RESUMO
The electrophoretic behaviors of five bases and corresponding nucleosides in the oil in water (o/w) microemulsion capillary electrophoresis, microemulsion electrokinetic chromatography (MEEKC), were examined in comparison with those in normal capillary zone electrophoresis (CZE). The microemulsion systems were composed of heptane, sodium dodecyl sulfate (SDS), 1-butanol and 10 mM phosphate buffer (pH 7.0) or toluene, SDS, 1-butanol and 5 mM carbonate buffer (pH 10.0). CZE was carried out in the range of pH 9.7-10.9, and the dissociation constants, pKa, of the bases and nucleosides and the electrophoretic mobilities of the anionic forms were determined. The electrophoretic behaviors of the solutes in the microemulsion systems were analyzed from their pKa, the electrophoretic mobilities of the anions determined by CZE, and the distribution constants, K(D), of the neutral forms between the microemulsion droplets and the outer aqueous phase. The importance of adsorption mechanism in MEEKC system was suggested from the correlation between log K(D) and log P.
Assuntos
Pareamento de Bases , Cromatografia Capilar Eletrocinética Micelar/métodos , Eletroforese Capilar/métodos , Nucleosídeos/análise , Ácidos/química , Ânions , Óleos , Fatores de Tempo , ÁguaRESUMO
Glycosylation inhibiting factor (GIF) and macrophage migration inhibitory factor (MIF) share an identical structure gene. Here we unravel two steps of posttranslational modifications in GIF/MIF molecules in human suppressor T (Ts) cell hybridomas. Peptide mapping and MS analysis of the affinity-purified GIF from the Ts cells revealed that one modification is cysteinylation at Cys-60, and the other is phosphorylation at Ser-91. Cysteinylated GIF, but not the wild-type GIF/MIF, possessed immunosuppressive effects on the in vitro IgE antibody response and had high affinity for GIF receptors on the T helper hybridoma cells. In vitro treatment of wild-type recombinant human GIF/MIF with cystine resulted in preferential cysteinylation of Cys-60 in the molecules. The cysteinylated recombinant human GIF and the Ts hybridoma-derived cysteinylated GIF were comparable both in the affinity for the receptors and in the immunosuppressive activity. Polyclonal antibodies specific for a stretch of the amino acid sequence in alpha2-helix of GIF bound bioactive cysteinylated GIF but failed to bind wild-type GIF/MIF. These results strongly suggest that cysteinylation of Cys-60 and consequent conformational changes in the GIF/MIF molecules are responsible for the generation of GIF bioactivity.
Assuntos
Linfocinas/genética , Linfocinas/metabolismo , Proteínas Secretadas pela Próstata , Processamento de Proteína Pós-Traducional , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Cisteína/metabolismo , Glicosilação , Humanos , Hibridomas , Imunoglobulina E/imunologia , Linfocinas/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Baço/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
We have shown previously that glycosylation-inhibiting factor (GIF) in culture supernatants of suppressor T cell (Ts) hybridomas had bioactivity, while the same cells contained a substantial quantity of inactive GIF in cytosol. Mass-spectrometric analysis of GIF in the culture supernatant and cytosol of a Ts hybridoma provided direct evidence that GIF protein was posttranslationally modified in the Ts cells, and that the GIF bioactivity is associated with the posttranslationally modified species. Assuming that conformational changes induced by the posttranslational modifications are responsible for generation of bioactivity, we constructed cysteine mutants of human rGIF (rhGIF) in which cysteine at position 57, 60, or 81 was replaced with Ala, and the mutants were expressed in Escherichia coli. Replacement of Cys57 or Cys60 with Ala resulted in generation of bioactivity, while replacement of Cys81 with Ala failed to do so. It was also found that replacement of Cys57 with Ala and carboxymethylation of a sulfhydryl group in Cys60 synergistically increased the GIF bioactivity of the GIF derivatives. A mutated GIF protein, in which Cys57 and Asn106 in the rhGIF were replaced with Ala and Ser, respectively, had immunosuppressive effects on the IgE and IgG1 Ab responses of BDF1 mice to DNP-OVA, while wild-type rhGIF did not. Evidence was obtained that the mutated GIF suppressed Ag priming of Th cells for the Ab responses and proliferative response.
Assuntos
Linfocinas/genética , Linfocinas/fisiologia , Mutagênese Sítio-Dirigida , Proteínas Secretadas pela Próstata , Proteínas Recombinantes/farmacologia , Fatores Supressores Imunológicos/genética , Fatores Supressores Imunológicos/fisiologia , Animais , Linhagem Celular , Feminino , Glicosilação , Humanos , Linfocinas/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Modelos Moleculares , Conformação Proteica , Processamento de Proteína Pós-Traducional/imunologia , Proteínas Recombinantes/química , Fatores Supressores Imunológicos/química , Linfócitos T Reguladores/químicaRESUMO
Thrombopoietin (TPO) is a recently isolated cytokine that primarily regulates megakaryocytopoiesis and thrombopoiesis. We recently reported the development of a variety of antibodies (Abs) to synthetic peptides of human (h)TPO and to recombinant human TPO (rhTPO). In this study, we characterized the Abs and mapped immunologically distinct areas of the molecule. Among the five different antipeptide polyclonal Abs, only one, raised against synthetic peptide D8 to Q28, neutralized the TPO-dependent growth of FDCP-2 cells expressing human Mpl (FDCP-hMpl5 cells). One out of seven anti-rhTPO monoclonal Abs, designated as TN1, also showed neutralizing activity. TN1 was found to be specifically reactive with two proteolytic fragments, residues S1 to R117 and A60 to K122 of hTPO, indicating that the epitope(s) of TN1 is localized in residues A60 to R117 of the molecule. These two neutralizing Abs inhibited the binding of biotinylated rhTPO to FDCP-hMpl5 cells. On the other hand, the other Abs, which reacted with five polypeptides of S47 to D62, L108 to A126, N172 to A190, S262 to T284, and P306 to G332 of hTPO, did not show either the neutralizing activity or the ability to inhibit the binding of biotinylated rhTPO to the cell surface hMpl. These findings indicate that two regions, residues D8 to Q28 and A60 to R117 of hTPO, may contain the domains associated with its receptor, C-Mpl. These Abs characterized here are valuable for studying the structural analysis and the biological function of hTPO mediated by its receptor.
