RESUMEN
OBJECTIVES: Tacrolimus (FK506) is an immunosuppressive drug with great clinical promise. There is a controversy regarding the role of tacrolimus metabolites in immunosuppression and toxicity, and immunoassays and immunophilin binding assays have not been adequately tested for metabolite cross-reactivity. Methods are limited to HPLC and HPLC-MS for quantifying the parent drug. Mixed lymphocyte culture assay (MLC) is the preferred functional bioassay for the measurement of parent drug and active metabolites but it is not practical for routine laboratory use. Due to differences in assay methods and reagent specificity, the concentration of tacrolimus in a given specimen may vary among different assay kit manufacturers. The objective of this study was to evaluate the degree of cross-reactivity or interference of the three first-generation tacrolimus metabolites [13-O-demethyl (M-I), 31-O-demethyl (M-II) and 15-O-demethyl (M-III)] among two different tacrolimus immunoassays (Immunoassay: PRO-Trac II FK506, Abbott IMx tacrolimus-II); and the radioreceptor assays (RRA) using minor immunophilins (14, 37, and 52 kDa immunophilins) and tacrolimus binding protein (FKBP12). METHODS: First-generation tacrolimus metabolites (M-I, M-II, and M-III) spiked in drug-free whole blood were assayed with RRA using three minor immunophilins (14, 37, and 52 kDa) and two commercial immunoassay procedures (Incstar PRO-Trac II tacrolimus, Abbott IMx tacrolimus II). The results were compared to previously published FKBP-12 RRA data and their immunosuppressive potency. RESULTS AND CONCLUSION: The first generation tacrolimus metabolites (M-I, M-II, and M-III) were tested using concentrations of 10 and 20 ng/mL. The significance of the metabolite interference (% of the total interference) was calculated based on the relative concentration of each metabolite present at steady-state trough concentrations in renal transplant recipients (22). Metabolite I, which has no functional immunosuppressive activity showed minimal interference compared to M-II and M-III in all assays except the 14 kDa RRA. The Incstar PRO-Trac II tacrolimus assay showed the least M-I interference. Metabolite-II, which has a pharmacologic potency similar to the parent drug, showed a significant interference in the immunoassays and significant interference in radioreceptor assays. Metabolite III, which is pharmacologically inactive, produces 3-10% interference in the different assays if its presence in the blood is 6% of the parent drug. The total interference from these three metabolites was greater in the immunoassays than in the receptor assays. Receptor assays for tacrolimus provide results closer to the target value than do immunoassays.
Asunto(s)
Inmunosupresores/metabolismo , Tacrolimus/metabolismo , Reacciones Cruzadas , Inmunoensayo , Inmunofilinas/aislamiento & purificación , Inmunofilinas/metabolismo , Trasplante de Riñón , Ensayo de Unión Radioligante , Tacrolimus/inmunologíaRESUMEN
OBJECTIVES: We have previously identified a minor immunophilin of 52 kDa molecular weight capable of binding tacrolimus and sirolimus. Because immunophilins are capable of binding both parent drug and metabolites and HPLC assays are typically used to assess parent drug in clinical situations, we used this immunophilin in a radioreceptor assay (RRA) to determine if any metabolites not included in the HPLC measurement would bind to the immunophilin and be associated with thrombocytopenia in patients receiving sirolimus. DESIGN AND METHODS: We tested 51 steady-state trough whole blood samples from non-thrombocytopenic patients and 51 steady-state trough samples from thrombocytopenic patients and compared them to HPLC measurements of parent drug in the same samples. We also tested whole blood samples spiked with authentic sirolimus metabolites using RRA to ascertain the effect these metabolites have on the technique. RESULTS: We found minimal cross-reactivity in this assay for sirolimus metabolites (binding ranged from <10% to 26%), and good correlation of the radioreceptor assay with HPLC (linear regression slope 0.92, y-intercept 0.79). There was no statistically significant difference between the RRA and HPLC results in two patient groups-thrombocytopenic and non-thrombocytopenic-using the paired t-test (p<0.005) and Bland-Altman analysis. CONCLUSIONS: These findings indicate that although the RRA could be substituted for HPLC in therapeutic drug monitoring, the 52 kDa immunophilin does not offer an advantage in terms of detecting metabolites associated with thrombocytopenia. However, the RRA offers the advantages of shorter turnaround time, smaller sample volume and potential for automation.
