RESUMEN
Human IFN-alpha is a family of structurally related proteins that exhibit a wide range of antiproliferative activities. To understand the structural basis for these different antiproliferative activities, eight recombinant human IFN-alpha hybrids (HY) of alpha21a/alpha2c (HY-4, HY-5) and mutants (site-directed mutagenesis (SDM)-1, 2 and cassette mutagenesis (CM)-1, 2, 3, and 4) have been expressed, purified, and characterized. The data showed that the amino acid region 81-95 is important for antiproliferative activity. Site-directed mutagenesis and cassette mutagenesis studies showed that if serine (S) 86 and asparagine (N) 90 were replaced by tyrosine (Y), the antiproliferative activity was increased. We have also observed that if Y86 was replaced by isoleucine (I), the antiproliferative activity was comparable. However, if Y86 was replaced by aspartic acid (D), lysine (K), or alanine (A), the antiproliferative activity was substantially decreased. Our results indicate that Y and/or I at position 86 and Y at position 90 are very important in antiproliferative activity of human IFN-alpha. Circular dichroism spectra showed that the amino acid replacements at position 86 did not change the secondary structure. Thus the biological activity changes among those mutants do not appear to be due to conformational changes. The results also suggest that hydrophobic residue(s) at position 86 may be important for the interaction of the molecule with its receptor. The competitive binding data correlated with the antiproliferative activity. The N-terminal region of the molecule and the hydrophobic residues (including Y and I) on the C-helix region at positions 86 and/or 90 are important for binding and antiproliferative activities of human IFN-alphas.
Asunto(s)
Sustitución de Aminoácidos , Inhibidores de Crecimiento/metabolismo , Inhibidores de Crecimiento/farmacología , Interferón-alfa/metabolismo , Interferón-alfa/farmacología , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Antivirales/farmacología , Unión Competitiva/genética , Bovinos , Línea Celular , Dicroismo Circular , Inhibidores de Crecimiento/genética , Humanos , Interferón-alfa/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Receptor de Interferón alfa y beta , Receptores de Interferón/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
Three human IFN-alpha hybrids, HY-1 [IFN-alpha21a(1-75)/alpha2c(76-165)], HY-2 [IFN-alpha21a(1-95)/alpha2c(96-165)], and HY-3 [IFN-alpha2c(1-95)/alpha21a(96-166)], were constructed, cloned, and expressed. The hybrids had comparable specific antiviral activities on Madin-Darby bovine kidney (MDBK) cells but exhibited very different antiproliferative and binding properties on human Daudi and WISH cells and primary human lymphocytes. Our data suggest that a portion of the N-terminal region of the molecule is important for interaction with components involved in binding of IFN-alpha2b while the C-terminal portion of IFN is critical for antiproliferative activity. A domain affecting the antiproliferative activity was found within the C-terminal region from amino acid residues 75-166. The signal transduction properties of HY-2 and HY-3 were evaluated by EMSA and RNase protection assays. Both HY-2 and HY-3 induced activation of STAT1 and 2. However, HY-2 exhibited essentially no antiproliferative effects at concentrations that activated STAT1 and 2. Additionally, at concentrations where no antiproliferative activity was seen, HY-2 induced a variety of IFN-responsive genes to the same degree as HY-3. RNase protection assays also indicate that, at concentrations where no antiproliferative activity was seen for HY-2, this construct retained the ability to induce a variety of IFN-inducible genes. These data suggest that the antiproliferative response may not be solely directed by the activation of the STAT1 and STAT2 pathway in the cells tested.
