythropoiesis. We show that short hairpin (shRNA)-mediated downregulation of VDAC1 accelerates erythroblast maturation. Thereafter, erythroblasts are blocked at the orthochromatic stage, exhibiting a significant decreased level of enucleation, concomitant with an increased cell death. We demonstrate that mitochondria clearance starts at the transition from basophilic to polychromatic erythroblast, and that VDAC1 downregulation induces the mitochondrial retention. In damaged mitochondria from non-erythroid cells, VDAC1 was identified as a target for Parkin-mediated ubiquitination to recruit the phagophore. Here, we showed that VDAC1 is involved in phagophore's membrane recruitment regulating selective mitophagy of still functional mitochondria from human erythroblasts. These findings demonstrate for the first time a crucial role for VDAC1 in human erythroblast terminal differentiation, regulating mitochondria clearance.


Asunto(s)
Mitocondrias , Mitofagia , Animales , Apoptosis , Diferenciación Celular , Eritroblastos/metabolismo , Eritropoyesis , Humanos , Mamíferos , Mitocondrias/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/genética , Canal Aniónico 1 Dependiente del Voltaje/metabolismo
2.
Br J Haematol ; 193(5): 988-993, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33754349

RESUMEN

Sickle cell disease (SCD) is characterised by chronic haemolysis and oxidative stress. Herein, we investigated 30 SCD patients and found 40% with elevated mitochondria levels (SS-mito+ ) in their mature red blood cells, while 60% exhibit similar mitochondria levels compared to the AA group (SS-mito- ). The SS-mito+ patients are characterised by higher reticulocytosis and total bilirubin levels, lower foetal haemoglobin, and non-functional mitochondria. Interestingly, we demonstrated decreased levels of mitophagy inducers, PINK1 and NIX, and higher levels of HSP90 chaperone in their red cells. Our results highlighted for the first time an abnormal retention of mitochondria in SCD linked with mitophagy-related proteins.


Asunto(s)
Anemia de Células Falciformes/sangre , Eritrocitos/metabolismo , Mitocondrias/metabolismo , Mitofagia , Adulto , Anemia de Células Falciformes/patología , Bilirrubina/sangre , Eritrocitos/patología , Femenino , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Mitocondrias/patología , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Reticulocitosis , Proteínas Supresoras de Tumor/metabolismo
3.
Biol Cell ; 112(4): 113-126, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-31989647

RESUMEN

BACKGROUND: 5-Aminolevulinic acid (ALA) is the first precursor of heme biosynthesis pathway. The exogenous addition of ALA to cells leads to protoporphyrin IX (PPIX) accumulation that has been exploited in photodynamic diagnostic and photodynamic therapy. Several types of ALA transporters have been described depending on the cell type, but there was no clear entry pathway for erythroid cells. The 18 kDa translocator protein (TSPO) has been proposed to be involved in the transport of porphyrins and heme analogs. RESULTS: ALA-induced PPIX accumulation in erythroleukemia cells (UT-7 and K562) was impaired by PK 11195, a competitive inhibitor of both transmembrane proteins TSPO (1 and 2). PK 11195 did not modify the activity of the enzymes of heme biosynthesis, suggesting that ALA entry at the plasma membrane was the limiting factor. In contrast, porphobilinogen (PBG)-induced PPIX accumulation was not affected by PK 11195, suggesting that plasma membrane TSPO2 is a selective transporter of ALA. Overexpression of TSPO2 at the plasma membrane of erythroleukemia cells increased ALA-induced PPIX accumulation, confirming the role of TSPO2 in the import of ALA into the cells. CONCLUSIONS: ALA-induced PPIX accumulation in erythroid cells involves TSPO2 as a selective translocator through the plasma membrane. SIGNIFICANCE: This is the first characterisation of molecular mechanisms involving a new actor in ALA transport in ALA-induced PPIX accumulation in erythroleukemia cells, which could be inhibited by specific drug ligands.