Asunto(s)
Cromatografía Líquida de Alta Presión/normas , Inmunosupresores/sangre , Ensayo de Unión Radioligante/normas , Sirolimus/sangre , Animales , Unión Competitiva , Bovinos , Reacciones Cruzadas , Ciclosporina/uso terapéutico , Inmunofilinas/aislamiento & purificación , Inmunofilinas/metabolismo , Trasplante de Riñón , Prueba de Cultivo Mixto de Linfocitos , Prednisona/uso terapéutico , Unión Proteica , Sirolimus/análogos & derivados , Trombocitopenia/sangre , Trombocitopenia/terapiaRESUMEN
OBJECTIVES: We have previously identified three minor immunophilins of molecular weights 37 kDa, 14 kDa, and 5-8 kDa capable of binding tacrolimus and sirolimus. DESIGN AND METHODS: When tested against pure preparations of five sirolimus metabolites, the 14 kDa protein had almost no cross-reactivity, the 37 kDa protein cross-reacted from a high of 23.2% to <10% and the 5-8 kDa protein cross-reacted from <10% to 46.4%. When the 5-8 kDa immunophilin was tested in whole blood samples to assess interference in clinical samples, the demethylated sirolimus metabolites showed about 25% less cross-reactivity while the hydroxylated metabolites reacted about the same. RESULTS: Since MLC data on sirolimus metabolites to date indicates that their pharmacologic potencies are < or =10% of the parent, the 14 kDa immunophilin appears to be the best candidate for a sirolimus radioreceptor assay. The 5-8 kDa immunophilin is newly identified and its cross-reactivity with tacrolimus metabolites had not been assessed. Binding of the 5-8 kDa immunophilin to pure preparations of three tacrolimus metabolites showed virtually no binding of the protein to 13-O-demethyl and 31-O-demethyl tacrolimus and binding to 15-O-demethyl tacrolimus at 121% relative to parent tacrolimus. Cross-reactivity of 15-O-demethyl tacrolimus with the 5-8 kDa protein was then assessed in whole blood samples, and it bound at a level of 163%. MLC data indicates that 31-O-demethyl tacrolimus is equipotent to parent tacrolimus in immunosuppressive activity, while the 13-O-demethyl and 15-O-demethyl have negligible immunosuppressive activity. CONCLUSIONS: Therefore, the 5-8 kDa immunophilin would have limitations in a radioreceptor assay for tacrolimus. In addition, we have evidence that the 5-8 kDa immunophilin is a subunit of a 52 kDa immunophilin previously identified by our group, and the cross-reactivity of the 5-8 kDa immunophilin with these metabolites is similar to that found previously with the 52 kDa, indicating that the two proteins could be related.
Asunto(s)
Inmunofilinas/metabolismo , Sirolimus/metabolismo , Tacrolimus/metabolismo , Animales , Antibacterianos/metabolismo , Bovinos , Reacciones Cruzadas , Humanos , Inmunofilinas/aislamiento & purificación , Inmunosupresores/metabolismo , Prueba de Cultivo Mixto de Linfocitos , Peso Molecular , Unión Proteica , Ensayo de Unión Radioligante , Sirolimus/análogos & derivados , Tacrolimus/análogos & derivadosRESUMEN
Fpr3 is the third member of the FKBP (FK506 binding protein) family in yeast. In this study, the fpr3 gene from Saccharomyces cerevisiae was overexpressed and the protein product was purified using different methods. The recombinant Fpr3 fusion protein (rFpr3) was then used as antigen to immunize BALB/c mice for the production of monoclonal antibodies (MAb). Western blot and ELISA results indicated that rFpr3 had specific binding ability to the MAbs, and isotyping results classified the MAb as the subclass IgG1 by antibody. The MAbs obtained in this study will be used as a molecular chaperone to obtain Fpr3 crystals.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/biosíntesis , Inmunoglobulina G/biosíntesis , Inmunofilinas/inmunología , Chaperonas Moleculares/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Proteínas de Saccharomyces cerevisiae/inmunología , Animales , Especificidad de Anticuerpos , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Hibridomas/metabolismo , Inmunofilinas/biosíntesis , Inmunofilinas/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , VolumetríaRESUMEN
