RESUMEN

Src, a nonreceptor tyrosine kinase, is an important regulator of osteoclast-mediated resorption. We have investigated whether compounds that bind to the Src SH2 domain inhibit Src activity in cells and decrease osteoclast-mediated resorption. Compounds were examined for binding to the Src SH2 domain in vitro using a fluorescence polarization binding assay. Experiments were carried out with compounds demonstrating in vitro binding activity (nmol/L range) to determine if they inhibit Src SH2 binding and Src function in cells, demonstrate blockade of Src signaling, and lack cellular toxicity. Cell-based assays included: (1) a mammalian two-hybrid assay; (2) morphological reversion and growth inhibition of cSrcY527F-transformed cells; and (3) inhibition of cortactin phosphorylation in csk-/- cells. The Src SH2 binding compounds inhibit Src activity in all three of these mechanism-based assays. The compounds described were synthesized to contain nonhydrolyzable phosphotyrosine mimics that bind to bone. These compounds were further tested and found to inhibit rabbit osteoclast-mediated resorption of dentine. These results indicate that compounds that bind to the Src SH2 domain can inhibit Src activity in cells and inhibit osteoclast-mediated resorption.

Asunto(s)

Resorción Ósea/metabolismo , Difosfonatos/metabolismo , Osteoclastos/metabolismo , Dominios Homologos src/fisiología , Familia-src Quinasas/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Transformada , Dentina/metabolismo , Difosfonatos/química , Difosfonatos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Fibroblastos/citología , Humanos , Ligandos , Mamíferos , Ratones , Datos de Secuencia Molecular , Osteoclastos/citología , Osteoporosis/metabolismo , Conejos , Ensayo de Unión Radioligante , Ratas , Tritio , Técnicas del Sistema de Dos Híbridos , Familia-src Quinasas/antagonistas & inhibidores

RESUMEN

Targeted disruption of the pp60(src) (Src) gene has implicated this tyrosine kinase in osteoclast-mediated bone resorption and as a therapeutic target for the treatment of osteoporosis and other bone-related diseases. Herein we describe the discovery of a nonpeptide inhibitor (AP22408) of Src that demonstrates in vivo antiresorptive activity. Based on a cocrystal structure of the noncatalytic Src homology 2 (SH2) domain of Src complexed with citrate [in the phosphotyrosine (pTyr) binding pocket], we designed 3',4'-diphosphonophenylalanine (Dpp) as a pTyr mimic. In addition to its design to bind Src SH2, the Dpp moiety exhibits bone-targeting properties that confer osteoclast selectivity, hence minimizing possible undesired effects on other cells that have Src-dependent activities. The chemical structure AP22408 also illustrates a bicyclic template to replace the post-pTyr sequence of cognate Src SH2 phosphopeptides such as Ac-pTyr-Glu-Glu-Ile (1). An x-ray structure of AP22408 complexed with Lck (S164C) SH2 confirmed molecular interactions of both the Dpp and bicyclic template of AP22408 as predicted from molecular modeling. Relative to the cognate phosphopeptide, AP22408 exhibits significantly increased Src SH2 binding affinity (IC(50) = 0.30 microM for AP22408 and 5.5 microM for 1). Furthermore, AP22408 inhibits rabbit osteoclast-mediated resorption of dentine in a cellular assay, exhibits bone-targeting properties based on a hydroxyapatite adsorption assay, and demonstrates in vivo antiresorptive activity in a parathyroid hormone-induced rat model.

Asunto(s)

Resorción Ósea/tratamiento farmacológico , Difosfonatos/farmacología , Diseño de Fármacos , Imitación Molecular , Osteoclastos/efectos de los fármacos , Dominios Homologos src/efectos de los fármacos , Adsorción , Sustitución de Aminoácidos/genética , Animales , Sitios de Unión , Huesos/efectos de los fármacos , Huesos/patología , Ácido Cítrico/química , Ácido Cítrico/metabolismo , Cristalografía por Rayos X , Dentina/efectos de los fármacos , Dentina/metabolismo , Difosfonatos/química , Difosfonatos/metabolismo , Difosfonatos/uso terapéutico , Femenino , Hidroxiapatitas , Concentración 50 Inhibidora , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Modelos Moleculares , Osteoclastos/patología , Hormona Paratiroidea/farmacología , Paratiroidectomía , Fosfotirosina/química , Fosfotirosina/metabolismo , Conformación Proteica , Proteínas Proto-Oncogénicas pp60(c-src)/química , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Conejos , Ratas , Ratas Wistar , Relación Estructura-Actividad , Especificidad por Sustrato

