In vivo neutralization of human IL-6 (hIL-6) achieved by immunization of hIL-6-transgenic mice with a hIL-6 receptor antagonist

Neutralization of IL-6 represents an attractive therapeutic option in several diseases, including B cell neoplasia, osteoporosis, and autoimmunity. Therapeutic attempts in humans have shown that administration of injectable doses of a mAb to IL-6 does not provide efficient neutralization of the cytokine in vivo. Therefore, alternative approaches are needed. In this study, we evaluated whether the Ab response to human IL-6 (hIL-6) elicited by vaccination with Sant1 (a hIL-6 variant with seven amino acid substitutions) was able to fully correct in vivo the clinical and biological effects of a chronic endogenous overproduction of hIL-6 in the hIL-6-transgenic NSE/hIL-6 mice. Because of the overexpression of hIL-6, occurring since birth, with circulating levels in the nanogram per milliliter range, NSE/hIL-6 mice have a marked decrease in growth rate, associated with decrease in insulin-like growth factor I levels, and represent an animal model of the growth impairment associated with human chronic inflammatory diseases. Following immunization with Sant1, but not with hIL-6, NSE/hIL-6 mice developed high titers of polyclonal Abs to hIL-6. The Abs, acquired by transplacental transfer, effectively neutralized IL-6 activities in vivo as shown by the complete correction of the growth defect and normalization of insulin-like growth factor levels in the hIL-6-transgenic offspring. Immunization with Sant1 could therefore represent a novel and simple therapeutic approach for the specific neutralization of IL-6 in humans.     

Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

IL-32 is a newly described cytokine in the human found to be an in vitro inducer of tumor necrosis factor alpha (TNFalpha). We examined the in vivo relationship between IL-32 and TNFalpha, and the pathologic role of IL-32 in the TNFalpha-related diseases - arthritis and colitis. We demonstrated by quantitative PCR assay that IL-32 mRNA was expressed in the lymphoid tissues, and in stimulated peripheral T cells, monocytes, and B cells. Activated T cells were important for IL-32 mRNA expression in monocytes and B cells. Interestingly, TNFalpha reciprocally induced IL-32 mRNA expression in T cells, monocyte-derived dendritic cells, and synovial fibroblasts. Moreover, IL-32 mRNA expression was prominent in the synovial tissues of rheumatoid arthritis patients, especially in synovial-infiltrated lymphocytes by in situ hybridization. To examine the in vivo relationship of IL-32 and TNFalpha, we prepared an overexpression model mouse of human IL-32beta (BM-hIL-32) by bone marrow transplantation. Splenocytes of BM-hIL-32 mice showed increased expression and secretion of TNFalpha, IL-1beta, and IL-6 especially in response to lipopolysaccharide stimulation. Moreover, serum TNFalpha concentration showed a clear increase in BM-hIL-32 mice. Cell-sorting analysis of splenocytes showed that the expression of TNFalpha was increased in resting F4/80+ macrophages, and the expression of TNFalpha, IL-1beta and IL-6 was increased in lipopolysaccharide-stimulated F4/80+ macrophages and CD11c+ dendritic cells. In fact, BM-hIL-32 mice showed exacerbation of collagen-antibody-induced arthritis and trinitrobenzen sulfonic acid-induced colitis. In addition, the transfer of hIL-32beta-producing CD4+ T cells significantly exacerbated collagen-induced arthritis, and a TNFalpha blockade cancelled the exacerbating effects of hIL-32beta. We therefore conclude that IL-32 is closely associated with TNFalpha, and contributes to the exacerbation of TNFalpha-related inflammatory arthritis and colitis.     

Human recombinant IL-10 reduces xenogenic cytotoxicity via macrophage M2 polarization