Asunto(s)
Membrana Celular/metabolismo , Leucemia Eritroblástica Aguda/metabolismo , Ácidos Levulínicos/farmacocinética , Receptores Citoplasmáticos y Nucleares/metabolismo , Transporte Biológico , Proteínas Fluorescentes Verdes/genética , Humanos , Isoquinolinas/farmacología , Células K562 , Leucemia Eritroblástica Aguda/patología , Protoporfirinas/metabolismo , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Transfección , Ácido Aminolevulínico
4.
Mol Cell Biochem ; 463(1-2): 1-11, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31531757

RESUMEN

We reported previously that the rat submandibular gland is able to release nanovesicles capable to hydrolyse millimolar concentrations of ATP, ADP and AMP in vitro. Here, we show that rat saliva also contains nanovesicles with the ability to hydrolyse ATP. Our aim was to identify and characterize vesicular nucleotidases by using kinetic, immunological and in silico approaches. Nucleotidase activity in the absence or presence of specific inhibitors allowed us to assume the participation of NTPDase1, -2 and -3, together with ecto-5'-nucleotidase, confirmed using specific antibodies. At neutral pH, initial ATPase activity would be mostly due to NTPDase2, which was thereafter inactivated, leaving NTPDase1 and NTPDase3 to hydrolyse ATP and ADP with an efficacy ATPase/ADPase around 2. Ecto-5'nucleotidase would be mainly responsible for AMP hydrolysis and adenosine accumulation. We proposed a kinetic model for NTPDase2 as a tool to isolate and analyse the turnover of this enzyme in the presence of different ATP concentrations, including those expected in extracellular media. Our study characterizes the ectonucleotidases carried by extracellular vesicles which contribute to modulate ATP and adenosine concentrations in the oral cavity, essential players in purinergic signalling.


Asunto(s)
5'-Nucleotidasa/metabolismo , Vesículas Extracelulares/metabolismo , Boca/metabolismo , Pirofosfatasas/metabolismo , Saliva/enzimología , Proteínas y Péptidos Salivales/metabolismo , Transducción de Señal , Adenosina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Ratas , Ratas Wistar
5.
Int J Mol Sci ; 21(23)2020 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-33260618

RESUMEN

Translocator protein (TSPO) and voltage dependent anion channels (VDAC) are two proteins forming a macromolecular complex in the outer mitochondrial membrane that is involved in pleiotropic functions. Specifically, these proteins were described to regulate the clearance of damaged mitochondria by selective mitophagy in non-erythroid immortalized cell lines. Although it is well established that erythroblast maturation in mammals depends on organelle clearance, less is known about mechanisms regulating this clearance throughout terminal erythropoiesis. Here, we studied the effect of TSPO1 downregulation and the action of Ro5-4864, a drug ligand known to bind to the TSPO/VDAC complex interface, in ex vivo human terminal erythropoiesis. We found that both treatments delay mitochondrial clearance, a process associated with reduced levels of the PINK1 protein, which is a key protein triggering canonical mitophagy. We also observed that TSPO1 downregulation blocks erythroblast maturation at the orthochromatic stage, decreases the enucleation rate, and increases cell death. Interestingly, TSPO1 downregulation does not modify reactive oxygen species (ROS) production nor intracellular adenosine triphosphate (ATP) levels. Ro5-4864 treatment recapitulates these phenotypes, strongly suggesting an active role of the TSPO/VDAC complex in selective mitophagy throughout human erythropoiesis. The present study links the function of the TSPO/VDAC complex to the PINK1/Parkin-dependent mitophagy induction during terminal erythropoiesis, leading to the proper completion of erythroid maturation.