RESUMEN

Chemically induced dimerization provides a general way to gain control over intracellular processes. Typically, FK506-binding protein (FKBP) domains are fused to a signaling domain of interest, allowing crosslinking to be initiated by addition of a bivalent FKBP ligand. In the course of protein engineering studies on human FKBP, we discovered that a single point mutation in the ligand-binding site (Phe-36 --> Met) converts the normally monomeric protein into a ligand-reversible dimer. Two-hybrid, gel filtration, analytical ultracentrifugation, and x-ray crystallographic studies show that the mutant (F(M)) forms discrete homodimers with micromolar affinity that can be completely dissociated within minutes by addition of monomeric synthetic ligands. These unexpected properties form the basis for a "reverse dimerization" regulatory system involving F(M) fusion proteins, in which association is the ground state and addition of ligand abolishes interactions. We have used this strategy to rapidly and reversibly aggregate fusion proteins in different cellular compartments, and to provide an off switch for transcription. Reiterated F(M) domains should be generally useful as conditional aggregation domains (CADs) to control intracellular events where rapid, reversible dissolution of interactions is required. Our results also suggest that dimerization is a latent property of the FKBP fold: the crystal structure reveals a remarkably complementary interaction between the monomer binding sites, with only subtle changes in side-chain disposition accounting for the dramatic change in quaternary structure.

Asunto(s)

Inmunofilinas/química , Ligandos , Dimerización , Humanos , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/química , Tacrolimus/química , Proteínas de Unión a Tacrolimus

RESUMEN

Using structure-based design and protein mutagenesis we have remodeled the FKBP12 ligand binding site to include a sizable, hydrophobic specificity pocket. This mutant (F36V-FKBP) is capable of binding, with low or subnanomolar affinities, novel synthetic ligands possessing designed substituents that sterically prevent binding to the wild-type protein. Using binding and structural analysis of bumped compounds, we show here that the pocket is highly promiscuous-capable of binding a range of hydrophobic alkyl and aryl moieties with comparable affinity. Ligand affinity therefore appears largely insensitive to the degree of occupancy or quality of packing of the pocket. NMR spectroscopic analysis indicates that similar ligands can adopt radically different binding modes, thus complicating the interpretation of structure-activity relationships.

Asunto(s)

Acetamidas/síntesis química , Acetamidas/metabolismo , Derivados del Benceno/síntesis química , Derivados del Benceno/metabolismo , Inmunofilinas/metabolismo , Acetamidas/química , Derivados del Benceno/química , Inmunofilinas/genética , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutación , Ingeniería de Proteínas , Relación Estructura-Actividad , Proteínas de Unión a Tacrolimus

RESUMEN

The A(280)/A(260) ratio of a purified protein is frequently used as an indication of the purity of the preparation with respect to nucleic acids. We show here that for low-molecular-weight recombinant proteins purified from Escherichia coli, a low A(280)/A(260) ratio can also result from contamination with UDP-linked murein precursors derived from bacterial cell wall metabolism. Although these precursors are small molecules of molecular weight 1000-1200, they comigrate in gel filtration with recombinant human FKBP (MW 11,820). This gel filtration behavior, which is distinct from that of unmodified mononucleotides, does not reflect binding interactions with FKBP, but is an intrinsic property of these precursors. Therefore, these molecules would be expected to copurify with other low-molecular-weight proteins, especially in the abbreviated purification protocols made possible by freeze-thaw release of recombinant proteins from E. coli (Johnson, B. H., and Hecht, M. H. (1994) BioTechnology 12, 1357-1360). Several alternative strategies are discussed for integrating these findings into the design of improved purification procedures for low-molecular-weight recombinant proteins.