Xenotransplantation has been considered an alternative to the moderate shortage of donor organs for transplantation. To achieve successful xenotransplatation, there is the need to overcome immune rejection. Although, hyperacute rejection has been overcome by α1,3-galactosyltransferase knockout pig, cellular immune rejection remains as a subsequent barrier. Interleukin-10 (IL-10) is known as an anti-inflammatory and immunomodulatory cytokine which has been shown to limit inflammatory responses by inhibiting macrophage activation in several animal experiments. To study the effect of human IL-10 (hIL-10) on pig-to-human xenotransplantation, porcine kidney epithelial cell line (PK(15)) expressing hIL-10 was established. The cytotoxicity of macrophages decreased by hIL-10 from transgenic cells. Furthermore, there is a decreased production of pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-23, and increased anti-inflammatory cytokines like IL-10, but not transforming growth factor beta, in the presence of hIL-10. Also, macrophage polarization toward M2-like phenotype were induced by hIL-10 from transgenic PK(15) cells. Finally, we suggest that the cytotoxicity of human macrophages was reduced by hIL-10 from transgenic cells, inducing M2-like macrophage polarization. Therefore, these results show that hIL-10 transgenic pig can be used as a model to overcome acute immune rejection in pig-to-human xenotransplantation.     

Prediction on the binding domain between human interleukin-6 and its receptor

Based on the spatial conformations of human interleukin-6 (hIL-6) derived from nuclear magnetic resonance analysis and human interleukin-6 receptor (hIL-6R) modeled with homology modeling method using human growth hormone receptor as template, the interaction between hIL-6 and its receptor (hIL-6R) is studied with docking program according to the surface electrostatic potential analysis and spatial conformation complement. The stable region structure composed of hIL-6 and hIL-6R is obtained on the basis of molecular mechanism optimization and molecular dynamics simulation. The binding domain between hIL-6 and hIL-6R is predicted theoretically. Furthermore, the especial binding sites that influence the interaction between hIL-6 and hIL-6R are confirmed. The results lay a theoretical foundation for confirming the active regions of hIL-6 and designing novel antagonist with computer-guided techniques.     

hIL-6与其受体作用的结构基础及拮抗剂的研究进展

通过对大量的分子生物学实验及晶体衍射结果的分析 ,从分子水平揭示人白细胞介素 6(hIL 6 )与其受体相互作用的结构模式及结合表位 .hIL 6属于促红细胞生成素受体超家族 ,首先和hIL 6受体α低亲和力结合 ,两者形成的复合物再与hIL 6受体 β(gp130 )的胞外区相互作用形成高亲和力三聚体 ,但是hIL 6不能单独和gp130结合 ,需要借助于hIL 6受体α的桥梁作用才能将二者联系起来进而促进六聚体的形成 .hIL 6是一种能够介导细胞表面信号转导 ,调节机体免疫及造血干细胞增殖和分化的细胞因子 ,许多疾病的发病机理及发展进程都和hIL 6过表达有关 .基于对hIL 6与其受体相互作用方式的探究 ,为hIL 6小分子拮抗剂的药物设计提供了理论模型 ,在此基础上已研究开发了许多不同种类的hIL 6新型分子拮抗剂 ,其中部分拮抗剂已应用于临床指导 .     

Viral Interleukin-6: Structure, pathophysiology and strategies of neutralization

Viral Interleukin-6 (vIL-6) is encoded by Human herpes virus 8 (HHV8), also known as Kaposi's sarcoma (KS)-associated herpes virus (KSHV). HHV8 infection is found in patients with KS, primary effusion lymphoma (PEL) and plasma cell-type of multicentric Castleman's disease (MCD), with a high incidence observed in HIV infected individuals. vIL-6 shares about 25% identity with its human counterpart. Human IL-6 (hIL-6) binds to the human IL-6 receptor (hIL-6R) and the hIL-6/hIL-6R complex associates with the signaling receptor subunit gp130. Upon dimerization of gp130 intracellular signaling is initiated. All cells in the body express gp130 but only some cell types express the hIL-6R. Human IL-6 does not stimulate cells, which do not express hIL-6R. However, a naturally occurring soluble form of the hIL-6R (shIL-6R) can bind hIL-6 and the complex of hIL-6/shIL-6R can stimulate cells, which only express gp130 but no hIL-6R. This process, which has been named trans-signaling, leads to a dramatic increase in the spectrum of hIL-6 target cells during inflammation and cancer. vIL-6, in contrast to hIL-6, can directly bind to and activate gp130 without the need of the hIL-6R. Therefore, at least in theory, vIL-6 can stimulate every cell in the human body. This review highlights the properties of vIL-6 regarding structural features, implications for pathophysiology, and strategies of neutralization. Furthermore, mechanisms of activation of gp130 by hIL-6, vIL-6, and by forced dimerization will be discussed.     