Our previous studies have shown that the His tag cleaved from fusion proteins contained two distinct components P1 and P2. P1 has been identified to be a His-tagged peptide of G-H-H-H-H-H-H-H-H-H-H-S-S-G-H-I-E-G-R resulted from initiator methionine deletion, and P2 contains an unknown moiety at the second residue glycine of the tag (x-G-H-H-H-H-H-H-H-H-H-H-S-S-G-H-I-E-G-R, x = 178.0 Da). This study aimed to determine the structure of the modification by using a combination of protein isotope labeling and mass spectrometry. His-tagged FKBP was expressed in (15)N and (13)C labeling growth media respectively. Isotopic labeled His-tagged proteins ((15)N-His-FKBP and (13)C-His-FKBP) were isolated by affinity chromatography and subjected to Xa digestions to release the labeled His tag. Subsequent analyses of the released His tag by MALDI-TOF-MS indicated a mass difference of 178.0 +/- 0.2 Da, between the two (15)N-labeled peptides P1 and P2, suggesting that the modification moiety contained no nitrogen. A mass difference of 184.0 +/- 0.2 Da was observed on MALDI between (13)C-labeled peptide P1 and P2, indicating six carbons in the modification group. Also, comparing the mass shift on MALDI spectra of P1 and P2 after hydrogen/deuterium exchange revealed that the modification moiety had five hydroxyl groups. It was concluded that the modification was a gluconic acid derivative attached to the N-terminus of His-tagged proteins expressed in bacteria. The proposed structure was further confirmed by MALDI analysis of periodate oxidation products of His-tagged peptides.
Asunto(s)
Gluconatos , Inmunofilinas/biosíntesis , Secuencia de Aminoácidos , Isótopos de Carbono , Cromatografía de Afinidad , Clonación Molecular , Histidina , Inmunofilinas/química , Inmunofilinas/aislamiento & purificación , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Proteínas de Unión a TacrolimusRESUMEN
The ryanodine receptor/calcium release channel (RyR1) of sarcoplasmic reticulum from rabbit skeletal muscle terminal cisternae (TC) contains four tightly associated FK506-binding proteins (FKBP12). Dissociation and reconstitution studies have shown that RyR1 can be modulated by FKBP12, which helps to maintain the channel in the quiescent state. In this study, we found that the association of FKBP with RyR1 of skeletal muscle is common to each of the five classes of vertebrates. TC from skeletal muscle representing animals from different vertebrates, i.e. mammals (rabbit), birds (chicken), reptiles (turtle), fish (salmon and rainbow trout), and amphibians (frog), were isolated. For each, we find the following: 1) FKBP12 is localized to the TC (there are four FKBP binding sites/ryanodine receptor); 2) soluble FKBP exchanges with the bound form on RyR1 of TC; 3) release of FKBP from terminal cisternae by drug (FK590) treatment leads to a significant reduction in the net calcium loading rate, consistent with channel activation (the calcium loading rate is restored to the control value by reconstitution with FKBP12); and 4) RyR1 of skeletal muscle TC can bind to and exchange with either FKBP12 or FKBP12.6 (FKBP12.6 is the novel FKBP isoform found selectively associated with RyR2 of dog cardiac sarcoplasmic reticulum). We conclude that FKBP is an integral part of the RyR1 of skeletal muscle in each of the classes of vertebrate animals. The studies are consistent with a role for FKBP in skeletal muscle excitation-contraction coupling.
