RESUMO
With the use of a partial sequence of the human genome, we identified a gene encoding a novel soluble receptor belonging to the class II cytokine receptor family. This gene is positioned on chromosome 6 in the vicinity of the IFNGR1 gene in a head-to-tail orientation. The gene consists of six exons and encodes a 231-aa protein with a 21-aa leader sequence. The secreted mature protein demonstrates 34% amino acid identity to the extracellular domain of the IL-22R1 chain. Cross-linking experiments demonstrate that the protein binds IL-22 and prevents binding of IL-22 to the functional cell surface IL-22R complex, which consists of two subunits, the IL-22R1 and the IL-10R2c chains. Moreover, this soluble receptor, designated IL-22-binding protein (BP), is capable of neutralizing IL-22 activity. In the presence of the IL-22BP, IL-22 is unable to induce Stat activation in IL-22-responsive human lung carcinoma A549 cells. IL-22BP also blocked induction of the suppressors of cytokine signaling-3 (SOCS-3) gene expression by IL-22 in HepG2 cells. To further evaluate IL-22BP action, we used hamster cells expressing a modified IL-22R complex consisting of the intact IL-10R2c and the chimeric IL-22R1/gammaR1 receptor in which the IL-22R1 intracellular domain was replaced with the IFN-gammaR1 intracellular domain. In these cells, IL-22 activates biological activities specific for IFN-gamma, such as up-regulation of MHC class I Ag expression. The addition of IL-22BP neutralizes the ability of IL-22 to induce Stat activation and MHC class I Ag expression in these cells. Thus, the soluble receptor designated IL-22BP inhibits IL-22 activity by binding IL-22 and blocking its interaction with the cell surface IL-22R complex.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/genética , Interleucinas/antagonistas & inibidores , Interleucinas/metabolismo , Receptores de Superfície Celular , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva/imunologia , Células CHO , Células COS , Proteínas de Transporte/biossíntese , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Clonagem Molecular , Cricetinae , DNA Complementar/isolamento & purificação , Humanos , Ligantes , Dados de Sequência Molecular , Receptores de Interleucina , Solubilidade , Células Tumorais Cultivadas , Interleucina 22RESUMO
Interleukin-10 (IL-10)-related T cell-derived inducible factor (IL-TIF; provisionally designated IL-22) is a cytokine with limited homology to IL-10. We report here the identification of a functional IL-TIF receptor complex that consists of two receptor chains, the orphan CRF2-9 and IL-10R2, the second chain of the IL-10 receptor complex. Expression of the CRF2-9 chain in monkey COS cells renders them sensitive to IL-TIF. However, in hamster cells both chains, CRF2-9 and IL-10R2, must be expressed to assemble the functional IL-TIF receptor complex. The CRF2-9 chain (or the IL-TIF-R1 chain) is responsible for Stat recruitment. Substitution of the CRF2-9 intracellular domain with the IFN-gammaR1 intracellular domain changes the pattern of IL-TIF-induced Stat activation. The CRF2-9 gene is expressed in normal liver and kidney, suggesting a possible role for IL-TIF in regulating gene expression in these tissues. Each chain, CRF2-9 and IL-10R2, is capable of binding IL-TIF independently and can be cross-linked to the radiolabeled IL-TIF. However, binding of IL-TIF to the receptor complex is greater than binding to either receptor chain alone. Sharing of the common IL-10R2 chain between the IL-10 and IL-TIF receptor complexes is the first such case for receptor complexes with chains belonging to the class II cytokine receptor family, establishing a novel paradigm for IL-10-related ligands similar to the shared use of the gamma common chain (gamma(c)) by several cytokines, including IL-2, IL-4, IL-7, IL-9, and IL-15.
