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1.
J N Snouwaert K Kariya D M Fowlkes 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(2):585-591
To examine structure-activity relationships of human IL-6, we have determined the effects of specific mutations on the biologic activity of a human rIL-6 expressed in bacteria. Three types of mutants were examined: 1) a variant that contains serines in place of the four naturally occurring cysteines; 2) a series of cysteine-containing deletion mutants, each having a single internal 20 amino acid deletion; and 3) a cysteine-free variant containing a single 20 amino acid deletion. The mutants of the second type constitute a set of nonoverlapping, adjacent deletions spanning amino acids 4 through 183 of the 184 amino acids in natural human IL-6. All of the mutants were expressed, along with the full length, cysteine-containing analogue, in Escherichia coli as fusion proteins, joined to beta-galactosidase through a collagen linker. This system allows microgram quantities of the rIL-6 variants to be partially purified from small bacterial cultures without chromatographic or refolding steps. Each of the rIL-6 variants was released from the beta-galactosidase fusion protein with collagenase, and the recovered rIL-6 was quantitated by laser densitometry of Coomassie-stained, SDS polyacrylamide gels. The sp. ac. of each of the rIL-6 variants was determined using four assays: induction of IgM secretion from an EBV transformed human B cell line, induction of fibrinogen secretion from a human hepatoma cell line, induction of fibrinogen secretion from a rat hepatoma cell line, and induction of proliferation of a murine hybridoma cell line. Replacement of cysteines with serines reduced activity relative to cysteine-containing rIL-6 to about 20% in the rat hepatoma assay and about 3% in the mouse hybridoma assay, whereas activity in both of the human cell lines was reduced to less than 0.1%. These data suggest that the murine and rat cell lines are less selective than the human cell lines in their requirements for recognition of biologically active IL-6. Each of the deletions, except that of amino acids 4 through 23, resulted in loss of activity in all four assays. These results suggest that the information necessary for activity is not contained within any one portion of the IL-6 molecule, but rather that multiple segments of the protein are required for each of the biologic activities that we tested. 相似文献
2.
Woodchuck is an important animal model for studying human hepatitis B virus (HBV) infection. Within the cytokine network, interleukin-6 (IL-6) plays an important role in immune responses that may lead to viral clearance. To further understand woodchuck IL-6 biology, we cloned and characterized the IL-6 gene from white blood cells. The complete woodchuck IL-6 gene is about 7 kb and consists of five exons and four introns. The IL-6 gene organization of the woodchuck is similar to those of the human, rat, and mouse. Also several elements are highly conserved in the 300 bp promoter region of the IL-6 gene, including a nuclear factor kappa B (NF-kappaB) binding site. The woodchuck IL-6 gene encodes a polypeptide of 207 amino acids in a precursor form and 189 amino acids in the mature form. The expressed protein was 23 kDa according to SDS-PAGE. To demonstrate biologic activity, we expressed woodchuck IL-6 and showed that the purified recombinant protein induced terminal differentiation, as reflected by upregulation of Fcgamma receptor expression, and substantially inhibited proliferation of M1 cells, a murine myeloid leukemia cell line. The inhibitory effect of woodchuck IL-6 on M1 cells was blocked by an anti-gp130 monoclonal antibody, suggesting that woodchuck IL-6 activity is specifically mediated by signaling through the IL-6 receptor complex. Cloning of the woodchuck IL-6 gene and demonstrating biologic activity of the gene product will facilitate studies of human hepatitis B virus using the woodchuck model. 相似文献
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Structure-activity studies of human IL-1 beta with mature and truncated proteins expressed in Escherichia coli 总被引:2,自引:0,他引:2
J S Lillquist P L Simon M Summers Z Jonak P R Young 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(6):1975-1981