Asunto(s)
Inmunofilinas/metabolismo , Proteínas Musculares/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Vertebrados/metabolismo , Animales , Sitios de Unión , Calcio/metabolismo , Perros , Inmunofilinas/aislamiento & purificación , Proteínas Musculares/aislamiento & purificación , Unión Proteica , Conejos , Canal Liberador de Calcio Receptor de Rianodina/aislamiento & purificación , Retículo Sarcoplasmático/química , Proteínas de Unión a TacrolimusRESUMEN
The in vitro protein folding activity of an FKBP (FK506 binding protein, abbreviated to MTFK) from a thermophilic archaeon, Methanococcus thermolithotrophicus, was investigated. MTFK exhibited FK506 sensitive PPIase (peptidyl prolyl cis-trans isomerase) activity which accelerated the speed of ribonuclease T1 refolding, which is rate-limited by isomerization of two prolyl peptide bonds. In addition, MTFK suppressed the aggregation of folding intermediates and elevated the final yield of rhodanese refolding. We called this activity of MTFK the chaperone activity. The chaperone activity of MTFK was also inhibited by FK506. Alignment of the amino acid sequences of MTFK with human FKBP12 showed that MTFK has two insertion sequences, consisting of 13 and 44 amino acids, at the N- and C-termini, respectively [Furutani, M., Iida, T., Yamano, S., Kamino, K., and Maruyama, T. (1998) J. Bacteriol. 180, 388-394]. To study the relationship between chaperone and PPIase activities of MTFK, mutant MTFKs with deletions of these insertion sequences or with amino acid substitutions were created. Their PPIase and chaperone activities were measured using a synthetic oligopeptide and denatured rhodanese as the substrates, respectively. The far-UV circular dichroism spectra of the wild type and the mutants were also analyzed. The results suggested that (1) the PPIase activity did not correlate with chaperone activity, (2) both insertion sequences were required for MTFK to take a proper conformation, and (3) the insertion sequence (44 amino acids) in the C-terminus was important for the chaperone activity.
Asunto(s)
Inmunofilinas/química , Methanococcus/química , Chaperonas Moleculares/química , Sitios de Unión , Escherichia coli/metabolismo , Expresión Génica , Inmunofilinas/genética , Inmunofilinas/aislamiento & purificación , Methanococcus/genética , Estructura Molecular , Mutación , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/farmacología , Pliegue de Proteína , Ribonucleasa T1/química , Tacrolimus/química , Proteínas de Unión a Tacrolimus , Tiosulfato Azufretransferasa/químicaRESUMEN
Immuonosuppressive drugs FK506 and rapamycin block a number of signal transduction pathways in eukaryotic systems. The 12 kDa FK506 binding protein (FKBP12) mediates the action of both FK506 and rapamycin against their functional targets. In this report, we cloned, sequenced and characterized a gene encoding FKBP12 in Vicia faba (VfFKBP12). While VfFKBP12 is highly homologous to animal and yeast FKBP12, it does not mediate the action of FK506 and rapamycin. There are unique features in plant FKBP12 sequences that cause the variation in their function. One lies in the domain that is critical for interaction with calcineurin (CaN), the mammalian and yeast target of FKBP12-FK506 complex. Protein-protein interaction assays revealed a low-affinity and unstable VfFKBP12-FK506-CaN ternary complex. In the genetic assay, VfFKBP12 did not restore the sensitivity of yeast FKBP12 mutant to rapamycin or FK506, supporting that plant FKBP12-ligand complexes are unable to block the function of the drug target. Also unique to plant FKBP12 proteins, a pair of cysteines is spatially adjacent to potentially form disulfide linkage. Treatment of VfFKBP12 with reductant dithiothreitol (DTT) abolished the formation of VfFKBP12-FK506-CaN ternary complex. Site-directed mutagenesis to substitute one of the cysteines, Cys26, with Ser produced a similar effect as DTT treatment. These results indicate that an intramolecular disulfide bond is a novel structural feature required for the low-affinity interaction between plant FKBP12 and CaN. In conclusion, plant FKBP12 proteins have evolved structural changes that modify their protein-protein interacting domains and cause loss of function against the drug targets.