Assuntos
Citocinas/metabolismo , Interleucinas/metabolismo , Receptores de Interleucina/isolamento & purificação , Sequência de Aminoácidos , Reagentes de Ligações Cruzadas , Humanos , Ligantes , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Receptores de Interleucina-10 , Transdução de Sinais , Interleucina 22RESUMO
Interferon-gamma (IFN-gamma) and its receptor complex are dimeric and bilaterally symmetric. We created mutants of IFN-gamma that bind only one IFN-gammaR1 chain per dimer molecule (called a monovalent IFN-gamma) to see if the interaction of IFN-gamma with one-half of the receptor complex is sufficient for bioactivity. Mutating a receptor-binding sequence in either AB loop of a covalent dimer of IFN-gamma yielded two monovalent IFN-gammas, gamma(m)-gamma and gamma-gamma(m), which cross-link to only a single soluble IFN-gammaR1 molecule in solution and on the cell surface. Monovalent IFN-gamma competes fully with wild type IFN-gamma for binding to U937 cells but only at a greater than 100-fold higher concentration than wild type IFN-gamma. Monovalent IFN-gamma had anti-vesicular stomatitis virus activity and antiproliferative activity, and it induced major histocompatibility complex class I and class II (HLA-DR) expression. In contrast, the maximal levels of activated Stat1alpha produced by monovalent IFN-gammas after 15 min were never more than half of those produced by either wild type or covalent IFN-gammas in human cell lines. These data indicate that while monovalent IFN-gamma activates only one-half of a four-chain receptor complex, this is sufficient for Stat1alpha activation, major histocompatibility complex class I surface antigen induction, and antiviral and antiproliferative activities. Thus, while interaction with both halves of the receptor complex is required for high affinity binding of IFN-gamma and efficient signal transduction, interaction with only one-half of the receptor complex is sufficient to initiate signal transduction.
Assuntos
Interferon gama/metabolismo , Receptores de Interferon/metabolismo , Transdução de Sinais , Sequência de Bases , Biopolímeros , Linhagem Celular , Cromatografia em Gel , Primers do DNA , Dimerização , Humanos , Interferon gama/química , Ligação Proteica , Receptor de Interferon gamaRESUMO
We identified a viral IL-10 homolog encoded by an ORF (UL111a) within the human cytomegalovirus (CMV) genome, which we designated cmvIL-10. cmvIL-10 can bind to the human IL-10 receptor and can compete with human IL-10 for binding sites, despite the fact that these two proteins are only 27% identical. cmvIL-10 requires both subunits of the IL-10 receptor complex to induce signal transduction events and biological activities. The structure of the cmvIL-10 gene is unique by itself. The gene retained two of four introns of the IL-10 gene, but the length of the introns was reduced. We demonstrated that cmvIL-10 is expressed in CMV-infected cells. Thus, expression of cmvIL-10 extends the range of counter measures developed by CMV to circumvent detection and destruction by the host immune system.
Assuntos
Citomegalovirus/genética , Substâncias de Crescimento/genética , Interleucina-10/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Citomegalovirus/química , Proteínas de Ligação a DNA/metabolismo , Genes Virais/genética , Genoma Viral , Substâncias de Crescimento/química , Humanos , Interleucina-10/química , Leucócitos , Complexo Principal de Histocompatibilidade/genética , Dados de Sequência Molecular , Ligação Proteica , Receptores de Interleucina/metabolismo , Receptores de Interleucina-10 , Fator de Transcrição STAT3 , Alinhamento de Sequência , Transdução de Sinais , Transativadores/metabolismo , Transfecção , Proteínas Virais/químicaRESUMO
Interleukin-10 (IL-10) is a pleiotropic cytokine with important immunoregulatory functions whose actions influence activities of many of the cell-types in the immune system. We report here identification and cloning of a gene and corresponding cDNAs encoding a novel homologue of IL-10, designated IL-19. IL-19 shares 21% amino acid identity with IL-10. The exon/intron structure of IL-19 is similar to that of the human IL-10 gene, comprising five exons and four introns within the coding region of the IL-19 cDNA. There are at least two distinct IL-19 mRNA species that differ in their 5'-sequences, suggesting the existence of an intron in the 5'-sequences of coding portion of the IL-19 gene. The longer 5'-sequence contains an alternative initiating ATG codon that is in-frame with the rest of the coding sequence. The expression of IL-19 mRNA can be induced in monocytes by LPS-treatment. The appearance of IL-19 mRNA in LPS-stimulated monocytes was slightly delayed compared to expression of IL-10 mRNA: significant levels of IL-10 mRNA were detectable at 2 h post-stimulation, whereas IL-19 mRNA was not detectable until 4 h. Treatment of monocytes with IL-4 or IL-13 did not induce de novo expression of IL-19, but these cytokines did potentiate IL-19 gene expression in LPS-stimulated monocytes. In addition, GM-CSF was capable of directly inducing IL-19 gene expression in monocytes. IL-19 does not bind or signal through the canonical IL-10 receptor complex, suggesting existence of an IL-19 specific receptor complex, the identity of which remains to be discovered.