IL-1 beta is synthesized as an inactive 31-kDa intracellular protein, which is then processed upon secretion to an active 17-kDa carboxyl-terminal fragment. To identify the minimal portion of IL-1 beta required for activity, we constructed several deletion mutants of mature IL-1 beta. These included three amino-terminal deletions of 10, 16, and 81 amino acids, two carboxyl-terminal deletions of 17 and 72 amino acids, and one internal fragment between amino acids 17 and 81. Expression of the mutants was monitored by Western blots and immunoprecipitation. With one exception, all of these mutants and the full length 17-kDa IL-1 beta were expressed as soluble protein in Escherichia coli and could be assayed for activity and receptor binding in lysates without further purification. Whereas the intact 17-kDa IL-1 beta retained full biologic activity (greater than 10(7) U/ml of lysate) and competed for binding with 125I-labeled IL-1 beta, none of the lysates containing IL-1 beta deletion mutant proteins had activity or competed for binding to receptor at significantly higher concentrations. The loss of function in the smallest C-terminal deletion mutant does not appear to be due to the direct involvement of these C-terminal residues in receptor binding because both monoclonal and polyclonal antisera directed to this region bind to IL-1 beta but do not neutralize its activity. Therefore, this region is probably indirectly involved in sustaining the structure of the receptor-binding site. 相似文献
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Purification to homogeneity and NH2-terminal amino acid sequence of a novel interleukin 1 species derived from a human B cell line 总被引:8,自引:0,他引:8
L Rimsky H Wakasugi P Ferrara P Robin J Capdevielle T Tursz D Fradelizi J Bertoglio 《Journal of immunology (Baltimore, Md. : 1950)》1986,136(9):3304-3310
A subclone, referred to as 3B6, derived from a DR-negative EBV-transformed B cell line, has been found to spontaneously produce IL 1. 3B6-IL 1 displays a pI of 5 on FPLC chromatofocusing. It has been purified to homogeneity by a sequence of ion-exchange chromatography and affinity chromatography on procion red agarose. The homogeneous material migrated with an apparent m.w. of 13,500 on SDS-PAGE. The overall recovery of IL 1 activity was estimated at 57%. The final material had a specific activity of 7.8 X 10(6) half-maximal units/mg and represented a 50,000-fold purification. A partial NH2-terminal amino acid sequence has been obtained that is different from those reported from monocytic IL 1. However, this molecule can formally be identified as IL 1 on its spectrum of biologic activities. In addition to inducing the proliferation of murine thymocytes in the co-stimulator assay. 3B6-IL 1 is active on both human T and B cells, respectively, in inducing IL 2 synthesis by cells from a subcloned HSB 2 line and promoting the proliferation of anti-IgM-stimulated human peripheral blood B lymphocytes. Furthermore, 3B6-IL 1 acts as a growth factor for normal human fibroblasts and for the 3B6 line itself. However, 3B6-IL 1 is not pyrogenic in rabbits. Thus, the 3B6 cell line was shown to produce a new molecular species of IL 1, with respect to its NH2-terminal sequence, which shared all of the studied biologic activities of monocytic IL 1 except for pyrogenicity. 相似文献
6.
Interleukin-6 (IL-6) is a 185 amino acid residue helical cytokine with various biological activities (e. g. B cell development, acute phase reaction). We have investigated the role of the 168-185 C-terminal region of IL-6 in the induction of fibrinogen synthesis and expression of junB mRNA using synthetic peptides corresponding to this region. Circular dichroism spectroscopy data suggest that even truncated peptides have a strong tendency to adopt an ordered conformation. Peptides were tested alone or in combination with recombinant hIL-6 on an IL-6 responsive human hepatoma HepG2 cell line. The expression of the protooncogene junB monitored by competitive RT-PCR represents an early, while the fibrinogen production detected by sandwich ELISA a late, marker of IL-6 initiated events. We found that peptides--depending on their structure--modulate spontaneous as well as IL-6 induced fibrinogen production and/or mRNA expression of junB by exhibiting inhibition (in the presence of IL-6) or stimulation (in the absence of IL-6). 相似文献
7.