Asunto(s)
Fabaceae/metabolismo , Inmunofilinas/genética , Inmunofilinas/metabolismo , Inmunosupresores/metabolismo , Plantas Medicinales , Sirolimus/metabolismo , Tacrolimus/metabolismo , Secuencia de Aminoácidos , Calcineurina/metabolismo , Clonación Molecular , Cisteína , Disulfuros , Ditiotreitol , Fabaceae/genética , Genes de Plantas/genética , Prueba de Complementación Genética , Inmunofilinas/química , Inmunofilinas/aislamiento & purificación , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Reactivos de Sulfhidrilo , Proteínas de Unión a TacrolimusRESUMEN
During embryogenesis, smooth muscle cells of the gut differentiate from mesenchymal cells derived from splanchnic mesoderm. We have isolated a gene involved in the differentiation of smooth muscle cells in the gut using differential display between the chicken proventriculus in which the smooth muscle layer develops poorly and the gizzard in which smooth muscles develop abundantly. The protein encoded by this gene showed highest similarity to mouse FK506 binding protein, FKBP65, and from the function of this protein it was designated chicken FKBP/smooth muscle activating protein (cFKBP/SMAP). cFKBP/SMAP was first expressed in smooth muscle precursor cells of the gut and, after smooth muscles differentiate, expression was restricted to smooth muscle cells. In organ culture of the gizzard, the differentiation of smooth muscle cells was inhibited by the addition of FK506, the inhibitor of FKBPs. Moreover, overexpression of cFKBP/SMAP in lung and gizzard mesenchymal cells induced smooth muscle differentiation. In addition, cFKBP/SMAP-induced smooth muscle differentiation was inhibited by FK506. We postulate therefore that cFKBP/SMAP plays a crucial role in smooth muscle differentiation in the gut and provides a powerful tool to study smooth muscle differentiation mechanisms, which have been poorly analyzed so far.
Asunto(s)
Inmunofilinas/fisiología , Músculo Liso/citología , Isomerasa de Peptidilprolil , Proteínas de Unión a Tacrolimus , Secuencia de Aminoácidos , Animales , Northern Blotting , Diferenciación Celular/efectos de los fármacos , Embrión de Pollo , Inmunofilinas/biosíntesis , Inmunofilinas/genética , Inmunofilinas/aislamiento & purificación , Inmunosupresores/farmacología , Mesodermo/metabolismo , Ratones , Datos de Secuencia Molecular , Músculo Liso/efectos de los fármacos , Tacrolimus/farmacologíaRESUMEN
We have identified an 8.4 kDa minor immunophilin from calf thymus and Jurkat T cells as ubiquitin. It binds tacrolimus and sirolimus with K(d)'s of 0.8 and 0.08 nM, respectively. The binding of this protein to cyclosporin A is negligible. Binding of tacrolimus to two commercial sources of ubiquitin, a bovine product, and a recombinant human ubiquitin, was also demonstrated after HPLC purification of the purchased preparations.
Asunto(s)
Inmunofilinas/química , Ubiquitinas/química , Ubiquitinas/fisiología , Secuencia de Aminoácidos , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Ciclosporina/metabolismo , Ciclosporina/farmacología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunofilinas/aislamiento & purificación , Inmunosupresores/metabolismo , Inmunosupresores/farmacología , Células Jurkat , Cinética , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes/metabolismo , Sirolimus/metabolismo , Sirolimus/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tacrolimus/metabolismo , Tacrolimus/farmacología , Timo/química , Ubiquitinas/aislamiento & purificaciónRESUMEN
A novel inositolphosphate-binding protein has been identified and shown to be an immunophilin. This protein, which was isolated from human erythrocyte membranes and from K562 (human erythroleukemia) cell membranes, has robust peptidylprolyl cis-trans isomerase activity that is strongly inhibited by nanomolar concentrations of FK506 or rapamycin, indicating a member of the FKBP (FK506-binding protein) class. However, unlike the cytosolic FKBP12, the isomerase activity of this membrane-associated immunophilin is strongly inhibited by nanomolar concentrations of inositol 1,4, 5-trisphosphate (IP(3)), inositol 1,3,4,5-tetrakisphosphate (IP(4)), and phosphatidylinositol 4- and 4,5-phosphates, which are suggested to be physiological ligands. The demonstration of a single 12-kD protein that binds both IP(4) or IP(3) and anti-FKBP12 provides strong support for the inositolphosphate-binding immunophilin having an apparent mass of 12 kD, and it is suggested that the protein might be called IPBP12 for 12-kD inositol phosphate binding protein. When an internal tryptic peptide derived from IPBP12 was sequenced, a sequence also present in human cytokeratin 10 was identified, suggesting a cytoskeletal localization for the immunophilin. While purifying IPBP12, it was found that it is immunoprecipitated with specific proteins that include a protein kinase and a phosphoprotein phosphatase. The latter is indicated to be phosphoprotein phosphatase 2A (PP-2A). It is suggested that immunophilins promote the assembly of multiprotein complexes that often include a protein kinase or a phosphoprotein phosphatase or both.