Asunto(s)

Peptidoglicano/metabolismo , Proteínas Recombinantes/química , Uridina Difosfato/metabolismo , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Escherichia coli/química , Escherichia coli/metabolismo , Humanos , Peso Molecular , Peptidoglicano/química , Resinas de Plantas , Espectrofotometría Ultravioleta , Uridina Difosfato/química

RESUMEN

pp60(c-src) is a prototypical nonreceptor tyrosine kinase and may play a role in diseases as diverse as cancer and osteoporosis. In Src, the SH3 domain (Src homology 3) binds proteins at specific, proline-rich sequences, while the SH2 domain (Src homology 2) binds phosphotyrosine-containing sequences. Inhibition of Src SH3 and SH2 domain function is of potential therapeutic value because of their importance in signaling pathways involved in disease states. We have developed dual-wavelength fluorescent peptide probes for both the Src SH3 and the Src SH2 domains, which allow the simultaneous measurement of compounds binding to each domain in assays based on the technique of fluorescence polarization. We demonstrate the utility of these probes in a dual-binding assay (suitable for high-throughput screening) to study the interactions of various peptides with these domains, including a sequence from the rat protein p130(CAS) which has been reported to bind simultaneously to both Src SH3 and SH2 domains. Utilizing this dual-binding assay, we confirm that sequences from p130(CAS) can simultaneously bind Src via both its SH3 and its SH2 domains. We also use the dual-binding assay as an internal control to identify substances which inhibit SH3 and SH2 binding via nonspecific mechanisms.

Asunto(s)

Polarización de Fluorescencia/métodos , Colorantes Fluorescentes , Proteínas , Dominios Homologos src , Animales , Proteína Sustrato Asociada a CrK , Escherichia coli , Colorantes Fluorescentes/síntesis química , Humanos , Proteína Oncogénica pp60(v-src)/metabolismo , Fosfoproteínas/metabolismo , Unión Proteica , Conformación Proteica , Ratas , Proteínas Recombinantes/metabolismo , Proteína p130 Similar a la del Retinoblastoma

RESUMEN

A series of 1,2,4-oxadiazole analogues has been shown to be potent and selective SH2 inhibitors of the tyrosine kinase ZAP-70, a potential therapeutic target for immune suppression. These compounds typically are 200-400-fold more potent than the native, monophosphorylated tetrapeptide sequences. When compared with the high-affinity zeta-1-ITAM peptide (Ac-NQL-pYNELNLGRREE-pYDVLD-NH(2), wherein pY refers to phosphotyrosine) some of the best 1,2, 4-oxadiazole analogues are approximately 1 order of magnitude less active. This series of compounds displays an unprecedented level of selectivity over the closely related tyrosine kinase Syk, as well as other SH2-containing proteins such as Src and Grb2. Gel shift studies using a protein construct consisting only of C-terminal ZAP-70 SH2 demonstrate that these compounds can effectively engage this particular SH2 domain.

Asunto(s)

Inhibidores Enzimáticos/síntesis química , Oxadiazoles/síntesis química , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Dominios Homologos src , Inhibidores Enzimáticos/química , Precursores Enzimáticos/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular , Modelos Moleculares , Oxadiazoles/química , Relación Estructura-Actividad , Quinasa Syk , Proteína Tirosina Quinasa ZAP-70

RESUMEN

Through an integrated study of the reactivity of a monoclonal antibody, 803-15.6, with synthetic peptides and native recombinant HIV-1 envelope glycoprotein gp120, we have obtained structure-functional information on a region of rgp120 not yet elucidated by X-ray crystallography. mAb 803-15.6 binds with high affinity and broad cross-clade specificity to the conserved C-terminal region (amino acids 502-516) of HIV-1 rgp120. Phage display selection from a random peptide library identified the core binding motif as AXXKXRH, homologous to residues 502-508. Using quantitative binding analyses, the affinity of mAb 803-15.6 for native, monomeric recombinant gp120HXB2 (rgp120) was found to be similar to that for the synthetic gp120 peptide (502-516). Circular dichroism studies indicate that the synthetic peptide largely has a random coil conformation in solution. The results therefore suggest that the 803-15.6 epitope is fully accessible on rgp120 and that this region of rgp120 is as flexible as the synthetic peptide. Residues 502-504 are on the edge of a putative gp41 binding site that has been postulated to change conformation on CD4 binding. However, the affinity of mAb 803-15.6 for rgp120 is not affected by binding of CD4 and vice-versa. These results suggest either that the 502-504 region does not change conformation upon CD4 binding, or that recombinant gp120 does not undergo the same changes as occur in the native viral gp120-gp41 oligomer. The detailed characterization of the 803-15.6 epitope may be useful for further study of the role of the C5 region of gp120 in the viral attachment and fusion process.