We have examined the functional importance of the two disulfide bonds formed by the four conserved cysteines of human interleukin (IL-6). Using a bacterial expression system, we have synthesized a series of recombinant IL-6 mutants in which the constituent cysteines of the first (Cys45-Cys51), second (Cys74-Cys84), or both disulfide bonds of recombinant human interleukin-6 were replaced by other amino acids. Each mutant was partially purified and tested in four representative bioassays. While mutants lacking Cys45 and Cys51 retained activity similar to nonmutated recombinant IL-6, the activity of mutants lacking Cys74 and Cys84 was significantly reduced, especially in assays involving human cell lines. These results indicate that the first disulfide bond of human interleukin-6 is not required for maintenance of normal biologic activity. However, the fact that mutants lacking Cys45 and Cys51 were more active than corresponding cysteine-free mutants indicates that the disulfide bond formed by these residues contributes to biologic activity in the absence of the second disulfide bond. Competition binding studies with representative mutants indicate that their affinity for the human IL-6 receptor parallels their biologic activities on human cells. 相似文献
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J P Brakenhoff E R de Groot R F Evers H Pannekoek L A Aarden 《Journal of immunology (Baltimore, Md. : 1950)》1987,139(12):4116-4121
Human monocytes produce a factor that supports the growth of B lymphocyte hybridoma cells, termed hybridoma growth factor (HGF). By using expression cloning in Escherichia coli of complementary DNA derived from human monocyte-poly(A+) RNA, we selected seven clones producing HGF activity as measured in a bioassay, based on the induction of proliferation of the HGF-dependent B cell hybridoma B9. Sequence analysis of the cDNA revealed that HGF is identical with interferon-beta 2, 26,000 protein, and B cell stimulatory factor-2. One of the active clones contained a cDNA that encoded a recombinant product lacking the 28-amino acid long signal peptide and the first 15 amino acids of the mature protein. Antibodies against the recombinant HGF inhibited the biologic activity of recombinant HGF as well as of monocyte-derived HGF. 相似文献
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M K Ganapathi D Rzewnicki D Samols S L Jiang I Kushner 《Journal of immunology (Baltimore, Md. : 1950)》1991,147(4):1261-1265
We have previously shown that induction of synthesis of the two major human acute phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP), can be accomplished in the human hepatoma cell line Hep 3B, in the presence of dexamethasone, either by conditioned medium from LPS-stimulated monocytes or by the combination of IL-6 and IL-1. Neither of these cytokines alone caused significant induction of either SAA or CRP. In the present study we extended our earlier observations by evaluating the role of dexamethasone, the effect of different concentrations of IL-6 and IL-1 alpha in combination, and the possible role of TNF-alpha in regulating synthesis of SAA and CRP. Dexamethasone alone had no effect on induction of SAA or CRP. Incubation of Hep 3B cells with conditioned medium from LPS-stimulated monocytes, in the absence of dexamethasone, led to modest induction of SAA or CRP, but addition of dexamethasone potentiated this response in a dose-dependent manner. Similar results were obtained for the effect of dexamethasone on the induction of SAA by IL-6 plus IL-1 alpha. Checkerboard titration of IL-6 and IL-1 alpha revealed that increases in concentration of either cytokine led to dose-related increases in synthesis of both SAA and CRP as long as a minimal amount of the other cytokine was present. TNF-alpha alone had no significant effect on synthesis of either SAA or CRP, but the combination of IL-6 plus TNF-alpha led to substantial induction of SAA. This combination was less effective than the combination of IL-6 plus IL-1 alpha. No detectable effect of IL-6 plus TNF-alpha was observed on CRP synthesis. Both combinations of cytokines, IL-6 plus IL-1 alpha, and IL-6 plus TNF-alpha, caused increased SAA mRNA accumulation that roughly paralleled increase in synthesis. These data indicate that IL-6, IL-1 alpha, TNF-alpha, and dexamethasone in various combinations are all capable of influencing synthesis of SAA in Hep 3B cells, whereas only IL-6, IL-1 alpha, and dexamethasone can influence CRP synthesis. 相似文献
11.