Assuntos
Interleucina-10/genética , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Humanos , Interleucinas , Dados de Sequência Molecular , Monócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
To expand our understanding of the role of Jak2 in cellular signaling, we used the yeast two-hybrid system to identify Jak2-interacting proteins. One of the clones identified represents a human homologue of the Schizosaccaromyces pombe Shk1 kinase-binding protein 1, Skb1, and the protein encoded by the Saccharomyces cerevisiae HSL7 (histone synthetic lethal 7) gene. Since no functional motifs or biochemical activities for this protein or its homologues had been reported, we sought to determine a biochemical function for this human protein. We demonstrate that this protein is a protein methyltransferase. This protein, designated JBP1 (Jak-binding protein 1), and its homologues contain motifs conserved among protein methyltransferases. JBP1 can be cross-linked to radiolabeled S-adenosylmethionine (AdoMet) and methylates histones (H2A and H4) and myelin basic protein. Mutants containing substitutions within a conserved region likely to be involved in AdoMet binding exhibit little or no activity. We mapped the JBP1 gene to chromosome 14q11.2-21. In addition, JBP1 co-immunoprecipitates with several other proteins, which serve as methyl group acceptors and which may represent physiological targets of this methyltransferase. Messenger RNA for JBP1 is widely expressed in human tissues. We have also identified and sequenced a homologue of JBP1 in Drosophila melanogaster. This report provides a clue to the biochemical function for this conserved protein and suggests that protein methyltransferases may have a role in cellular signaling.
Assuntos
Metiltransferases , Proteínas Metiltransferases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Sequência de Aminoácidos , Proteínas de Transporte/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 14 , Clonagem Molecular , Proteínas de Drosophila , Histonas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Janus Quinase 2 , Metilação , Dados de Sequência Molecular , Mutação Puntual , Testes de Precipitina , Ligação Proteica , Proteínas Quinases/genética , Proteínas Metiltransferases/genética , Proteína-Arginina N-Metiltransferases , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Técnicas do Sistema de Duplo-HíbridoRESUMO
Type I IFNs activate the Jak-Stat signal transduction pathway. The IFN-alpha receptor 1 (IFN-alphaR1) subunit and two splice variants of the IFN-alphaR2 subunit, IFN-alphaR2c and IFN-alphaR2b, are involved in ligand binding. All these receptors have been implicated in cytokine signaling and, specifically, in Stat recruitment. To evaluate the specific contribution of each receptor subunit to Stat recruitment we employed chimeric receptors with the extracellular domain of either IFN-gammaR2 or IFN-gammaR1 fused to the intracellular domains of IFN-alphaR1, IFN-alphaR2b, and IFN-alphaR2c. These chimeric receptors were expressed in hamster cells. Because human IFN-gamma exhibits no activity on hamster cells, the use of the human IFN-gamma receptor extracellular domains allowed us to avoid the variable cross-species activity of the type I IFNs and eliminate the possibility of contributions of endogenous type I IFN receptors into the Stat recruitment process. We demonstrate that Stat recruitment is solely a function of the IFN-alphaR2c intracellular domain. When chimeric receptors with the human IFN-gammaR1 extracellular domain and various human IFN-alpha receptor intracellular domains were expressed in hamster cells carrying the human IFN-gammaR2 subunit, only the IFN-alphaR2c subunit was capable of supporting IFN-gamma signaling as measured by MHC class I induction, antiviral protection, and Stat activation. Neither the IFN-alphaR2b nor the IFN-alphaR1 intracellular domain was able to recruit Stats or support IFN-gamma-induced biological activities. Thus, the IFN-alphaR2c intracellular domain is necessary and sufficient to activate Stat1, Stat2, and Stat3 proteins.