Asunto(s)

Epítopos/química , Proteína gp120 de Envoltorio del VIH/química , VIH-1/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Antígenos CD4/metabolismo , Dicroismo Circular , Secuencia Conservada , Mapeo Epitopo , Epítopos/metabolismo , Femenino , Proteína gp120 de Envoltorio del VIH/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie

RESUMEN

The two-domain form of recombinant soluble human CD4 (rsCD4(183)) has been used for structural studies and to probe the interaction of CD4 with its ligands. rsCD4(183) has generally been produced in Escherichia coli in the form of inclusion bodies. The generation of conformationally native protein from these inclusion bodies is a time-consuming and inefficient process, requiring a refolding step. Here, we describe a procedure for producing 2-4 mg of secreted, conformationally native rsCD4(183) per liter of E. coli, completely bypassing the requirement for protein refolding in vitro. Furthermore, the yield of active protein is comparable to that reported for expression systems that generate inclusion bodies.

Asunto(s)

Antígenos CD4/biosíntesis , Escherichia coli , Proteínas Recombinantes de Fusión/biosíntesis , Antígenos CD4/genética , Antígenos CD4/inmunología , Antígenos CD4/aislamiento & purificación , Extractos Celulares , Humanos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Solubilidad

RESUMEN

We compared insect cell production levels of secreted HIV-1 gp120 glycoprotein encoded by five different baculovirus expression constructs. Combinations consisting of one of two baculovirus promoters (very late or hybrid late/very late) and one of three different signal sequences [human tissue plasminogen activator (tpa), human placental alkaline phosphatase (pap), or baculovirus envelope glycoprotein (gp67)] were constructed. Production of secreted gp120 from these constructs was analyzed in two enzyme-linked immunosorbent assay formats, one detecting the total amount of secreted gp120 protein and the other measuring the level of "active" gp120 (as defined by the ability to bind to CD4). We found that for all of the constructs, approximately 50 to 90% of the secreted gp120 protein was active. Furthermore, our results indicated that expression from either promoter yielded comparable production of secreted protein, despite the fact that transcription from the hybrid promoter begins at an earlier time. By contrast, the signal sequence had a much greater effect on the levels of secreted gp120: the tpa leader yielded the highest level of secreted protein, followed by the gp67 and pap sequences. This result suggests that transcription is not a limiting factor in the production of secreted gp120, but rather that downstream processing of the protein is more critical. Furthermore, these results confirm the notion that the "optimal" signal sequence is protein dependent and that an insect-derived signal sequence is not optimal in all cases.

Asunto(s)

Proteína gp120 de Envoltorio del VIH/biosíntesis , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/genética , Fosfatasa Alcalina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Femenino , Expresión Génica , Genes Virales , Vectores Genéticos , Humanos , Nucleopoliedrovirus/genética , Placenta/enzimología , Plásmidos/genética , Embarazo , Regiones Promotoras Genéticas , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Spodoptera , Activador de Tejido Plasminógeno/genética , Proteínas Virales de Fusión/genética

RESUMEN

Recombinant soluble human CD4 (rsCD4) has been used in iodinated form to study the interaction of CD4 with its ligands. However, the utility of [125I]-rsCD4 is limited because rsCD4 is inefficiently iodinated and the iodinated protein is poorly active. The iodination properties of rsCD4 most likely reflect the poor accessibility of the tyrosine residues, apparent from the available X-ray structures. We have generated an iodinatable mutant of rsCD4 by substituting Tyr for Phe(179) in the flexible, solvent-exposed C-terminal region of rsCD4(183), a truncated form of CD4 that consists of the first 183 residues of CD4 and includes the binding sites for HIV-1 gp120 and MHC class II molecules. When F179Y rsCD4(183) is iodinated under trace-labeling conditions, the efficiency of 125I incorporation and the percentage of iodinated molecules that are active are much enhanced compared with WT rsCD4. Moreover, trace-labeled [125I]-F179Y rsCD4(183) has the same affinity for HIV-1 rgp120 as unlabeled WT rsCD4. The improved activity of trace-labeled [125I]-F179Y rsCD4(183) appears to be due to effective competition by Y179 for reactive iodine species that, in WT rsCD4, react with traces of denatured protein and/or with residues critical for activity or conformational integrity. The incorporation of accessible tyrosine residues may improve the iodinatibility of a protein both by introducing a readily iodinatable residue and by protecting sensitive proteins from adverse reactions.