Effect of in situ expression of human interleukin-6 on antibody responses against Salmonella typhimurium antigens

In an attempt to trigger increased mucosal secretory immune responses against bacterial surface antigens, we constructed an optimized human interleukin (hIL)-6-secreting Salmonella typhimurium strain (X4064(pCH1A+pYL3E)), utilizing the hemolysin (Hly) exporter for secretory delivery of a functional hIL-6-hemolysin fusion protein (hIL-6-HlyA(s)). Through stable introduction of a second hIL-6-HlyA(s) expression plasmid (pYL3E) in the previously described X4064(pCH1A) strain, hIL-6-HlyA(s) secretion efficiencies were increased by at least 10-fold. As pCH1A in the parental strain, pYL3E was stable in vitro in the absence of antibiotic selection and in vivo neither did plasmids interfere in their stabilities. Increased hIL-6-HlyA(s) expression did not adversely interfere with bacterial growth. Comparative immunization experiments in mice with oral application of the different hIL-6-secreting strains revealed that increased in situ hIL-6-production influenced systemic antibody responses against Salmonella antigens but had no marked effect on mucosal responses. In mice immunized with X4064(pCH1A+pYL3E) significantly higher sera IgG and IgA titers for lipopolysaccharide (LPS) were found compared to mice immunized with X4064(pCH1A) and a hIL-6-negative control strain. Higher sera antibody titers were accompanied by increased numbers of IgG- and IgA-specific antibody-secreting cells in spleens and Peyer's patches, respectively. These data suggest that systemic antibody responses against Salmonella LPS are largely effected by IL-6 and, moreover, the amount and the cellular location of recombinantly expressed IL-6 appears to be crucial for enhancement of immune responses.     

The design of antagonist peptide of hIL-6 based on the binding epitope of hIL-6 by computer-aided molecular modeling

The interaction between human interleukin-6 (hIL-6) and human interleukin-6 receptor (hIL-6R) is the initial and most specific step in the hIL-6 signaling pathway. Understanding its binding core and interaction mechanism at amino acid level is the basis for designing small IL-6 inhibiting molecules, such as peptides or lead compounds. With Docking method, the complex structure composed of hIL-6 and its alpha-subunit receptor (hIL-6R) was analyzed theoretically. By using structure-based analysis and phage display methods, the loop AB (from Lys67 to Glu81) of hIL-6 was found to be the important binding epitope of hIL-6R. By means of computer-aided design, the mimic antagonist peptide (14 residues) was designed and synthesized. Using multiple myeloma cell line (XG7), IL-6 dependent cell line, as test model, the influence of antagonist peptides on the proliferation of XG7 cells was investigated. The results showed that the synthetic peptide could be competitive to bind to hIL-6R with hIL-6, and the effect was concentration dependent. The theoretical design approach is a powerful alternative to phage peptide library for protein mimics. Such mini-peptide is more amenable to synthetic chemistry and thus may be useful starting points for the design of small organic mimics.     

Identification of amino acid residues of gp130 signal transducer and gp80 alpha receptor subunit that are involved in ligand binding and signaling by human herpesvirus 8-encoded interleukin-6

Human herpesvirus 8-encoded interleukin-6 (vIL-6) signals through the gp130 signal transducer but is not dependent on the IL-6 receptor alpha subunit (IL-6R, gp80) that is required for signaling by endogenous IL-6 proteins; however, IL-6R can enhance vIL-6 activity and can enable signaling through a gp130 variant, gp130.PM5, that is itself unable to support vIL-6 signaling. These findings suggest that the vIL-6-gp130 interactions are qualitatively different from those of human IL-6 (hIL-6) and that vIL-6 signaling may be more promiscuous than that of hIL-6 but that IL-6R may play a role in vIL-6 signaling in vivo. To examine the receptor binding requirements of vIL-6, we have undertaken mutational analyses of regions of gp130 and IL-6R potentially involved in interactions with ligand or in functional complex formation and used these variants in functional, ligand-binding, and receptor dimerization assays. The data presented identify positions within two interstrand loops of the gp130 cytokine-receptor homology domain that are important for vIL-6 signaling and vIL-6-induced receptor dimerization and show that vIL-6, like hIL-6, can form complexes with IL-6R and gp130 but that the roles of putative cytokine-binding residues of IL-6R in ligand-induced functional complex formation are qualitatively different in the case of vIL-6 and hIL-6.     