Asunto(s)
Interferón Tipo I/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/inmunología , Secuencia de Aminoácidos , Animales , Antivirales/farmacología , Bovinos , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Inhibidores de Crecimiento/farmacología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Interferón Tipo I/química , Interferón Tipo I/aislamiento & purificación , Interferón Tipo I/farmacología , Interferón-alfa , Datos de Secuencia Molecular , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/fisiología , Proteínas Recombinantes , Factor de Transcripción STAT1 , Factor de Transcripción STAT2 , Transducción de Señal/efectos de los fármacos , Transactivadores/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/inmunología , Células Tumorales CultivadasRESUMEN
The cDNA encoding the extracellular domain of the human interferon-alpha (IFN-alpha) receptor (Uzé, G., Lutfalla, G., and Gresser, I. Cell 1990;60:225-234) lacking the signal peptide has been expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The fusion protein represented 12% of total bacterial proteins and was found exclusively within cytoplasmic inclusion bodies. Inclusion body material was completely solubilized by 8 M urea; 20% solubilization was achieved by cell lysis in the presence of 0.45% cholamidopropyl dimethylammoniol-propane sulfonate and 1% Triton X-100. The soluble fusion protein was purified by gel filtration and affinity chromatography. Overall recovery of affinity purified fusion protein was approximately 100-200 micrograms/liter of cell culture. The affinity purified and refolded fusion protein exhibited the expected amino terminal sequence and M(r) of 68,000 on reduced sodium dodecylsulfate gel electrophoresis. The protein reacted with antibodies specific for the cloned IFN-alpha receptor and inhibited the antiviral and antiproliferative activities of recombinant IFN-alpha B. We have demonstrated that the fusion protein binds to IFN-alpha B and competes with the cell surface receptor for binding to this IFN-alpha species.
Asunto(s)
Glutatión Transferasa/genética , Receptores de Interferón/genética , Antivirales/farmacología , Unión Competitiva , División Celular/efectos de los fármacos , Línea Celular , Escherichia coli , Glutatión Transferasa/biosíntesis , Humanos , Interferón Tipo I/farmacología , Interferón-alfa , Pliegue de Proteína , Receptor de Interferón alfa y beta , Receptores de Interferón/biosíntesis , Receptores de Interferón/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes , SolubilidadRESUMEN
The colchicine analog 3-chloroacetyl-3-demthylthio-colchicine (3CTC) is a competitive inhibitor of colchicine binding to tubulin, binds to tubulin at 37 degrees C, but not at 0 degree C, and covalently reacts with beta-tubulin at 37 degree C, but not at 0 degree C, in a reaction inhibited by colchicine site drugs. The approximate intramolecular distance between the oxygen at position C-3 in 3CTC and the chlorine atom of the 3-chloroacetyl group is 3 A. using decylagarose chromatography, we purified beta-tubulin that had reacted with 3-(chloromethyl-[14C] Carbonyl)-3- demethylthiocolchicine ([14C]3CTC). This beta-tubulin that had reacted with 3-(chloromethyl-[14C]carbonyl)- 3-demethythiocolchicine ([14C]3CTC). This beta-tubulin was digested with formic acid, cyanogen bromide, endoproteinase Glu-C, or endoproteinase Lys-C, and the radio-labeled peptide(s) were isolated. The sequences of these peptides indicated that as much as 90% of the covalent reaction between the [14C]3CTC and beta-tubulin occurred at cysteine 354. This finding indicates that the C-3 oxygen atom of colchicinoids is within 3 A of the sulfur atom of the Cys-354 residue, suggests that the colchicine A ring lies between Cys-354 and Cys-239, based on the known 9 A distance between these residues, and may indicate that the tropolone C ring lies between the peptide region containing Cys-239 and the amino-terminal beta-tubulin sequence, based on the labeling pattern observed following direct photoactivation of tubulin-bound colchicine.
Asunto(s)
Colchicina/metabolismo , Cisteína/metabolismo , Tubulina (Proteína)/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Colchicina/análogos & derivados , Colchicina/antagonistas & inhibidores , Colchicina/farmacología , Bromuro de Cianógeno , Datos de Secuencia Molecular , Unión Proteica , Tubulina (Proteína)/químicaRESUMEN
Twenty-two components of human interferon-alpha (IFN-alpha) derived from Sendai virus-induced Namalwa cells were purified by sequential immunoadsorbent affinity chromatography using four monoclonal antibody affinity columns followed by ultrafiltration and reversed-phase high-performance liquid chromatography. The specific activity ranged from 0.2 to 2.6 x 10(8) IU/mg protein on Madin-Darby bovine kidney cells, 0.3 to 4.6 x 10(8) IU/mg protein on human WISH cells, and 10(4) to 7 x 10(5) units/mg protein on mouse L929 cells. The apparent molecular weights of the components ranged from 17,500 to 23,300 using nonreducing sodium dodecyl polyacrylamide-gel electrophoresis and 17,500 to 27,600 using reducing sodium dodecyl polyacrylamide-gel electrophoresis. The amino-terminal amino acid sequences were similar among the components as well as to those reported for the cloned human IFN-alpha genes (Pestka, S. (1986) Methods Enzymol. 119, 3-14). However, four components, f, i, l, and m, have amino-terminal amino acid sequences which appear to be unique when compared to those predicted from the cDNA clones. One component, pre-a, has a potential N-linked glycosylation site on the Asn of residues 2 through 4, Asn-Leu-Ser.