Assuntos
Proteínas de Ligação a DNA/genética , Receptores de Interferon/genética , Transdução de Sinais , Transativadores/genética , Animais , Linhagem Celular , Cricetinae , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Células Híbridas , Interferon-alfa/metabolismo , Receptor de Interferon alfa e beta , Receptores de Interferon/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Transdução de Sinais/genética , Transativadores/metabolismoRESUMO
Interleukin-10 (IL-10) is a pleiotropic cytokine which signals through a specific cell surface receptor complex. Only one chain, that for ligand binding (IL-10Ralpha or IL-10R1), was identified previously. We report here that, although human IL-10 binds to the human IL-10R1 chain expressed in hamster cells, it does not induce signal transduction. However, the co-expression of CRFB4, a transmembrane protein of previously unknown function belonging to the class II cytokine receptor family, together with the IL-10R1 chain renders hamster cells sensitive to IL-10. The IL-10:CRFB4 complex was detected by cross-linking to labeled IL-10. In addition, the IL-10R1 chain was able to be co-immunoprecipitated with anti-CRF antibody when peripheral blood mononuclear cells were treated with IL-10. These results demonstrate that the CRFB4 chain is part of the IL-10 receptor signaling complex. Thus, the CRFB4 chain, which we designate as the IL-10R2 or IL-10Rbeta chain, serves as an accessory chain essential for the active IL-10 receptor complex and to initiate IL-10-induced signal transduction events.
Assuntos
Glicoproteínas de Membrana , Receptores de Citocinas/metabolismo , Receptores de Interleucina/metabolismo , Animais , Células COS , Cricetinae , Reagentes de Ligações Cruzadas/farmacologia , Haplorrinos , Humanos , Subunidade beta de Receptor de Interleucina-10 , Janus Quinase 1 , Monócitos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Receptores de Citocinas/química , Receptores de Citocinas/genética , Receptores de Interleucina/química , Receptores de Interleucina/genética , Receptores de Interleucina-10 , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Succinimidas/farmacologia , TYK2 QuinaseRESUMO
With the purification and cloning of the interferon gamma (IFN-gamma) receptor chains the mechanism of IFN-gamma action and the resultant signal transduction events were delineated in remarkable detail. The interferon gamma (IFN-gamma) receptor complex consists of two chains: IFN-gammaR1, the ligand-binding chain, and IFN-gammaR2, the accessory chain. Binding of IFN-gamma causes oligomerization of the two IFN-gamma receptor subunits, IFN-gammaR1 and IFN-gammaR2, which initiates the signal transduction events: activation of Jak1 and Jak2 receptor associated protein tyrosine kinases, phosphorylation of the IFN-gammaR1 intracellular domain on Tyr440 followed by phosphorylation and activation of Stat1alpha, the latent transcriptional factor. With all these steps established, the IFN-gamma receptor complex has provided the basic model for understanding the receptors for other members of the family of class II cytokine receptors.
Assuntos
Interferon gama/metabolismo , Receptores de Interferon/genética , Receptores de Interferon/fisiologia , Animais , Humanos , Modelos BiológicosRESUMO
Each cytokine which utilizes the Jak-Stat signal transduction pathway activates a distinct combination of members of the Jak and Stat families. Thus, either the Jaks, the Stats, or both could contribute to the specificity of ligand action. With the use of chimeric receptors involving the interferon gamma receptor (IFN-gammaR) complex as a model system, we demonstrate that Jak2 activation is not an absolute requirement for IFN-gamma signaling. Other members of the Jak family can functionally substitute for Jak2. IFN-gamma can signal through the activation of Jak family members other than Jak2 as measured by Statlalpha homodimerization and major histocompatibility complex class I antigen expression. This indicates that Jaks are interchangeable and indiscriminative in the Jak-Stat signal transduction pathway. The necessity for the activation of one particular kinase during signaling can be overcome by recruiting another kinase to the receptor complex. The results may suggest that the Jaks do not contribute to the specificity of signal transduction in the Jak-Stat pathway to the same degree as Stats.