P T Van Hal J P Hopstaken-Broos J M Wijkhuijs A A Te Velde C G Figdor H C Hoogsteden 《Journal of immunology (Baltimore, Md. : 1950)》1992,149(4):1395-1401
IL-4 has multiple biologic activities and it has been shown to have effects on B and T lymphocytes, mast cells, NK cells, and monocytes. We studied the influence of IL-4 on the expression of cell membrane determinants, in particular aminopeptidase-N (CD13) and Fc epsilon RIIb (CD23), on human peripheral blood monocytes. We compared the response of monocytes with the response of human alveolar macrophages and monocytic cell lines (U937 and THP1), as mature and more immature representatives of the mononuclear phagocyte system, respectively. A dose-dependent increase of the expression of CD13 Ag was observed when monocytes were cultured with IL-4. Kinetic analyses revealed that this induction was maximal after 2 to 3 days of culture and resembled the kinetics of IL-4-induced expression of Fc epsilon RIIb on monocytes. This IL-4-induced increase was absent when monocytes were cultured with IL-4 and an anti-IL-4 antiserum. Concomitantly, an IL-4-induced increase in leucine-aminopeptidase activity could be observed. Northern blot analysis showed that incubation of monocytes with IL-4 induced a marked increase in CD13 mRNA. Alveolar macrophages also exhibited an increase in CD13 Ag expression when exposed to IL-4. Surprisingly, IL-4 was unable to induce expression of Fc epsilon RIIb on alveolar macrophages. U937 and THP1 cells did not show an induction of CD13 Ag when cultured in the presence of IL-4. However, IL-4 did induce the expression of Fc epsilon RIIb on both cell lines, suggesting the presence of functional IL-4R. Our data demonstrate that IL-4 increases the expression of CD13 Ag on monocytes. This IL-4-induced increase can also be observed in more mature monocytic cells such as alveolar macrophages, but is absent in immature cells such as U937 or THP1 cells. This is functionally accompanied by an increase in leucine-aminopeptidase activity and may be part of the general activation of monocytes/macrophages by IL-4. In conclusion, the data suggest that IL-4 responsiveness, in particular the induction of CD13 Ag and Fc epsilon RIIb expression, may be dependent on the stage of maturation of monocytes/macrophages. 相似文献
12.
IL-6 has multiple biologic activities in different cell systems including both the ability to support cell proliferation and to induce differentiation. We reported previously the isolation and functional expression of a mouse IL-6 (mIL-6) cDNA clone derived from bone marrow stromal cells. In this paper, we show that mIL-6 is a potent inducer of terminal macrophage differentiation for a mouse myeloid leukemic cell line, M1. Addition of mIL-6 to cultures of M1 cells rapidly inhibits their proliferation and induces phagocytic activity and morphologic changes characteristic of mature macrophages. These phenotypic changes are accompanied at the molecular level by a decrease in proto-oncogene c-myc mRNA accumulation and increases in Fc gamma R, proto-oncogenes c-fos and c-fms (CSF-1R) mRNA expression. Furthermore, IL-6 enhances the expression of Fc gamma R and c-fms in differentiation-responsive D+, but not unresponsive D- sublines of mouse myelomonocytic leukemic WEHI-3B cells. Together with our previous observation that IL-6 stimulates colony formation by normal myeloid progenitors, these results strongly suggest an important regulatory role for IL-6 in myeloid cell growth and differentiation. 相似文献
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When amino acids or ammonia are added to plant systems, the effects on the development of nitrate-dependent nitrate reductase activity are variable. In addition, amino acids added singly or as casein hydrolysate may not support a normal growth. A physiologically correct mixture of amino acids, one similar in composition to amino acids released by the endosperm, has been shown to support normal growth and protein synthesis in corn (Zea mays) embryos. In this investigation, we have used the mixture of corn amino acids to determine whether amino acids have an effect on the appearance or disappearance of nitrate reductase activity. The results show that these amino acids partially inhibit the induction of nitrate reductase in corn roots. The effect is more pronounced in mature root than in root tip sections. When glutamine and asparagine are included along with the "corn amino acid mixture," the inhibition is more severe. Amino acids or amino acid analogues added singly to the induction medium have a similar effect: i.e. when the induction of nitrate reductase is inhibited in the root tips (lysine, canavanine, azaserine, azetidine-2-carboxylic acid, dl-4-azaleucine, asparagine, and glutamine), that inhibition is more severe in mature root sections. Arginine enhanced the recovery of nitrate reductase in root tips but inhibited it in mature root sections. The effect of the amino acids is apparently on some phase of the induction processes (i.e. the uptake or distribution of nitrate or a direct effect on the synthesis of the enzyme) and not on the turnover of the enzyme. 相似文献
14.