Assuntos
Proteínas de Neoplasias , Testes de Neutralização/métodos , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Citocinas , Trombopoetina/metabolismo , Anticorpos , Anticorpos Monoclonais , Divisão Celular , Linhagem Celular , Mapeamento de Epitopos , Humanos , Fragmentos de Peptídeos , Peptídeos/síntese química , Receptores de Trombopoetina , Proteínas Recombinantes , Deleção de Sequência , Trombopoetina/imunologiaRESUMO
A heterogeneity in the molecular weight (Mr) of thrombopoietin (TPO) has been reported. We found several thrombin cleavage sites in human, rat, murine, and canine TPOs, and also found that human TPO undergoes selective proteolysis by thrombin. Recombinant human TPO (rhTPO) was incubated with human platelets in the presence of calcium ions to allow the generation of thrombin, and was cleaved into low Mr peptide fragments. The cleavage was completely inhibited by hirudin, indicating that the proteolysis was mediated by thrombin. In a platelet-free system, analyses of thrombin cleavage by immunoblotting using anti-human TPO peptide antibodies revealed that the four major thrombin-cleaved peptide fragments were selectively generated depending on the digestion time. The amino acid sequences of the thrombin-polypeptides were further analyzed, and two major thrombin cleavage sites were determined. One of them was at AR191-T192 in the C-terminal domain of TPO, and thrombin cleaved first at this site. The other site at GR117-T118 in the N-terminal domain was subsequently cleaved by prolonged thrombin digestion. As a result, the biological activity of TPO was modulated. The generation of truncated forms of TPO by thrombin may be a notable event in view of the platelet-related metabolism of TPO.
Assuntos
Plaquetas/metabolismo , Fragmentos de Peptídeos/metabolismo , Trombina/metabolismo , Trombopoetina/metabolismo , Sequência de Aminoácidos , Especificidade de Anticorpos , Antitrombinas/farmacologia , Plaquetas/efeitos dos fármacos , Hirudinas/farmacologia , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes/metabolismo , Análise de Sequência , Trombopoetina/química , Trombopoetina/imunologiaRESUMO
Escherichia coli-derived recombinant human glycosylation inhibiting factor (rhGIF) contains three cysteine residues (Cys-57, -60, and -81). All SH groups in the cysteine residues are free, and the GIF molecule had no biologic activity. Carboxymethylation of the SH group of Cys-60 in the molecule resulted in the generation of bioactivity, although the activity of the carboxymethylated GIF was 10- to 20-fold less than that of suppressor T cell (Ts)-derived GIF. However, treatment of the inactive rhGIF with ethylmercurithiosalicylate or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) resulted in the generation of derivatives whose bioactivity was comparable to that of the Ts-derived bioactive GIF. The activity of these derivatives was lost by treatment with DTT. Isolation and chemical analysis of the DTNB-treated GIF derivative revealed that binding the 5-thio-2-nitrobenzoic acid group with Cys-60 was responsible for the generation of the highly bioactive derivative. Inactive cytosolic GIF from mammalian cells could also be converted to bioactive derivative by treatment with the SH reagent, while Ts-derived bioactive GIF was inactivated by DTT. These results, together with an x-ray crystal structure of GIF molecules, strongly suggest that the generation of bioactivity of GIF in Ts cells is due to posttranslational modifications that result in conformational changes in the molecule.
Assuntos
Linfocinas/química , Linfocinas/farmacologia , Proteínas Secretadas pela Próstata , Sequência de Aminoácidos , Bioensaio , Dissulfetos , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Glicosilação , Humanos , Linfocinas/efeitos dos fármacos , Linfocinas/genética , Espectrometria de Massas , Dados de Sequência Molecular , Mapeamento de Peptídeos , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Reagentes de Sulfidrila/farmacologiaRESUMO
Glycosylation-inhibiting factor (GIF) is a cytokine that is involved in the regulation of IgE synthesis. The crystal structure of recombinant human GIF was determined by the multiple isomorphous replacement method. The structure was refined to an R factor of 0.168 at 1.9 angstrom resolution. The overall structure is seen to consist of three interconnected subunits forming a barrel with three 6-stranded beta-sheets on the inside and six alpha-helices on the outside. There is a 5-angstrom-diameter "hole" through the middle of the barrel. The barrel structure of GIF in part resembles other "trefoil" cytokines such as interleukin 1 and fibroblast growth factor. Each subunit has a new class of alpha + beta sandwich structure consisting of two beta-alpha-beta motifs. These beta-alpha-beta motifs are related by a pseudo-twofold axis and resemble both interleukin 8 and the peptide binding domain of major histocompatibility complex protein, although the topology of the polypeptide chain is quite different.