Asunto(s)
Núcleo Celular/metabolismo , Regulación hacia Abajo , Eritropoyesis , Mitocondrias/metabolismo , Mitofagia , Receptores de GABA/metabolismo , Benzodiazepinonas/farmacología , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Eritropoyesis/efectos de los fármacos , Humanos , Cinética , Mitocondrias/efectos de los fármacos , Mitofagia/efectos de los fármacos , Fenotipo , ARN Interferente Pequeño/metabolismo
6.
Int J Mol Sci ; 19(10)2018 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-30308949

RESUMEN

Two main isoforms of the Translocator Protein (TSPO) have been identified. TSPO1 is ubiquitous and is mainly present at the outer mitochondrial membrane of most eukaryotic cells, whereas, TSPO2 is specific to the erythroid lineage, located at the plasma membrane, the nucleus, and the endoplasmic reticulum. The design of specific tools is necessary to determine the molecular associations and functions of TSPO, which remain controversial nowadays. We recently demonstrated that TSPO2 is involved in a supramolecular complex of the erythrocyte membrane, where micromolar doses of the classical TSPO ligands induce ATP release and zinc protoporphyrin (ZnPPIX) transport. In this work, three newly-designed ligands (NCS1016, NCS1018, and NCS1026) were assessed for their ability to modulate the functions of various erythrocyte's and compare them to the TSPO classical ligands. The three new ligands were effective in reducing intraerythrocytic Plasmodium growth, without compromising erythrocyte survival. While NCS1016 and NCS1018 were the most effective ligands in delaying sorbitol-induced hemolysis, NCS1016 induced the highest uptake of ZnPPIX and NCS1026 was the only ligand inhibiting the cholesterol uptake. Differential effects of ligands are probably due, not only, to ligand features, but also to the dynamic interaction of TSPO with various partners at the cell membrane. Further studies are necessary to fully understand the mechanisms of the TSPO's complex activation.


Asunto(s)
Adenosina Trifosfato/metabolismo , Colesterol/metabolismo , Eritrocitos/metabolismo , Protoporfirinas/metabolismo , Receptores de GABA/metabolismo , Transporte Biológico , Hemólisis , Humanos , Ligandos , Plasmodium falciparum/efectos de los fármacos , Unión Proteica , Especies Reactivas de Oxígeno , Sorbitol/farmacología
7.
Purinergic Signal ; 11(1): 107-16, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25523180

RESUMEN

Extracellular nucleotides modulate a wide number of biological processes such as neurotransmission, platelet aggregation, muscle contraction, and epithelial secretion acting by the purinergic pathway. Nucleotidases as NTPDases and ecto-5'-nucleotidase are membrane-anchored proteins that regulate extracellular nucleotide concentrations. In a previous work, we have partially characterized an NTPDase-like activity expressed by rat submandibular gland microsomes, giving rise to the hypothesis that membrane NTPDases could be released into salivary ducts to regulate luminal nucleotide concentrations as was previously proposed for ovarian, prostatic, and pancreatic secretions. Present results show that rat submandibular glands incubated in vitro release membrane-associated NTPDase and ecto-5'-nucleotidase activities. Electron microscopy images show that released membranes presenting nucleotidase activity correspond to exosome-like vesicles which are also present at microsomal fraction. Both exosome release and nucleotidase activities are raised by adrenergic stimulation. Nucleotidase activities present the same kinetic characteristics than microsomal nucleotidase activity, corresponding mainly to the action of NTPDase2 and NTPDase3 isoforms as well as 5'-nucleotidase. This is consistent with Western blot analysis revealing the presence of these enzymes in the microsomal fraction.


Asunto(s)
5'-Nucleotidasa/metabolismo , Adenosina Trifosfatasas/metabolismo , Vesículas Secretoras/enzimología , Glándula Submandibular/metabolismo , Animales , Transporte Biológico , Ratas , Ratas Wistar , Glándula Submandibular/enzimología
8.
Anal Biochem ; 438(1): 82-9, 2013 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-23541519