Asunto(s)
Inmunofilinas/aislamiento & purificación , Fosfatos de Inositol/metabolismo , Isomerasa de Peptidilprolil/aislamiento & purificación , Secuencia de Aminoácidos , Células Precursoras Eritroides/química , Humanos , Inmunofilinas/antagonistas & inhibidores , Inmunofilinas/metabolismo , Fosfatos de Inositol/farmacología , Células K562/química , Sustancias Macromoleculares , Datos de Secuencia Molecular , Proteínas de Neoplasias/aislamiento & purificación , Proteínas de Neoplasias/metabolismo , Isomerasa de Peptidilprolil/antagonistas & inhibidores , Isomerasa de Peptidilprolil/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Quinasas/metabolismo , Proteína Fosfatasa 2 , Sistemas de Mensajero Secundario , Tacrolimus/farmacología , Proteínas de Unión a TacrolimusRESUMEN
Reactive oxygen species have been implicated in the pathogenesis of atherosclerosis and hypertension, in part by promoting vascular smooth muscle cell (VSMC) growth. We have previously shown that LY83583, a generator of O-(2), activated extracellular signal-regulated kinases (ERK1/2) with early (10 min) and late (2 h) peaks and stimulated VSMC growth. To investigate whether secreted oxidative stress-induced factors (termed SOXF) from VSMC were responsible for late ERK1/2 activation in response to LY83583, we purified putative SOXF proteins from conditioned medium (2 h of LY83583 exposure) by sequential chromatography based on activation of ERK1/2. Proteins identified by capillary chromatography, electrospray ionization tandem mass spectrometry, and data base searching included heat shock protein 90-alpha (HSP90-alpha) and cyclophilin B. Western blot analysis of conditioned medium showed specific secretion of HSP90-alpha but not HSP90-beta. Immunodepletion of HSP90-alpha from conditioned medium significantly inhibited conditioned medium-induced ERK1/2 activation. Human recombinant HSP90-alpha reproduced the effect of conditioned medium on ERK1/2 activation. These results show that brief oxidative stress causes sustained release of protein factors from VSMC that can stimulate ERK1/2. These factors may be important mediators for the effects of reactive oxygen species on vascular function.
Asunto(s)
Ciclofilinas , Proteínas HSP90 de Choque Térmico/aislamiento & purificación , Inmunofilinas/aislamiento & purificación , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/fisiología , Estrés Oxidativo/fisiología , Sal Disódica del Ácido 1,2-Dihidroxibenceno-3,5-Disulfónico/farmacología , Aminoquinolinas/farmacología , Animales , Comunicación Autocrina , Medios de Cultivo Condicionados , Proteínas HSP90 de Choque Térmico/metabolismo , Inmunofilinas/metabolismo , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Isomerasa de Peptidilprolil , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Tripsina/farmacologíaRESUMEN
BACKGROUND: Five birch pollen allergens have been identified so far. In a previous study we detected new birch pollen allergens with an isoelectric point in the range 9.0 to 9.3, present only in extracts prepared at controlled basic pH. OBJECTIVE: The purpose of the current study was to purify and characterize those allergens. METHODS: The target allergens were purified by ion exchange and hydrophobic interaction chromatography. Analyses were carried out by SDS-PAGE, isoelectric focusing, immunoblotting, and amino acid sequencing. The in vivo reactivity of the allergens was evaluated by skin testing. RESULTS: An 18-kd protein, which we named Bet v 7, was purified. This 18-kd protein corresponded to 3 bands on isoelectric-focusing immunoblots that probably represent isoforms. On immunoblots up to 20.8% of birch pollen-allergic patients recognized those allergens. The clinical relevance of Bet v 7 was demonstrated by positive immediate-type skin testing on a patient allergic to birch pollen. Sequencing of an internal peptide yielded an amino acid sequence showing high homology with various plant cyclophilins. The rotamase activity of the protein, inhibited by cyclosporin A, further confirmed that Bet v 7 belongs to the group of cyclophilins. CONCLUSION: We have purified a novel allergen of birch pollen, Bet v 7, belonging to the cyclophilin family. Because cyclophilins are highly conserved proteins over the phylogeny, we may postulate that Bet v 7 is a member of a new family of panallergens.