《Bioscience, biotechnology, and biochemistry》2013,77(11):1858-1861
We constructed an efficient system for synthesis and secretion of human interleukin-2 (IL-2) by Bacillus brevis. The secretion vector we constructed had strong promoters and contained the region coding for the signal peptide of the gene for B. brevis 47 cell-wall protein, followed directly by the gene encoding mature IL-2. Modification of the signal peptide and use of a protease-deficient mutant of B. brevis HPD31 increased productivity. When the signal peptide was more basic near its amino terminal and more hydrophobic in the middle region, IL-2 production increased 20 fold. Production by the mutant harboring the secretion vector was four fold that of the parent harboring the same plasmid. The yield of IL-2 increased further to 0.12 g/liter, when cultural conditions were made optimal, such by the addition of Tween 40 to the medium. The IL-2 produced by B. brevis had the same biological activity as authentic IL-2. Biologically active human IL-2 was produced efficiently and secreted directly into the medium by B. brevis. 相似文献
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S Takaki A Tominaga Y Hitoshi S Mita E Sonoda N Yamaguchi K Takatsu 《The EMBO journal》1990,9(13):4367-4374
Murine interleukin-5 (IL-5) is known to play an essential role in Ig production of B cells and proliferation and differentiation of eosinophils. Here, we have isolated cDNA clones encoding a murine IL-5 receptor by expression screening of a library prepared from a murine IL-5 dependent early B cell line. A cDNA library was expressed in COS7 cells and screened by panning with the use of anti-IL-5 receptor monoclonal antibodies. The deduced amino acid sequence analysis demonstrates that the receptor is a glycoprotein of 415 amino acids (Mr 45,284), including an N-terminal hydrophobic region (17 amino acids), a glycosylated extracellular domain (322 amino acids), a single transmembrane segment (22 amino acids) and a cytoplasmic tail (54 amino acids). COS7 cells transfected with the cDNA expressed a 60 kd protein that bound IL-5 with a single class of affinity (KD = 2-10 nM). FDC-P1 cells transfected with the cDNA for murine IL-5 receptor showed the expression of IL-5 binding sites with both low (KD = 6 nM) and high affinity (KD = 30 pM) and acquired responsiveness to IL-5 for proliferation, although parental FDC-P1 cells did not show any detectable IL-5 binding. In addition, several cDNA clones encoding soluble forms of the IL-5 receptor were isolated. Northern blot analysis showed that two species of mRNAs (5.0 kb and 5.8 kb) were detected in cell lines that display binding sites for murine IL-5. Homology search for the amino acid sequence of the IL-5 receptor reveals that the IL-5 receptor contains a common motif of a cytokine receptor family that is recently identified. 相似文献
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The processing of interleukin-1 beta studied with antibodies raised against synthetic peptides from the precursor N-terminal region 总被引:1,自引:0,他引:1
The exact sequence of events during processing of human interleukin-1 beta (IL-1 beta) and the fate of the N-terminal region are unknown. We have used anti-peptide sera specific for the precursor and mature regions of IL-1 beta to study biosynthesis. These were raised against peptides corresponding to amino acids 1-15, 17-32, and 43-54 of the precursor and a peptide corresponding to the C-terminal 33 amino acids of mature human IL-1 beta. Antiserum to the mature region peptide immunoprecipitated the 35-kD precursor from cell lysates and 17-kD mature IL-1 beta and a 31-kD protein from the culture supernatants from radiolabeled human peripheral blood monocytes stimulated with lipopolysaccharide (LPS). Antisera to peptides from the precursor region also immunoprecipitated the 35-kD IL-1 beta precursor but not the 31-kD or 17-kD forms. Of the precursor-specific sera, only antiserum to amino acids 1-15 specifically recognized any other proteins; a peptide of 18 kD and a low molecular weight peptide, both of which accumulated in the medium. The 18-kD protein was not recognized by any of the other antisera and is unlikely to be the N-terminal region of the precursor removed during processing. Pulse-chase experiments indicated that the 31-kD protein could be a processing intermediate and also that it was itself an end product along with full-length precursor. Only 17-kD mature IL-1 beta had biological activity. 相似文献
17.