RESUMEN

The preparation of a V(H)H (nanobody) named IH4 that recognizes human glycophorin A (GPA) is described. IH4 was isolated by screening a library prepared from the lymphocytes of a dromedary immunized by human blood transfusion. Phage display and panning against GPA as the immobilized antigen allowed isolating this V(H)H. IH4, representing 67% of the retrieved V(H)H sequences, was expressed as a soluble correctly folded protein in SHuffle Escherichia coli cells, routinely yielding approximately 100 mg/L fermentation medium. Because IH4 recognizes GPA independently of the blood group antigens, it recognizes red cells of all humans with the possible exception of those with some extremely rare genetic background. The targeted linear epitope comprises the GPA Y52PPE55 sequence. Based on surface plasmon resonance results, the dissociation constant of the IH4-GPA equilibrium is 33 nM. IH4 is a stable protein with a transition melting temperature of 75.8 °C (measured by differential scanning calorimetry). As proof of concept, we fused HIV p24 to IH4 and used the purified construct expressed in E. coli to show that IH4 was amenable to the preparation of autologous erythrocyte agglutination reagents: reconstituted blood prepared with serum from an HIV-positive patient was readily agglutinated by the addition of the bifunctional reagent.


Asunto(s)
Agregación Eritrocitaria , Glicoforinas/inmunología , Proteínas Recombinantes de Fusión/inmunología , Anticuerpos de Dominio Único/inmunología , Secuencia de Aminoácidos , Infecciones por VIH/sangre , Humanos , Oligopéptidos/química , Oligopéptidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/aislamiento & purificación , Anticuerpos de Dominio Único/metabolismo
9.
Biochim Biophys Acta Biomembr ; 1864(10): 183980, 2022 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-35654147

RESUMEN

Plasmodium falciparum, a dangerous parasitic agent causing malaria, invades human red blood cells (RBCs), causing hemolysis and microvascular obstruction. These and other pathological processes of malaria patients are due to metabolic and structural changes occurring in uninfected RBCs. In addition, infection activates the production of microparticles (MPs). ATP and byproducts are important extracellular ligands modulating purinergic signaling within the intravascular space. Here, we analyzed the contribution of uninfected RBCs and MPs to the regulation of extracellular ATP (eATP) of RBCs, which depends on the balance between ATP release by specific transporters and eATP hydrolysis by ectonucleotidases. RBCs were cultured with P. falciparum for 24-48 h prior to experiments, from which uninfected RBCs and MPs were purified. On-line luminometry was used to quantify the kinetics of ATP release. Luminometry, colorimetry and radioactive methods were used to assess the rate of eATP hydrolysis by ectonucleotidases. Rates of ATP release and eATP hydrolysis were also evaluated in MPs. Uninfected RBCs challenged by different stimuli displayed a strong and transient activation of ATP release, together with an elevated rate of eATP hydrolysis. MPs contained ATP in their lumen, which was released upon vesicle rupture, and were able to hydrolyze eATP. Results suggest that uninfected RBCs and MPs can act as important determinants of eATP regulation of RBCs during malaria. The comparison of eATP homeostasis in infected RBCs, ui-RBCs, and MPs allowed us to speculate on the impact of P. falciparum infection on intravascular purinergic signaling and the control of the vascular caliber by RBCs.


Asunto(s)
Malaria , Plasmodium falciparum , Adenosina Trifosfato/metabolismo , Eritrocitos/metabolismo , Homeostasis , Humanos , Malaria/metabolismo , Plasmodium falciparum/metabolismo
10.
Cell Mol Life Sci ; 67(19): 3371-87, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20458517

RESUMEN

Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. K (D) of CA52-DARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs' potentialities to target, to purify, and to modulate the function of cellular markers.