Cloning and hemolysin-mediated secretory expression of a codon-optimized synthetic human interleukin-6 gene in Escherichia coli

Previously, we constructed human interleukin-6 (hIL-6)-secreting Escherichia coli and Salmonella typhimurium strains by fusion of the hIL-6 cDNA to the HlyA(s) secretional signal, utilizing the hemolysin export apparatus for extracellular delivery of a bioactive hIL-6-hemolysin (hIL-6-HlyA(s)) fusion protein. Molecular analysis of the secretion process revealed that low secretion levels were due to inefficient gene expression. To adapt the codon usage in hIL-6 cDNA to the E. coli codon bias, a synthetic hIL-6Ec gene variant was constructed from 20 overlapping oligonucleotides, yielding a 561-bp fragment, which comprises the complete hIL-6 cDNA sequence. Genetic fusion of the hIL-6Ec gene with the hlyA(s) secretional signal as an integral part of the hemolysin operon resulted in 3-fold higher hIL-6-HlyA(s) secretion levels in E. coli, compared to a strain expressing the original hIL-6-hlyA(s) fusion gene. An increase in the electrophoretic mobility of secreted hIL-6-HlyA(s) in non-reducing SDS-PAGE, similar to that found for recombinant mature hIL-6, and the absence of such a mobility shift in the intracellular hIL-6-HlyA(s) protein fraction indicated that in hIL-6-HlyA(s) most probably correct intramolecular disulfide bond formation occurred during the secretion step. To confirm the disulfide bond formation, hIL-6-HlyA(s) was purified by a single-step immunoaffinity chromatography from culture supernatant in yields of 18 microg/L culture supernatant with purity in the range of 60%. These results demonstrate that codon usage has an impact on the hemolysin-mediated secretion of hIL-6 and, furthermore, provide evidence that the hemolysin system enables secretory delivery of disulfide-bridged proteins.     

Molecular analysis of hemolysin-mediated secretion of a human interleukin-6 fusion protein in Salmonella typhimurium

Previously, we reported a plasmid-bearing Salmonella typhimurium strain capable of secreting human interleukin-6 (hIL-6) when genetically fused to the Escherichia coli hemolysin transport signal (HlyA(S)). Stationary phase culture supernatants of this strain revealed three major forms of hIL-6-HlyA(S) fusion protein (apparent molecular masses 32.4, 30.3, 27.0 kDa), at which the largest protein presumably represented full-length hIL-6-HlyA(S). The biological activity of the hIL-6-HlyA(S) protein mixture was similar to that of mature hIL-6. Accumulation of hIL-6-HlyA(S) in the culture supernatant occurred only during the initial growth phase, whereas in stationary phase and under in vitro conditions successive cleavage into the two truncated forms was observed. On the other hand, in whole cell lysates only full-length hIL-6-HlyA(S) could be detected, accounting for more than 50% of the totally synthesized protein. Upon cell fractionation, cellular hIL-6-HlyA(S) was exclusively found in the membrane fraction. These results suggest, that in S. typhimurium production and secretion of hIL-6-HlyA(S) is restricted to growing cells. A specific processing by a Salmonella-derived protease did not affect the biological activity of the fusion protein.     

Production of soluble human interleukin-6 in cytoplasm by fed-batch culture of recombinant E. coli

The major objective of this study is to identify fed-batch culture conditions optimal for the production of human interleukin-6 (hIL-6) in a soluble form. Five different expression vectors were constructed for the expression of hIL-6 and hIL-6s fused with NusA, maltose binding protein (MBP), thioredoxin (Trx) or ubiquitin (Ubi). A series of flask cultures were conducted in LB medium at 37 degrees C. The intact hIL-6 was expressed mostly in the form of inclusion body. More than 95% of the hIL-6 fused with NusA (NusA/hIL-6) and about 90% of MBP/hIL-6 were expressed in a soluble form, whereas Trx/hIL-6 and Ubi/hIL-6 were expressed mostly in the form of inclusion body. Based on this result, NusA was selected as the fusion partner for the production of hIL-6 in the subsequent experiments. A series of pH-stat fed-batch cultures of an E. coli BL21(DE3) transformed with a NusA/hIL-6 expression vector were conducted in a bioreactor with a working volume of about 3 L. As the amount of nitrogen source was increased in the feeding medium, more soluble NusA/hIL-6 was produced, while the total amount was not significantly changed. Under the best conditions tested, about 90% of NusA/hIL-6 was produced in the soluble form. In this case, the concentration of soluble NusA/hIL-6 was 7.5 g/L with a volumetric productivity of 0.43 g/L-h.     