Asunto(s)
Interferón-alfa/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Bovinos , Células Cultivadas , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Humanos , Interferón-alfa/genética , Interferón-alfa/metabolismo , Ratones , Datos de Secuencia Molecular , Peso Molecular , Especificidad de la Especie , UltrafiltraciónRESUMEN
Human interferon-beta (IFN-beta) induces in human embryonic foreskin fibroblasts a cytoplasmic protein with antigenic similarities to mouse Mx protein, a nuclear protein implicated in inhibition of influenza virus replication. The human protein was purified to virtual homogeneity by immunoaffinity chromatography using a monoclonal antibody to mouse Mx protein. The purified protein has an apparent Mr of 78,000 and displays a strong tendency to self-aggregate. It can be resolved on two-dimensional gels into four spots with pIs between 6.0 and 6.2, each of which reacts with antibodies to mouse Mx protein. The partial amino-terminal sequence was determined for the affinity-purified protein. Cytoplasmic microinjection of the affinity-purified protein does not lead to efficient protection against infection with influenza virus. Cytoplasmic microinjection of the monoclonal Mx antibody, which increases suceptibility of IFN-treated mouse Mx cells to influenza virus, does not alter the viral susceptibility of IFN-treated human cells. These results suggest that, unlike the mouse Mx protein, the human Mx protein studied in this communication may not be sufficient to confer to cells a high degree of protection against influenza virus.
Asunto(s)
Proteínas de Unión al GTP , Proteínas/aislamiento & purificación , Secuencia de Aminoácidos , Anticuerpos/administración & dosificación , Antivirales/aislamiento & purificación , Antivirales/fisiología , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Humanos , Virus de la Influenza A/fisiología , Interferón Tipo I/farmacología , Microinyecciones , Datos de Secuencia Molecular , Peso Molecular , Proteínas de Resistencia a Mixovirus , Proteínas/antagonistas & inhibidores , Proteínas/fisiología , Replicación ViralRESUMEN
Some patients treated with interferon (IFN) have developed antibodies (ABs) to that IFN. We examined the incidence of such ABs in hairy cell leukemia (HCL) patients treated with IFN-alpha 2a and IFN-alpha 2b. In the initial enzyme-linked immunosorbent assay (ELISA) assays, the serum samples were tested against their corresponding IFNs. Of 73 evaluable patients treated with IFN-alpha 2b, 6 patients, who tested negative prior to treatment, were positive by ELISA following treatment with IFN. Two of the samples tested positive in the neutralization assay. Regarding the samples from patients treated with IFN-alpha 2a, 2 of the 44 patients were ELISA positive following IFN treatment (while being negative prior to treatment) and 2 others became positive in the neutralization assay following IFN treatment. Five serum samples with neutralizing activity were tested for their ability to bind to each of eight natural IFN-alpha species derived from human lymphoblastoid cells induced with Sendai virus; a different cross-reactivity profile was seen for each sample. In conclusion, a low incidence of neutralizing and nonneutralizing ABs against IFN-alpha 2 was observed in patients with HCL treated with recombinant DNA-derived IFN-alpha 2a or IFN-alpha 2b. Finally, ABs against these IFNs cross-react by ELISA with several naturally occurring IFN-alpha s.