Asunto(s)

Antígenos CD4/metabolismo , Radioisótopos de Yodo , Marcaje Isotópico , Animales , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Ratones , Mutación , Proteínas Recombinantes/metabolismo

RESUMEN

Erythrocyte membrane-associated antigens of Plasmodium falciparum have been of long-standing interest as potential adherence receptors and vaccine candidates. We recently identified in trophozoite-stage infected erythrocytes a novel high molecular weight erythrocyte membrane-associated protein of P. falciparum, PfEMP3, defined by Western blotting with the rat monoclonal antibody 12C11. Genomic clone lambda 12.1.3 and cDNA clone p12.2 contain nucleic acid sequences encoding PfEMP3. Analysis of Malayan Camp strain parasites by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 5% gels revealed that PfEMP3, defined by Western blot, has the same relative molecular weight (M(r)) as the surface-exposed protein PfEMP1 defined by cell surface iodination. We show here that PfEMP3 is distinct from PfEMP1 by three criteria. First, 125I-labeled PfEMP1 was resolved from PfEMP3 by extended migration on 4% gels. Second, in two strains of P. falciparum in which 125I-PfEMP1 has a different M(r), PfEMP3 had the same M(r). Third, immunization studies were performed with fusion proteins derived from clones lambda 12.1.3 and p12.2. Although one rabbit, Rb 05.75, immunized with the PfEMP3-derived fusion protein beta gal12.1.3, produced a serum that strongly immunoprecipitated PfEMP1 as well as PfEMP3, most sera immunoprecipitated only PfEMP3. Furthermore, immunoprecipitation of PfEMP3 by Rb 05.75 serum was blocked by the glutathione S-transferase 12.1.3 fusion protein, whereas immunoprecipitation of PfEMP1 was unaffected. Therefore, we conclude that PfEMP1 and PfEMP3 are antigenically distinct.

Asunto(s)

Proteínas de la Membrana/análisis , Plasmodium falciparum/química , Proteínas Protozoarias/análisis , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Western Blotting , Adhesión Celular , Ensayo de Inmunoadsorción Enzimática , Membrana Eritrocítica/metabolismo , Humanos , Sueros Inmunes/inmunología , Inmunohistoquímica , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Ratones , Datos de Secuencia Molecular , Plasmodium falciparum/inmunología , Pruebas de Precipitina , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Conejos

RESUMEN

The rat monoclonal antibody, mAb 12C11, reacts with numerous proteins from mature asexual stages of Plasmodium falciparum. The largest is 315 kDa and is designated PfEMP3. A lambda gt11 expression library, generated from genomic DNA of Malayan Camp strain parasites, was screened with mAb 12C11. One positive clone, lambda 12.1.3, contained a 1.4-kb fragment in frame with the beta-galactosidase gene of lambda gt11. The deduced 455-amino acid sequence is a novel, highly charged sequence encoding two 15-amino acid repeats at the N-terminus followed by 27 repeats of 13 amino acids. The last 59 C-terminal residues are non-repetitive. Two in-frame stop codons at the 3' end of the DNA suggests that this DNA fragment encodes the C-terminus of the protein. Southern blotting with the cloned fragment identified two copies of this fragment per haploid genome in knob-positive, parasitized erythrocytes (K+PE). Both DNA fragments are absent from K - PE. Northern blotting of trophozoite-stage PE total RNA revealed mRNAs of 10, 4.4 and 2 kb in K+PE, but no hybridization with K - PE. Immune sera were elicited against the lambda 12.1.3 beta-galactosidase fusion protein and peptides generated from the predicted lambda 12.1.3 amino acid sequence. These sera and mAb 12C11 reacted specifically with PfEMP3 in Western blots of mature K+PE but not with K - PE. Rat and mouse sera against the recombinant protein produced an immunofluorescence pattern in fixed mature K+PE almost identical to the pattern produced by a monoclonal antibody against the knob-associated protein, Histidine Rich Protein 1. The same antibodies were immunofluorescence negative with fixed K - PE. Mouse antibodies against the recombinant protein reacted on immunoelectron microscopy with the erythrocyte membrane of K+PE, labeling knobs as well as the membrane between knobs. In contrast, a mAb against Histidine Rich Protein 1 reacted only under the electron dense material of knobs. We conclude that the lambda 12.1.3 clone encodes the C-terminal portion of the 315 kD PfEMP3 antigen and that PfEMP3 may be involved in knob formation or other perturbations of the erythrocyte membrane.