Altered expression of inflammatory cytokine receptors in response to LPS challenge through interaction between intestinal epithelial cells and lymphocytes of Peyer's patch

The intense innate immunological activities occurring at the enteric mucosal surface involve interactions between intestinal epithelial cells and immune cells. Our previous studies have indicated that Peyer's patch lymphocytes may modulate intestinal epithelial barrier and ion transport function in homeostasis and host defense via cell-cell contact as well as cytokine signaling. The present study was undertaken using the established co-culture system of Caco-2 epithelial cells with lymphocytes of Peyer's patch to investigate the expression of IL-8 and IL-6 cytokines and cytokine receptors in the co-culture system after challenge with Shigella F2a-12 lipopolysaccharide (LPS). The human colonic epithelial cell line Caco-2 was co-cultured with freshly isolated lymphocytes from the murine Peyer's patch either in the mixed or separated (isolated but permeable compartments) co-culture configuration, and was challenged with Shigella F2a-12 LPS for 8 h. The level of mRNA expressions of human interleukin-8 (hIL-8), human interleukin-8 receptor (hIL-8R), mouse interleukin-8 receptor (mIL-8R), mouse interleukin-6 (mIL-6), mouse interleukin-6 receptor (mIL-6R) and human interleukin-6 receptor (hIL-6R) was examined by semi-quantitative PCR. In both co-culture groups, hIL-8 expression of Caco-2 cells was decreased, and hIL-8R expression was increased compared to the Caco-2 alone group. Upon LPS challenge, hIL-8 expression from the Caco-2 cells of both co-culture groups was higher than in the Caco-2 control group. The increased hIL-8 expression of Caco-2 cells in the separated co-culture group is correlated with a decreased hIL-8R expression and an increased mIL-8R expression. In the mixed co-culture group, the increased expression of hIL-8 was associated with the upregulated hIL-8R expression on Caco-2 cells and downregulated mIL-8R on murine Peyer's patch lymphocytes (PPL). mIL-6 expression from mouse PPL was also upregulated by LPS in mixed co-culture. However, upon the treatment with LPS, hIL-6R expression of Caco-2 cells was decreased in the mixed co-culture, but increased in separated co-culture. The data suggest that release of hIL-8 from epithelial cells may act on lymphocytes through a paracrine pathway, but it may also act on the epithelial cells themselves via an autocrine pathway. The data also suggest that the release of mIL-6 from Peyer's patch lymphocytes affects epithelial cells in a paracrine fashion.     

Three-dimensional structure and function study on the active region in the extracellular ligand-binding domain of human IL-6 receptor

In this study the three-dimensional (3-D) model of the ligand-binding domain (V106-P322) of human interleukin-6 receptor (hIL-6 R) was constructed by computer-guided homology modeling technique using the crystal structure of the ligand-binding domain (K52-L251) of human growth hormone receptor (hGHR) as templet. Furthermore, the active binding region of the 3-D model of hIL-6R with the ligand (hIL-6) was predicted. In light of the structural characteristics of the active region, a hydrophobic pocket shielded by two hydrophilic residues (E115 and E505) of the region was identified by a combination of molecular modelling and the site-directed or double-site mutation of the twelve crucial residues in the ligand-binding domain of hIL-6R (V106-P322). We observed and analyzed the effects of these mutants on the spatial conformation of the pocket-like region of hIL-6 R. The results indicated that any site-directed mutation of the five Cys residues (four conservative Cys residues: Cys121, Cys132, Cys165, Cys176; near membrane Cys residue: Cys193) or each double-site mutation of the five residues in WSEWS motif of hIL-6R (V106-P322) makes the corresponding spatial conformation of the pocket region block the linkage between hIL-6 R and hIL-6. However, the influence of the site-directed mutation of Cys211 and Cys277 individually on the conformation of the pocket region benefits the interaction between hIL-6R and hIL-6. Our study suggests that the predicted hydrophobic pocket in the 3-D model of hIL-6R (V106-P322) is the critical molecular basis for the binding of hIL-6R with its ligand, and the active pocket may be used as a target for designing small hIL-6R-inhibiting molecules in our further study.     