Assuntos
Antígenos CD/fisiologia , Proteínas de Ligação a DNA/metabolismo , Interferon gama/farmacologia , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Receptores de Interferon/fisiologia , Transdução de Sinais , Animais , Antígenos CD/biossíntese , Sequência de Bases , Células CHO , Cricetinae , Primers do DNA , Ativação Enzimática , Antígeno HLA-B7/biossíntese , Humanos , Células Híbridas , Janus Quinase 2 , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Fosfotirosina/metabolismo , Reação em Cadeia da Polimerase , Receptores de Interferon/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor de Interferon gamaRESUMO
Interferon gamma (IFN-gamma) signals through a multimeric receptor complex consisting of two different chains: the IFN-gamma receptor binding subunit (IFN-gamma R, IFN-gamma R1), and a transmembrane accessory factor (AF-1, IFN-gamma R2) necessary for signal transduction. Using cell lines expressing different cloned components of the IFN-gamma receptor complex, we examined the function of the receptor components in signal transduction upon IFN-gamma treatment. A specific IFN-gamma R2:IFN-gamma cross-linked complex was observed in cells expressing both IFN-gamma R1 and IFN-gamma R2 indicating that IFN-gamma R2 (AF-1) interacts with IFN-gamma and is closely associated with IFN-gamma R1. We show that the intracellular domain of IFN-gamma R2 is necessary for signaling. Cells coexpressing IFN-gamma R1 and truncated IFN-gamma R2, lacking the COOH-terminal 51 amino acids (residues 286-337), or cells expressing IFN-gamma R1 alone were unresponsive to IFN-gamma treatment as measured by MHC class I antigen induction. Jak1, Jak2, and Stat1 alpha were activated, and IFN-gamma R1 was phosphorylated only in cells expressing both IFN-gamma R1 and IFN-gamma R2. Jak2 kinase was shown to associate with the intracellular domain of the IFN-gamma R2.
Assuntos
Proteínas Proto-Oncogênicas , Receptores de Interferon/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Linhagem Celular , Cricetinae , Proteínas de Ligação a DNA/metabolismo , Humanos , Células Híbridas , Janus Quinase 1 , Janus Quinase 2 , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Fosforilação , Ligação Proteica , Proteínas Tirosina Quinases/metabolismo , Receptores de Interferon/química , Fator de Transcrição STAT1 , Transdução de Sinais , Transativadores/metabolismo , Receptor de Interferon gamaRESUMO
The gene of human mutant (serine-17) fibroblast interferon was isolated with the use of highly efficient oligonucleotide-directed mutagenesis. On the basis of the constructed expression plasmid pPR-IFN Ser17 a strain producing human mutant beta-interferon (VKPM V-4678) was developed. It was shown that the specific activity of the human mutant (serine-17) fibroblast interferon was 1 order of magnitude higher than that of the recombinant interferon which reaches the specific activity of natural fibroblast interferon.
Assuntos
Biotecnologia/métodos , Escherichia coli , Escherichia coli/metabolismo , Interferon beta/biossíntese , Mutagênese Sítio-Dirigida/genética , Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli/genética , Técnicas In Vitro , Interferon beta-1a , Interferon beta-1b , Interferon beta/genética , Interferon beta/isolamento & purificaçãoRESUMO
An epitope of human leukocyte alpha interferon A (IFN-A), which is recognized by the murine monoclonal antibody NK2, has been mapped by using four successive approaches. Limited proteolysis of the IFN-A chain, followed by electrophoresis, Western blotting, and probing of the proteolytic fragments with NK2 showed that an epitope was located within the sequence residues 110-140. A panel of human IFN subtypes bearing substitutions within the sequence 110-140 was tested for reactivity with NK2 in enzyme-linked immunosorbent assays. The results from these assays suggested that the epitope is within the sequence 112-121. Analysis of a hybrid protein IFN-A(1-92)/F(93-166) revealed that the N-terminal region of IFN-A played no significant role in NK2 binding. Three residues of IFN-F (Asn113, Val114, and Lys121) were substituted for the corresponding residues from IFN-A (Lys113, Glu114, and Arg121) by site-directed mutagenesis of the gene encoding IFN-F. NK2 was able to bind the mutated protein, IFN-F(A 113, 114, 121), as well as unmodified IFN-A. The data show that the epitope recognized by NK2 is located within the C-terminal region of IFN-A (residues 112-121). This epitope consists of the essential residues 114 and 116, and residues 112, 113, 115, 117, and 121 presumably contribute the configuration of the epitope.