Asunto(s)

Genes Protozoarios , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Clonación Molecular , ADN Protozoario/genética , Membrana Eritrocítica/parasitología , Humanos , Malaria Falciparum/parasitología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología

RESUMEN

Stable transformants of Chinese hamster ovary (CHO) cell lines expressing high levels of human CD36 or intercellular adhesion molecule-1 (ICAM-1) have been produced as target cells for cytoadherence of Plasmodium falciparum-infected erythrocytes. An improved adherence microassay has been designed using small sample volumes and allowing convenient and reliable measurements on a large number of samples. The assay can be used both with purified proteins spotted on plastic and with the stably transformed CHO cell lines. The same assay plate can be evaluated either microscopically or by scintillation counting after use of 3H-hypoxanthine-labeled parasites. Using the microassay, functional expression of the transfected receptor molecules on CHO-CD36 and CHO-ICAM was confirmed using parasites with different cytoadherence phenotypes and cytoadherence inhibition experiments with a panel of anti-CD36 antibodies. The use of isolates from The Gambia confirmed the applicability of these assays for laboratory studies of these isolates.

Asunto(s)

Antígenos CD/inmunología , Moléculas de Adhesión Celular/inmunología , Eritrocitos/parasitología , Plasmodium falciparum/citología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD/biosíntesis , Antígenos CD/química , Western Blotting , Antígenos CD36 , Células CHO , Adhesión Celular , Moléculas de Adhesión Celular/biosíntesis , Separación Celular , Cricetinae , Criopreservación , Ensayo de Inmunoadsorción Enzimática , Eritrocitos/citología , Citometría de Flujo , Humanos , Sueros Inmunes/inmunología , Molécula 1 de Adhesión Intercelular , Melanoma , Datos de Secuencia Molecular , Plasmodium falciparum/inmunología , Pruebas de Precipitina , Transfección , Células Tumorales Cultivadas

RESUMEN

Plasmodium falciparum-infected erythrocytes (parasitized red blood cells [PRBCs]) can adhere to uninfected erythrocytes (RBCs) to form rosettes, and adhere to the endothelial cell (EC) surface antigen CD36. These adherence phenomena have previously been considered quite different. We show that anti-CD36 monoclonal antibodies (MoAbs) reverse rosetting of PRBCs from both a culture-adapted line (Malayan Camp [MC] strain) and a natural isolate, GAM425. Three MoAbs that block adherence of PRBCs to ECs or C32 melanoma cells also reversed rosetting by greater than 50% at levels of less than 1 microgram/mL (OKM5, OKM8, and 8A6). Two other MoAbs that react with purified CD36 (1D3 and 1B1), but do not react with the surface of C32 cells, failed to reverse rosetting. When rosettes were disrupted and the RBCs and PRBCs were pretreated separately with antibodies before mixing to allow rosette reformation, only pretreatment of RBCs had an effect. MoAb 8A6 pretreatment of RBCs blocked rosette reformation, while MoAb 1B1 pretreatment did not. Rosetting was also reversed by purified human platelet CD36. In conjunction with evidence that CD36 is expressed on normal human erythrocytes (van Schravendijk et al, Blood 80:2105, 1992), we conclude that this CD36 is able to act as a host receptor for rosetting in the MC strain and some natural isolates of P falciparum.

Asunto(s)

Antígenos CD/inmunología , Eritrocitos/inmunología , Eritrocitos/parasitología , Plasmodium falciparum/inmunología , Receptores de Superficie Celular/inmunología , Formación de Roseta , Animales , Anticuerpos Monoclonales/inmunología , Plaquetas/inmunología , Antígenos CD36 , Epítopos/inmunología , Humanos