Asunto(s)
Quimiocinas/metabolismo , Animales , Antígenos de Grupos Sanguíneos/inmunología , Antígenos de Grupos Sanguíneos/metabolismo , Camelus/inmunología , Camelus/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Quimiocinas/inmunología , Sistema del Grupo Sanguíneo Duffy , Eritrocitos/inmunología , Eritrocitos/metabolismo , Humanos , Inmunoglobulinas/inmunología , Inmunoglobulinas/metabolismo , Interleucina-8/inmunología , Interleucina-8/metabolismo , Plasmodium vivax/inmunología , Plasmodium vivax/metabolismo , Receptores de Antígenos/inmunología , Receptores de Antígenos/metabolismo , Receptores de Superficie Celular , Receptores de Quimiocina/inmunología , Receptores de Quimiocina/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo
11.
Sci Rep ; 10(1): 9069, 2020 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-32494000

RESUMEN

During inflammatory response, blood leukocytes adhere to the endothelium. This process involves numerous adhesion molecules, including a transmembrane chemokine, CX3CL1, which behaves as a molecular cluster. How this cluster assembles and whether this association has a functional role remain unknown. The analysis of CX3CL1 clusters using native electrophoresis and single molecule fluorescence kinetics shows that CX3CL1 is a homo-oligomer of 3 to 7 monomers. Fluorescence recovery after photobleaching assays reveal that the CX3CL1-transmembrane domain peptide self-associates in both cellular and acellular lipid environments, while its random counterpart (i.e. peptide with the same residues in a different order) does not. This strongly indicates that CX3CL1 oligomerization is driven by its intrinsic properties. According to the molecular modeling, CX3CL1 does not associate in compact bundles but rather with monomers linearly assembled side by side. Finally, the CX3CL1 transmembrane peptide inhibits both the CX3CL1 oligomerization and the adhesive function, while its random counterpart does not. This demonstrates that CX3CL1 oligomerization is mandatory for its adhesive potency. Our results provide a new direction to control CX3CL1-dependent cellular adherence in key immune processes.


Asunto(s)
Adhesión Celular/fisiología , Quimiocina CX3CL1/metabolismo , Animales , Células CHO , Células COS , Línea Celular , Chlorocebus aethiops , Cricetulus , Células HEK293 , Humanos , Proteínas de la Membrana/metabolismo
12.
Sci Rep ; 8(1): 11384, 2018 07 30.
Artículo en Inglés | MEDLINE | ID: mdl-30061676

RESUMEN

We previously demonstrated that the translocase protein TSPO2 together with the voltage-dependent anion channel (VDAC) and adenine nucleotide transporter (ANT) were involved in a membrane transport complex in human red blood cells (RBCs). Because VDAC was proposed as a channel mediating ATP release in RBCs, we used TSPO ligands together with VDAC and ANT inhibitors to test this hypothesis. ATP release was activated by TSPO ligands, and blocked by inhibitors of VDAC and ANT, while it was insensitive to pannexin-1 blockers. TSPO ligand increased extracellular ATP (ATPe) concentration by 24-59% over the basal values, displaying an acute increase in [ATPe] to a maximal value, which remained constant thereafter. ATPe kinetics were compatible with VDAC mediating a fast but transient ATP efflux. ATP release was strongly inhibited by PKC and PKA inhibitors as well as by depleting intracellular cAMP or extracellular Ca2+, suggesting a mechanism involving protein kinases. TSPO ligands favoured VDAC polymerization yielding significantly higher densities of oligomeric bands than in unstimulated cells. Polymerization was partially inhibited by decreasing Ca2+ and cAMP contents. The present results show that TSPO ligands induce polymerization of VDAC, coupled to activation of ATP release by a supramolecular complex involving VDAC, TSPO2 and ANT.


Asunto(s)
Adenosina Trifosfato/metabolismo , Conexinas/metabolismo , Eritrocitos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Multimerización de Proteína , Canales Aniónicos Dependientes del Voltaje/metabolismo , Translocador 1 del Nucleótido Adenina/metabolismo , Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Cinética , Ligandos , Modelos Biológicos , Polimerizacion , Proteína Quinasa C/metabolismo , Receptores de GABA/metabolismo
13.
Brain Res ; 1147: 12-24, 2007 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-17379193