Assuntos
Anticorpos Monoclonais/química , Epitopos/química , Interferon-alfa/química , Mapeamento de Peptídeos , Sequência de Aminoácidos , Animais , Reações Antígeno-Anticorpo , Sequência de Bases , Endopeptidases , Epitopos/imunologia , Humanos , Hidrólise , Interferon-alfa/imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , CoelhosRESUMO
A new dual-replicon recombinant plasmid, pPR53-tsr, has been constructed; it is a derivative of the expression vector pPR-TGATG-1 [Mashko et al., Gene 88 (1990) 121-126]. In contrast to its progenitor, pPR53-tsr is a low-copy-number (low-Cop) plasmid amplifiable in temperature-dependent fashion. In addition to both the replicon and the par locus from plasmid pSC101, providing segregational stability and a low Cop at 28 degrees C, the new plasmid contains a mutant ColE1 replicon whose RNAII is synthesized under the control of the pL promoter. The presence of a thermolabile repressor, cIts857, allows the thermo-inducible amplification of pPR53-tsr; the increased plasmid Cop is estimated at approx. 200 per genome 6 h after thermal induction at 42 degrees C. Thus, pPR53-tsr can be used as a donor of the thermo-inducible dual-replicon fragment for recombinant plasmids. Here, we employ such an approach for optimization of production of human interleukin-1 beta (hIL-1 beta) in Escherichia coli at a high level. The thermo-induced level of recombinant hIL-1 beta (re-hIL-1 beta) biosynthesis was around 9% of total cellular protein when the dual-replicon high-Cop vector was used. A method based on acidification of the water-soluble protein fraction to pH 4.0 has been developed that allows for the isolation of 80%-pure re-hIL-1 beta. The homogeneous material was obtained by two subsequent hydrophobic sorbent chromatographies. The protein yield ranged between 3-5 mg of re-hIL-1 beta/g of wet cells. The re-hIL-1 beta specific activity was about 2 x 10(8) units/mg, coinciding with that of the authentic hIL-1 beta.
Assuntos
Escherichia coli/genética , Expressão Gênica , Interleucina-1/genética , Replicon , Sequência de Bases , Clonagem Molecular , Replicação do DNA , Eletroforese em Gel de Poliacrilamida , Humanos , Interleucina-1/biossíntese , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas , Mapeamento por Restrição , TemperaturaRESUMO
Recombinant human angiogenin has been synthesized in Escherichia coli with the aid of a human angiogenin gene (hAng) cloned by Neznanov et al (1990) from a human complementary DNA (cDNA) library. The gene has been expressed by use of a new type of expression vector called a 'TGATG vector' (plasmid pPR-TGATG-1; Mashko et al 1990a). The highest level of accumulation of the recombinant angiogenin (6%-8% of the total cell protein) was observed in E. coli strain BL21 carrying a temperature-amplifiable version of the plasmid. The synthesized polypeptide carries an additional serine residue at its N terminus in comparison with natural angiogenin. Furthermore, the initiator methionine residue of the recombinant protein is removed with high efficiency by E. coli terminal aminopeptidase. Simple procedures for purification of the recombinant angiogenin from the insoluble fraction of cell protein, and for refolding the protein allowed the isolation of almost 5 mg recombinant angiogenin g-1 wet bacterial biomass. The recombinant Ser-(-1) angiogenin displayed the same biological properties (specific RNAase activity and the ability to induce blood vessel growth on the sclera of experimental animals) as its natural counterpart isolated from human blood.