RESUMEN

We have recently shown that rosetting of Plasmodium falciparum (MC R+ line)-infected erythrocytes (parasitized red blood cells [PRBCs]) with uninfected erythrocytes (RBCs) is blocked by coating of the RBCs with anti-CD36 monoclonal antibodies (MoAbs; Handunnetti et al, Blood 80:2097, 1992). Adult RBCs have previously been considered negative for CD36. However, using fluorescence-activated cell sorter analysis with the anti-CD36 MoAbs 8A6, OKM5, and OKM8, which reverse rosetting, we consistently detect CD36 on the majority of normal adult RBCs. Absorption of the MoAb solutions with CD36-transfected Chinese hamster ovary (CHO-CD36) cells removed the reactivity against both CHO-CD36 cells and RBCs, whereas absorption with CHO cells had no effect. By comparison with staining for glycophorin A, LFA-3, and CR1, the level of expression of CD36 appeared to be low. Nevertheless, normal RBCs were capable of adhering to plastic coated with anti-CD36 MoAbs. RBCs from one African malaria patient were identified as deficient in CD36 and these RBCs did not rosette with the patient's own P falciparum PRBCs, even though these PRBCs were capable of rosetting with RBCs from a normal donor in a CD36-dependent manner. Therefore, the level of expression of CD36 on normal RBCs is sufficient to be important in cell adherence, and may have a biologic role in normal individuals as well as in the pathology of P falciparum malaria.

Asunto(s)

Antígenos CD/análisis , Plaquetas/inmunología , Eritrocitos/inmunología , Monocitos/inmunología , Animales , Anticuerpos Monoclonales , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos CD36 , Células CHO , Adhesión Celular , Cricetinae , Endotelio/inmunología , Eritrocitos/parasitología , Eritrocitos/fisiología , Citometría de Flujo , Humanos , Técnicas de Inmunoadsorción , Malaria Falciparum/sangre , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Plasmodium falciparum/inmunología , Formación de Roseta , Transfección

RESUMEN

We prepared rat monoclonal antibodies (mAb) specific for very large Plasmodium falciparum proteins to assist in their characterization. Hybridomas prepared from rats immunized with parasitized erythrocyte (PE) proteins of greater than 200 kDa exhibited two patterns of Western blot reactivity with PE SDS extracts: one represented by clone 41E11 (IgM, kappa), the other by clone 12C11 (IgM, lambda). MAb 41E11 reacted by Western blotting with at least 15 antigens, most of which comigrated with antigens identified by the 33G2 human IgM mAb. The stage specificity of mAb 41E11 reactivity and indirect immunofluorescence (IFA) pattern closely resemble those previously described for antigens that share the EEXXEE sequence motif. Unlike mAb 33G2, MAb 41E11 immunoprecipitated a biosynthetically radiolabeled protein of 320 kDa. MAb 41E11 did not immunoprecipitate any cell surface 125I proteins. MAb 12C11 reacted on Western blotting with a different group of malarial antigens of approximately 44, 95, 117, 145, and 310 kDa, as well as with some low-molecular-weight, uninfected erythrocyte antigens. MAb 12C11 did not immunoprecipitate any cell surface 125I or biosynthetically labeled proteins. The 310-kDa antigen recognized by mAb 12C11 (denoted Ag 12A) does not correspond to PfEMP2 or the 320-kDa antigen recognized by mAbs 33G2 or 41E11. With trophozoites and more mature stages, fixed IFA reactivity of mAb 12C11 was at the parasite and in antigen aggregates in the host cell cytoplasm that extended to the PE plasma membrane. Indirect results suggest that Ag 12A does not correspond to cell surface-exposed PfEMP1 and is most likely a hitherto unidentified malarial protein.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Plasmodium falciparum/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Antiprotozoarios/biosíntesis , Western Blotting , Electroforesis en Gel de Poliacrilamida , Eritrocitos/inmunología , Eritrocitos/parasitología , Técnica del Anticuerpo Fluorescente , Humanos , Peso Molecular , Pruebas de Precipitina , Ratas , Ratas Endogámicas Lew

RESUMEN

We have developed methods for in vitro selection of Plasmodium falciparum parasites that bear knob protrusions (K+) and are either of the rosette-positive (K+R+) or rosette-negative (K+R-) phenotypes. Cryopreserved parasites from spleen-intact Aotus monkeys that were K+, C32 cell adherence-positive (C+), CD36 adherence-positive, and R- with Aotus erythrocytes were adapted to continuous growth in human erythrocytes, and selected initially for adherence to C32 melanoma cells. In the absence of independent selection for rosettes, K+R-C+ parasites were produced that adhered to both C32 cells and CD36. Without selection for the C+ phenotype, K+R-C- parasites eventually predominated in such cultures. The R+ parasites were selected using differences in sedimentation behavior of rosette-infected cells versus non-rosette-infected cells. Methods were devised for selection of the R+ or R- phenotypes and for the purification of R+ or R- infected cells of high parasitemia that were suitable for molecular studies. With the repeated selection for K+R+ parasites, we were able to maintain the K+R+ phenotype for several months in vitro. These methods will allow systematic study of the molecular basis of the K+R+ and K+R- phenotypes.