Asunto(s)
Anticuerpos/inmunología , Interferón Tipo I/inmunología , Interferón-alfa/inmunología , Leucemia de Células Pilosas/inmunología , Anticuerpos/análisis , Humanos , Interferón alfa-2 , Interferón-alfa/uso terapéutico , Leucemia de Células Pilosas/sangre , Leucemia de Células Pilosas/terapia , Proteínas RecombinantesRESUMEN
Species lacking either 8 or 10 residues at the amino terminus of recombinant human interferon-gamma (Hu-IFN-gamma) were generated by limited digestion with Staphylococcus aureus V8 protease. A crude digest, consisting predominantly of these species, were completely inactive in inducing antiviral activity and the expression of HLA-DR antigens on HL-60 cells. The NH2-terminal deletion fragments were separated from residual intact IFN-gamma and from smaller polypeptides by reverse phase high performance liquid chromatography (HPLC) at pH 2.2. Intact IFN-gamma, purified by HPLC and subsequently refolded by dilution in 0.1 M sodium phosphate buffer (pH 7.5, 0.1% bovine serum albumin) was similar to untreated IFN-gamma in terms of binding to its cell surface receptor and in inducing antiviral activity and the expression of HLA-DR molecules. Conversely, biological activity was not detected in purified fragments 8-139 and 10-139. Examination of fragments 8-139 and 10-139 by far-UV circular dichroism revealed that cleavage of 8-10 residues at the amino terminus accompanied a dramatic change in secondary structure (6% alpha-helical and 36% beta-sheet content) as compared to untreated or HPLC-purified IFN-gamma (66% alpha-helix and 0% beta-sheet content). In summary, these results indicate that the amino terminus contributes to the structural integrity of the IFN-gamma molecule.
Asunto(s)
Interferón gamma/farmacología , Secuencia de Aminoácidos , Línea Celular , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Antígenos HLA-DR/biosíntesis , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/farmacología , Conformación Proteica , Desnaturalización Proteica , Proteínas Recombinantes , Serina Endopeptidasas , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/inmunologíaRESUMEN
After human IgG binds to antigen, it attains biological functions that are not properties of monomeric, uncomplexed IgG, including the ability to activate complement and to bind to cellular receptors. Associated with antigen binding, we have recently demonstrated that IgG itself has neoantigenic epitopes. Antibodies to these neoantigens on immune-complexed IgG may represent a significant proportion of circulating anti-human IgG in rabbits immunized with immune complexes. In contrast, mice immunized in an identical fashion have very little circulating anti-neoantigen antibody. This is true whether the mice are genetically easy to tolerize to monomeric human IgG (DBA/2 and C57Bl/6) or difficult to tolerize (BALB/c). Fusions were made between the NS-1 myeloma cell line and spleen cells from mice of each strain, which had been made tolerant to monomeric human IgG and then immunized with immune complexes containing IgG. Like the serum antibody, antibodies made by these fusions showed little specificity for immune complexes since 99% of the hybridoma antibodies that recognized IgG in immune complexes also bound to uncomplexed IgG. Only 1 hybridoma produced antibody that preferentially recognized human IgG in immune complexes. This antibody, called CE9, is an IgM that binds to IgG in plate-bound immune complexes with 100-1000-fold greater avidity than it does to plate-bound uncomplexed IgG. Because CE9 will not bind to immune complexes made with F(ab')2 antibody, the epitope it recognizes requires the Fc fragment of IgG. The minimal binding of CE9 to uncomplexed IgG is easily inhibited by soluble aggregates of IgG, but binding to immune complexes is not inhibited by aggregated IgG. CE9 does recognize fluid-phase immune complexes as well as solid-phase immune complexes. We conclude that, while mice produce much less anti-immune complex antibody than rabbits, anti-neoantigen is still a component of their response to immunization with immune complexes. Using hybridoma techniques to amplify these anti-neoantigen antibodies, we have shown that they resemble rheumatoid factors in their isotype and binding properties.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Inmunoglobulina G/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones EndogámicosRESUMEN
We raised rabbit antibodies that specifically recognize antigen-bound but not monomeric human IgG. These rabbit IgG antibodies (RAb) were induced in rabbits that were made tolerant to monomeric human IgG. They bound to immune complexes (IC) made with human IgG and various antigens including tetanus toxoid, sheep erythrocytes (E), rabbit E, or human Rh(D) + E, and were very poorly inhibitable with monomeric IgG compared to conventional rabbit anti-human IgG. RAb did not recognize complement components bound to the IgG containing IC. Cleavage of the Fc domain from human IgG markedly decreased binding of RAb to IC. Surprisingly, RAb did not bind to heat-aggregated human IgG (agg-IgG) better than to monomeric IgG. We conclude that human IgG expresses an Fc neoantigen when it binds its own antigen, and that this determinant is not expressed by agg-IgG. The implications of these findings for the biologic functions of antigen-complexed IgG are discussed.