RESUMEN

The Kell blood group is constituted by two covalently linked antigens at the surface of red blood cells, Kell and Kx. Whereas Kell is a metalloprotease with demonstrated in vitro enzymatic activity, the role of Kx thereon, and/or alone, remains unknown, although its absence is linked to the McLeod syndrome, a neuroacanthocytosis. In the central nervous system, the expression of Kell and XK has been suggested, but their expression patterns remain uncharacterized, as are the post-translational pathogenic mechanisms involved in the development of the McLeod syndrome. The distributions of Kell and XK were thus studied by in situ hybridization as well as immunohistochemistry in rodent and human brain. The results reveal an independent localization of the two constituents of the Kell blood group, XK (Kx) being expressed throughout this tissue, whereas Kell expression is restricted to red blood cells in cerebral vessels. The XK protein is shown to be neuronal, located mainly in intracellular compartments, suggesting a cell specific trafficking pattern, possibly associated with specific physiological functions.


Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Antígenos de Grupos Sanguíneos/metabolismo , Encéfalo/metabolismo , Eritrocitos/metabolismo , Sistema del Grupo Sanguíneo de Kell/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Antígenos de Grupos Sanguíneos/genética , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Humanos , Inmunohistoquímica , Sistema del Grupo Sanguíneo de Kell/genética , Masculino , Metaloendopeptidasas/metabolismo , Ratones , ARN Mensajero/análisis , Ratas , Ratas Wistar , Distribución Tisular
14.
Methods Mol Biol ; 1635: 1-25, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28755361

RESUMEN

TSPO is a 18 kDa membrane protein that exists in mammalian as two isoforms 1 and 2. They are involved in different functions and are located in different membranes. TSPO1 is mainly located in outer mitochondrial membrane, whereas TSPO2 is encountered in plasma membrane of red blood cells. Determination of their structures is a milestone to understand their function. Their natural abundance is not sufficient to get large amounts usually required for structural studies. We described heterologous overexpression in both bacterial and cell-free system and purification on immobilized-metal affinity chromatography (IMAC) of both proteins. Using the same vector, TSPO1 is mostly recovered in bacterial inclusion bodies whereas TSPO2 is found in both bacterial cytosol and inclusion bodies, but in low amounts. Cell-free expression was the best system to overexpress pure TSPO2.


Asunto(s)
Escherichia coli/genética , Receptores de GABA/genética , Proteínas Recombinantes/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Sistema Libre de Células , Citosol/metabolismo , Escherichia coli/metabolismo , Humanos , Cuerpos de Inclusión/metabolismo , Ratones , Ingeniería de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de GABA/metabolismo
15.
PLoS One ; 10(2): e0116472, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25706384

RESUMEN

Duffy Antigen Receptor for Chemokines (DARC) plays multiple roles in human health as a blood group antigen, a receptor for chemokines and the only known receptor for Plasmodium vivax merozoites. It is the target of the murine anti-Fy6 monoclonal antibody 2C3 which binds to the first extracellular domain (ECD1), but exact nature of the recognized epitope was a subject of contradictory reports. Here, using a set of complex experiments which include expression of DARC with amino acid substitutions within the Fy6 epitope in E. coli and K562 cells, ELISA, surface plasmon resonance (SPR) and flow cytometry, we have resolved discrepancies between previously published reports and show that the basic epitope recognized by 2C3 antibody is 22FEDVW26, with 22F and 26W being the most important residues. In addition, we demonstrated that 30Y plays an auxiliary role in binding, particularly when the residue is sulfated. The STD-NMR studies performed using 2C3-derived Fab and synthetic peptide corroborated most of these results, and together with the molecular modelling suggested that 25V is not involved in direct interactions with the antibody, but determines folding of the epitope backbone.


Asunto(s)
Anticuerpos Monoclonales , Sistema del Grupo Sanguíneo Duffy/inmunología , Epítopos/inmunología , Receptores de Superficie Celular/inmunología , Animales , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Escherichia coli/metabolismo , Humanos , Ratones
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