Assuntos
Escherichia coli/genética , Proteínas de Neoplasias/biossíntese , Biossíntese de Proteínas , Ribonuclease Pancreático , Sequência de Aminoácidos , Animais , Sequência de Bases , Capilares/efeitos dos fármacos , Feminino , Vetores Genéticos , Humanos , Masculino , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Plasmídeos/genética , Proteínas/genética , Proteínas/fisiologia , Ratos , Proteínas Recombinantes , Ribonucleases/biossíntese , Ribonucleases/efeitos dos fármacos , Esclera/irrigação sanguínea , TransfecçãoRESUMO
Using stepwise ion-exchange and gel-permeation high performance liquid chromatography and SDS-PAAG gel electrophoresis, it was demonstrated that the non-reduced gene-engineered interferon alpha A is represented by multiple forms, namely, four monomers, four dimers, two trimers and one tetramer. All the protein forms were obtained in an individual state and characterized in terms of antiviral activity and immunochemical properties. The heterogeneity of the protein is due both to the formation of anomalous intermolecular disulfide bonds and to the existence of reduced S-S bonds. The antiviral activity of the dimers, trimers and tetramers expressed as units per mole of protein is equal to that for the monomeric form, i.e., the interaction of one monomeric subunit of the covalently-linked oligomer is sufficient for the manifestation of the protein antiviral activity. This suggests that the antiviral status of the cell does not depend on the amount internalized interferon molecules of their processing products but is controlled by the cell receptor whose internalization and, possibly, processing stimulate the transcription of genes involved in the triggering of the immune response.
Assuntos
Engenharia Genética , Interferon Tipo I/isolamento & purificação , Pseudomonas/genética , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Humanos , Interferon Tipo I/análise , Interferon Tipo I/farmacologia , Peso Molecular , Conformação Proteica , Radioimunoensaio , Proteínas RecombinantesRESUMO
Using SDS-PAAG electrophoresis, gel-permeation HPLC and immunoblotting, it was demonstrated that homogeneous preparations of human leukocyte interferons (alpha-INF)-A, -N and -I1 obtained from the biomass of the corresponding producer strains (Pseudomonas sp.) contained several oligomeric forms produced by way of S-S intermolecular cross-linkage and making up to 10-15%, 4-7% and 2-5% of the total monomeric form content in the protein preparations. Immunologic testing with the use of MAB NK-2 and [125I]NK-2 showed that the oligomeric forms of alpha-INF-A, -N and -I1 were present in the protein preparations at all purification stages and seemed to be formed at early steps of interferon synthesis in the cell. The effects of limited proteolysis as well as of acid, alkaline and thermodenaturation on the aggregation and oligomerization of alpha-INF-A were studied. SDS-PAAG electrophoresis performed in the absence of the reducing agents showed that upon denaturation of 10% TCA, the amount of the oligomeric forms in the preparations of homogeneous and especially partly proteolytic INF was significantly increased. The causes and the putative mechanisms of aggregation and oligomerization of INF are discussed.
Assuntos
Interferon Tipo I/análise , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Humanos , Hidrólise , Conformação Proteica , Desnaturação Proteica , Radioimunoensaio , Proteínas Recombinantes/análiseRESUMO
Highly sensitive, effective and simple methods for determination of leukocytic interferons were developed. The methods are based on formation of immune complexes of interferon with anti-interferon monoclonal and polyclonal antibodies immobilized on an insoluble basis. As a result of interferon alpha 2 purification from the biomass of the interferon-producing bacteria the preparative amounts of the protein were obtained. These amounts were sufficient for the interferon structural and physico-chemical analysis. The isolated interferons are characterized by high stability on storage and may serve a basis for preparation of pharmaceutical agents.
Assuntos
Interferon Tipo I/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Aminoácidos/análise , Animais , Anticorpos Monoclonais/análise , Cromatografia Líquida de Alta Pressão , Estabilidade de Medicamentos , Eletroforese em Gel de Poliacrilamida , Humanos , Interferon Tipo I/análise , Interferon Tipo I/biossíntese , Coelhos , Radioimunoensaio , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , TemperaturaRESUMO
Using radioimmunological methods based on the use of mono- and polyclonal antibodies raised against interferon alpha A, it was shown that polyclonal antibodies quantitatively reacted not only with this protein, but also with interferons alpha F and alpha N, whereas all the variants of monoclonal antibodies studied reacted only with interferons alpha A and alpha N. Monoclonal antibodies 5A6, 11E9, 19C10, 258 and 268 are directed against overlapping epitopes of the interferon alpha A molecule, which simultaneously binds not more than two molecules of antibodies with different specificity. The correlation between immunochemical and biological activities of interferon alpha A during temperature denaturation and proteolytic degradation and its ability to form oligomeric complexes were investigated.