Rabbit IL-1. Cloning, expression, biologic properties, and transcription during endotoxemia 总被引:15,自引:0,他引:15
J G Cannon B D Clark P Wingfield U Schmeissner C Losberger C A Dinarello A R Shaw 《Journal of immunology (Baltimore, Md. : 1950)》1989,142(7):2299-2306
The cloning, sequencing, expression, and biologic activities of rabbit IL-1 alpha and beta are described. A cDNA library was constructed in lambda gt10 by using polyadenylated RNA extracted from rabbit adherent splenic macrophages 4 h after stimulation with endotoxin. By using the cDNA for human IL-1 beta and IL-1 alpha as hybridization probes, cDNA for both forms of rabbit IL-1 were isolated. The cDNA for rabbit IL-1 beta encodes a precursor polypeptide of 268 amino acids with an overall homology to human IL-1 beta of 74% (81% in the mature region coding for a 17.5 kDa carboxyl-terminal protein). The similarity between the two rabbit IL-1 forms is 31% for the entire molecule and 34% for the mature protein. The mature polypeptides of both forms were expressed in Escherichia coli. The recombinant proteins were purified to homogeneity and tested in a variety of biologic assays. Both forms produced typical endogenous pyrogen fevers in rabbits and augmented murine thymocyte and Th cell proliferation. Rabbit IL-1 alpha and beta were more pyrogenic in rabbits than human rIL-1 beta, whereas human rIL-1 alpha and beta were slightly more potent lymphocyte-activating factors. The recombinant rabbit proteins induced PGE and IL-1 production from human PBMC in vitro. A RIA for human IL-1 alpha did not recognize rabbit IL-1 alpha or beta, but rabbit IL-1 beta cross-reacted (as much as 30%) in a RIA for human IL-1 beta. Rabbits were injected with endotoxin and mRNA for both forms of IL-1 were observed primarily in the spleen and liver. The mRNA reached maximal levels after 60 min, then declined rapidly over the next 3 h, but were still present after 24 h. Liver tissue removed 4 h after endotoxin infusion produced lymphocyte-activating factors which were neutralized by more than 90% with a combination of goat anti-rabbit IL-1 alpha and anti-IL-1 beta. 相似文献
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The mechanism by which interleukin-1 alpha (IL-1 alpha) activates NF-kappa B DNA-binding activity is not completely understood. While it is well established that protein kinase C can activate NF-kappa B, neither protein kinase C nor protein kinase A appears to be critical in the induction of NF-kappa B by IL-1 alpha. Since a number of growth factors signal via protein tyrosine kinase, in this study we examined a possible involvement of protein tyrosine kinase in the IL-1 alpha-induced NF-kappa B. The results showed that in the murine pre-B cell line 70Z/3 and in the murine T cell line EL-4 6.1 C10 IL-1 alpha-induced NF-kappa B was associated with transient increase in protein tyrosine kinase activity. Pre-treatment of these cell lines with herbimycin A, an inhibitor of tyrosine kinase activity, blocked the IL-1 alpha-enhanced protein tyrosine kinase activity and the IL-1 alpha-induced NF-kappa B DNA-binding activity. Herbimycin A at concentrations sufficient to block IL-1 alpha-induced NF-kappa B did not block the phorbol 12-myristate 13-acetate (PMA)-induced NF-kappa B. The data suggest that IL-1 alpha and PMA activate NF-kappa B by different pathways and that induction of NF-kappa B DNA-binding activity by IL-1 might be dependent on protein tyrosine phosphorylation. 相似文献