Asunto(s)
Alérgenos/aislamiento & purificación , Inmunofilinas/aislamiento & purificación , Proteínas de Plantas/aislamiento & purificación , Polen/química , Árboles/química , Alérgenos/química , Alérgenos/inmunología , Alérgenos/metabolismo , Secuencia de Aminoácidos , Antígenos de Plantas , Ciclosporina/metabolismo , Humanos , Hipersensibilidad/sangre , Hipersensibilidad/inmunología , Immunoblotting , Inmunoglobulina E/sangre , Inmunofilinas/inmunología , Inmunofilinas/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Isomerasa de Peptidilprolil/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Polen/inmunología , Pruebas Cutáneas , Árboles/inmunologíaRESUMEN
In animal cell lysates the multiprotein heat-shock protein 90 (hsp90)-based chaperone complexes consist of hsp70, hsp40, and p60. These complexes act to convert steroid hormone receptors to their steroid-binding state by assembling them into heterocomplexes with hsp90, p23, and one of several immunophilins. Wheat germ lysate also contains a hsp90-based chaperone system that can assemble the glucocorticoid receptor into a functional heterocomplex with hsp90. However, only two components of the heterocomplex-assembly system, hsp90 and hsp70, have thus far been identified. Recently, purified mammalian p23 preadsorbed with JJ3 antibody-protein A-Sepharose pellets was used to isolate a mammalian p23-wheat hsp90 heterocomplex from wheat germ lysate (J.K. Owens-Grillo, L.F. Stancato, K. Hoffmann, W.B. Pratt, and P. Krishna [1996] Biochemistry 35: 15249-15255). This heterocomplex was found to contain an immunophilin(s) of the FK506-binding class, as judged by binding of the radiolabeled immunosuppressant drug [3H]FK506 to the immune pellets in a specific manner. In the present study we identified the immunophilin components of this heterocomplex as FKBP73 and FKBP77, the two recently described high-molecular-weight FKBPs of wheat. In addition, we present evidence that the two FKBPs bind hsp90 via tetratricopeptide repeat domains. Our results demonstrate that binding of immunophilins to hsp90 via tetratricopeptide repeat domains is a conserved protein interaction in plants. Conservation of this protein-to-protein interaction in both plant and animal cells suggests that it is important for the biological action of the high-molecular-weight immunophilins.
Asunto(s)
Proteínas HSP90 de Choque Térmico/aislamiento & purificación , Inmunofilinas/aislamiento & purificación , Proteínas de Plantas/aislamiento & purificación , Tacrolimus/metabolismo , Triticum/química , Secuencia de Aminoácidos , Animales , Benzoquinonas , Sitios de Unión , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Inmunofilinas/química , Técnicas de Inmunoadsorción , Lactamas Macrocíclicas , Sustancias Macromoleculares , Peso Molecular , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Unión Proteica/efectos de los fármacos , Quinonas/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas de Unión a Tacrolimus , Triticum/metabolismoRESUMEN
Like other nuclear receptors, steroid hormone receptors form large protein hetero-complexes in their inactive, ligand-friendly state. Several heat-shock proteins, immunophilins and others have been identified as members of these highly dynamic complexes. The interaction kinetics and dynamics of hsp90, hsp70, p60 (Hop), FKBP52, FKBP51, p48 (Hip) and p23 have been assessed by a biosensor approach measuring the complex formation in real time. A core chaperone complex has been reconstituted from p60, hsp90 and hsp70. p60 forms a molecular bridge between hsp90 and hsp70 with an affinity in the range of 10(5) M(-1). Dynamics of hsp90-p60 complex formation is modulated by ATP through changes in the co-operativity of interaction. At low protein concentrations ATP stabilizes the complex. Binding of p23 to hsp90 did not change the affinity of the hsp90-p60 complex and the stabilizing effect of ATP. Saturation of the p48-hsp70 interaction could not be achieved, suggesting multiple binding sites. A picture of the protein complex, including stoichiometric coefficients, co-operativity of interaction and equilibrium-binding constants, has been formed.