Asunto(s)

Eritrocitos/parasitología , Plasmodium falciparum/aislamiento & purificación , Formación de Roseta , Animales , Aotus trivirgatus , Adhesión Celular , Células Cultivadas , Centrifugación por Gradiente de Densidad , Humanos , Microscopía Electrónica , Fenotipo , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/ultraestructura

RESUMEN

The malaria-induced surface antigens on Plasmodium falciparum-infected erythrocytes from West African patients were characterized by agglutination of infected cells by human sera, surface immunofluorescence of live infected cells, inhibition of cytoadherence to C32 melanoma cells by human sera, immunoelectron microscopy (immunoEM), and immunoprecipitation. In a nonimmune individual, serum antibody reactivity to surface antigens of infected cells was acquired during convalescence, as tested by all five methods, and was generally parasite isolate-specific. By contrast, adult hyperimmune West African sera reacted with many isolates, including isolates from geographically distinct regions. A quantitative correlation was established between agglutination and surface immunofluorescence assay titers, and between surface immunofluorescence assay and immunoEM reactivity, suggesting that a single antigen or a set of coexpressed antigens is being detected. Surface iodination of infected cells identified trypsin-sensitive high M, antigens in the sodium dodecyl sulfate extract. All sera tested that agglutinated infected cells also immunoprecipitated these antigens. The same surface antigens were immunoprecipitated by the homologous convalescent serum as by adult sera. By immunoEM these antigens were localized exclusively at the knob-like protrusions of infected cells, where they may participate in adherence to vascular endothelium.

Asunto(s)

Antígenos de Protozoos/análisis , Eritrocitos/inmunología , Malaria/inmunología , Plasmodium falciparum/inmunología , África Occidental/epidemiología , Aglutinación , Animales , Antígenos de Superficie/análisis , Adhesión Celular , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Eritrocitos/química , Eritrocitos/ultraestructura , Técnica del Anticuerpo Fluorescente , Gambia/epidemiología , Humanos , Malaria/sangre , Malaria/epidemiología , Malaria/patología , Microscopía Inmunoelectrónica , Pruebas de Precipitina

RESUMEN

To understand the molecular mechanisms that lead to sequestration of red blood cells infected with mature stages of Plasmodium falciparum and to examine the relevance of earlier studies on adherence properties of laboratory-derived P falciparum parasites to the natural parasite population, we analyzed Gambian and Tanzanian isolates for in vitro cytoadherence and antibody-mediated microagglutination. Eighteen cryopreserved isolates of ring-stage parasites were cultured for 20 to 30 hours in vitro, in the patients original erythrocytes, to the trophozoite and schizont stage. All parasites were positive in the microagglutination assay with at least one of four African hyperimmune sera. In a rosetting assay, only 2 of the 18 isolates were strongly positive (35% and 41% of parasitized erythrocytes with more than two uninfected cells bound). Thirteen isolates showed either intermediate (5% to 18%) or low (less than 5%) rosetting while three isolates did not form rosettes. Infected cell-binding of the different isolates to immobilized CD36 or thrombospondin, or C32 melanoma cells correlated with the percentage of mature parasites in the blood samples (r = .932 for CD36, r = .946 for thrombospondin, and r = .881 for C32 melanoma cells). There was a high correlation between binding to CD36 and thrombospondin (r = .982). The extent of infected cell rosetting with uninfected cells in these blood samples was not correlated with these other receptor properties. We also observed coexpression of rosetting and cytoadherence receptors on the same parasitized erythrocytes.

Asunto(s)

Aglutinación/fisiología , Agregación Eritrocitaria/fisiología , Eritrocitos/fisiología , Malaria/sangre , Plasmodium falciparum/aislamiento & purificación , Formación de Roseta , Animales , Bioensayo , Adhesión Celular/fisiología , Eritrocitos/microbiología , Gambia , Humanos , Melanoma/patología , Melanoma/fisiopatología , Fenotipo , Tanzanía