Human interleukin-6 is in vivo an autocrine growth factor for human herpesvirus-8-infected malignant B lymphocytes

Human interleukin-6 (hIL-6) acts as a growth factor in several human B lymphoid cancers. As human herpesvirus-8 (HHV-8) encodes for a viral IL-6 (vIL-6), the viral cytokine may be responsible for several manifestations of HHV-8-related disorders. Using an anti-hIL-6 mAb (B-E8) which does not recognize vIL-6, we investigated the involvement of the human cytokine in the proliferation of HHV-8-positive primary effusion lymphoma (PEL) cells. In vitro, 5/5 PEL cell lines produced hIL-6 (4 to 1,200 pg/ml). The EBV- HHV-8+ cell line (BCBL-1) was adapted to grow in SCID mice. hIL-6 was detected in the serum of mice with grafts, as well as human soluble CD138 (sCD138) and human IL-10 (hIL-10). The serum level of these mediators increased with tumor progression. The effect of treatment with the B-E8 mAb on the tumor progression and survival was evaluated. This treatment significantly slowed down the tumor development: on day 54, there were more mice with low levels of sCD138 and hIL-10 in the treated group than in controls (p = 0.03 and 0.02, respectively); treatment also delayed death (median date of death was day 65 for control mice and day 84 for anti-hIL-6 mAb-treated mice; p < 0.02). Thus, hIL-6 is expressed in addition to vIL-6 in HHV-8-positive malignant B lymphocytes, and the viral cytokine does not totally substitute for human IL-6 in promoting tumor progression.     

Preparation of functionally active recombinant human interleukin-6

The gene of human interleukin-6 (hIL-6) with an additional 20 amino acids on the N-end, including six histidine residues, was cloned into the expression plasmid pET28b(+). The conditions were elaborated for preparing highly active protein both using denaturing agents and without them. Application of a dialysis cascade allowed us to prepare a functionally active hIL-6 of 90-95% purity with the yield of 3 mg from liter of the cell culture. The highest activity was detected by ELISA in the preparation obtained without denaturing agents. The functional activity of hIL-6 was studied by flow cytofluorimetry. Addition of hIL-6 to the cells of immortal lines of human multiple myeloma resulted in dimerization of the gp130 receptor molecule.     

Engineering human interleukin-6 to obtain variants with strongly enhanced bioactivity.

Interleukin-6 (IL-6) triggers the formation of a high affinity receptor complex with the ligand binding subunit IL-6Ralpha and the signal transducing chain gp130. Since the intracytoplasmic region of the IL-6Ralpha does not contribute to signaling, soluble forms of the extracytoplasmic domain (sIL-6Ralpha), potentiate IL-6 bioactivity and induce a cytokine-responsive status in cells expressing gp130 only. This observation, together with the detection of high levels of circulating soluble human IL-6Ralpha (shIL-6Ralpha) in sera, suggests that the hIL-6-shIL-6Ralpha complex is an alternative form of the cytokine. Here we describe the generation of human IL-6 (hIL-6) variants with strongly enhanced shIL-6Ralpha binding activity and bioactivity. Homology modeling and site-directed mutagenesis of hIL-6 suggested that the binding interface for hIL-6Ralpha is constituted by the C-terminal portion of the D-helix and residues contained in the AB loop. Four libraries of hIL-6 mutants were generated by each time fully randomizing four different amino acids in the predicted AB loop. These libraries were displayed monovalently on filamentous phage surface and sorted separately for binding to immobilized shIL-6Ralpha. Mutants were selected which, when expressed as soluble proteins, showed a 10- to 40-fold improvement in shIL-6Ralpha binding; a further increase (up to 70-fold) was achieved by combining variants isolated from different libraries. Interestingly, high affinity hIL-6 variants show strongly enhanced bioactivity on cells expressing gp13O in the presence of shIL-6Ralpha at concentrations similar to those normally found in human sera.     

In-vivo processing of the initiator methionine from recombinant methionyl human interleukin-6 synthesized in Escherichia coli overproducing aminopeptidase-P

Summary Human interleukin 6 (hIL-6) overproduced in Escherichia coli HB101 was found to partially retain the initiator methionine (Met) residue (Met-hIL-6). In order to remove the residual N-terminal Met in vivo, an attempt was made to express hIL-6 in aminopeptidase-P (Ap-P)-hyperproducing strains, since the N-terminus Met-Pro- structure of nascent recombinant hIL-6 has been shown to be a favoured substrate of the enzyme in vitro. Using a mutant with duplicated Ap-P genes (pepP) on a chromosome or some recombinant strains overproducing Ap-P, we have succeeded in removing the initiator Met form Met-hIL-6 in vivo. The content of the mature product without the initiator Met in the pepP recombinant strains could be increased to approximately 99% from 85%. Offprint requests to: H. Yasueda     

Internal deletions in human interleukin-6: structure-function analysis

By cDNA mutagenesis, we have constructed internal and C-terminal deletions (delta 21-51, delta 52-97, delta 97-104, delta 127-174, delta 97-184 and delta 134-184) in human interleukin-6 (hIL-6). All those deletion-carrying hIL-6 (delta hIL-6) proteins were then produced in Xenopus laevis oocytes and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results show that, at least in frog oocytes, the first potential N-glycosylation site (Asn45) is utilized exclusively. The IL-6 conformation of these deletion-carrying proteins has been studied by immunoprecipitation with two kinds of monoclonal antibodies (mAb's): mAb's that show preference towards denatured hIL-6, or conformation-specific mAb's. The binding pattern of these two series of mAb's indicated that the IL-6 conformation has been largely destroyed for four of our delta-proteins. Proteins delta 21-51 and delta 127-174 have kept a part of the IL-6 tertiary structure since they are still recognized by some conformation-specific mAb's. All of these delta hIL-6 proteins were inactive in the IL-6 hybridoma growth factor (HGF) assay and unable to inhibit the HGF activity of the recombinant human wild-type IL-6 (wt hIL-6). Moreover, the oocyte-synthesized delta hIL-6 (delta 21-51, delta 127-174, delta 97-184, delta 134-184) did not bind to the IL-6 receptor. Finally, we have produced two proteins with aa 29-33 or 97-104 substituted by corresponding murine IL-6 (mIL-6) sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversal of some viral IL-6 electrostatic properties compared to IL-6 contributes to a loss of alpha receptor component recruitment

Human interleukin-6 (hIL-6) is a pleiotropic mediator of activation and proliferation across a large number of different cell types. Human herpesvirus-8 (HHV-8) has been associated with classical and AIDS-related Kaposi's sarcoma (KS). HHV-8 encodes viral IL-6 (vIL-6), a functional homolog of human interleukin-6, that promotes the growth of KS and of some lymphoma cells. Signaling induced by human IL-6 requires recruitment of the glycoprotein gp130, which acts as the signal transducing chain, and of IL-6Ralpha, which is necessary for cognate recognition and high affinity receptor complex formation. In contrast, the formation of a functional complex between vIL-6 and gp130 does not require the presence of IL-6Ralpha. The physico-chemical properties of vIL-6 have been analyzed and compared to those of hIL-6 and of the receptor chains, gp130 and IL-6Ralpha. Interaction sites on vIL-6 involve more hydrophobic residues than those of hIL-6. The electrostatic fields induced by vIL-6 and IL-6Ralpha are repulsive and prevent interaction between vIL-6 and IL-6Ralpha, whereas the electrostatic field induced by hIL-6 steers the complex formation with IL-6Ralpha. Subsequently, electrostatic binding free energy in the vIL-6/IL-6Ralpha complex is destabilizing, whereas it is stabilizing in the complex comprising hIL-6. These properties result from charge reversals between viral and human IL-6, an unusual phenomenon of amino acid substitutions within a homologous protein family. This suggests a selection pressure for vIL-6 to by-pass the IL-6Ralpha control of host defense against virus infection. This selection pressure has yielded the reversal of electrostatic properties of vIL-6 when compared to hIL-6.