Molecular characterization of woodchuck interleukin 15 (wIL-15) and detection of its expression in liver samples of woodchucks infected with woodchuck hepatitis virus (WHV)

Interleukin 15 (IL-15) is a member of the four-helix bundle cytokine family and has T cell growth factor activity. IL-15 plays a unique role in both innate and adaptive immune cell homeostasis, particularly for the development of NK cells and CD8+memory cells. It may be useful for stimulation of specific immune responses in chronic viral infection such as hepatitis B virus infection. The woodchuck model is an informative animal model for studies on hepadnavirus infection and therapeutic interventions. Here, the complete coding sequence of woodchuck IL-15 (wIL-15) was cloned and sequenced. wIL-15 shows a high homology (>70%) to its counterparts of other mammalian species. His-tagged recombinant wIL-15 protein was expressed and purified and showed the ability to promote the proliferation of activated mouse splenocytes and woodchuck peripheral blood lymphocytes. Further, examination of mRNA amounts in liver samples of woodchucks by semi-quantitative RT-PCR showed a slightly increased expression of wIL-15 in woodchuck livers during chronic woodchuck hepatitis virus infection. This available information will provide a basis for further studies on the function of IL-15 in the context of acute and chronic hepadnavirus infection and its potential therapeutic use for chronic hepatitis B virus infection in the woodchuck model.     

Analysis of human IL-6 mutants expressed in Escherichia coli. Biologic activities are not affected by deletion of amino acids 1-28

We have constructed and analyzed amino terminally deleted analogs of IL-6. Progressively shortened variants of mature IL-6 were constructed at the cDNA level and expressed in Escherichia coli. Mutant proteins were recovered from refractile bodies by solubilizing in 6 M guanidine-HCl. The mutant protein concentration in these preparations was estimated by Western blotting by using an IL-6-specific mAb and the biologic activity was measured in the B9 (hybridoma growth factor) assay. The first 28 amino acids of mature IL-6 could be removed without significantly affecting biologic activity. A further removal of amino acids 29 and 30 resulted in an approximately 50-fold decrease, whereas removal of amino acids 31 to 34 virtually abolished the activity. The mutants showed the same reaction pattern in three other IL-6 assays: induction of murine thymocyte proliferation, induction of fibrinogen synthesis by a human hepatoma cell line (HepG2), and the induction of IgM synthesis by an EBV-transformed B cell line. This suggests that a single functional domain might be responsible for all four activities of IL-6.     

Molecular characterization of woodchuck interleukin 15 (wIL-15) and detection of its expression in liver samples of woodchucks infected with woodchuck hepatitis virus (WHV)

Interleukin 15 (IL-15) is a member of the four-helix bundle cytokine family and has T cell growth factor activity. IL-15 plays a unique role in both innate and adaptive immune cell homeostasis, particularly for the development of NK cells and CD8 + memory cells. It may be useful for stimulation of specific immune responses in chronic viral infection such as hepatitis B virus infection.The woodchuck model is an informative animal model for studies on hepadnavirus infection and therapeutic interventions. Here, the complete coding sequence of woodchuck IL-15 (wIL-15) was cloned and sequenced. wIL-15 shows a high homology (>70%) to its counterparts of other mammalian species. His-tagged recombinant wIL-15 protein was expressed and purified and showed the ability to promote the proliferation of activated mouse splenocytes and woodchuck peripheral blood lymphocytes. Further, examination of mRNA amounts in liver samples of woodchucks by semi-quantitative RT-PCR showed a slightly increased expression of wIL-15 in woodchuck livers during chronic woodchuck hepatitis virus infection. This available information will provide a basis for further studies on the function of IL-15 in the context of acute and chronic hepadnavirus infection and its potential therapeutic use for chronic hepatitis B virus infection in the woodchuck model.     

Prokaryotic expression of woodchuck cytotoxic T lymphocyte antigen 4 (wCTLA-4) and preparation of polyclonal antibody to wCTLA-4

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is an inhibitory T cell receptor predominately expressed on activated T cells and plays an important role in regulation of specific T cell responses to viral infection. The woodchuck model is an informative animal model for hepatitis B virus (HBV) infection. In this study, the extracellular region of woodchuck CTLA-4 (wCTLA-4) was cloned and the fusion protein of GST-wCTLA-4 was expressed and purified. Polyclonal antibody against GST-wCTLA-4 (anti-GST-wCTLA-4) was prepared. The full length wCTLA-4 protein expressed in transfected baby hamster kidney cells was detected by anti-GST-wCTLA-4 in western blot analysis and immunofluorescence staining. Anti-GST-wCTLA-4 provides a useful tool to study the role of CTLA-4 in T-cell response in the woodchuck model. Further, the blocking of CTLA-4 with anti-GST-wCTLA-4, as a novel therapy approach for chronic hepatitis B virus infection, could be studied in woodchuck model now.     

Phenotypic mixing between different hepadnavirus nucleocapsid proteins reveals C protein dimerization to be cis preferential.

Hepadnaviruses encode a single core (C) protein which assembles into a nucleocapsid containing the polymerase (P) protein and pregenomic RNA during viral replication in hepatocytes. We examined the ability of heterologous hepadnavirus C proteins to cross-oligomerize. Using a two-hybrid assay in HepG2 cells, we observed cross-oligomerization among the core proteins from hepatitis B virus (HBV), woodchuck hepatitis virus, and ground squirrel hepatitis virus. When expressed in Xenopus oocytes, in which hepadnavirus C proteins form capsids, the C polypeptides from woodchuck hepatitis virus and ground squirrel hepatitis virus, but not duck hepatitis B virus, can efficiently coassemble with an epitope-tagged HBV core polypeptide to form mixed capsids. However, when two different core mRNAs are coexpressed in oocytes the core monomers show a strong preference for forming homodimers rather than heterodimers. This holds true even for coexpression of two HBV C proteins differing only by an epitope tag, suggesting that core monomers are not free to diffuse and associate with other monomers. Thus, mixed capsids result from aggregation of different species of homodimers.     

Porcine interleukin 18: cloning, characterization of the cDNA and expression with the baculovirus system

We have isolated and sequenced a cDNA that contains the coding sequence of porcine interleukin 18 (IL-18) and the recombinant protein of porcine IL-18 was expressed using the baculovirus system. The open reading frame (ORF) of the porcine IL-18 cDNA is 579 base pairs (bp) in length and encodes 192 amino acids. The predicted amino acid sequence is 76.7%, 64.7% and 61.6% homologous to the predicted human, murine and rat amino acid sequences, respectively. The porcine precursor and mature IL-18 protein were expressed respectively in Trichoplusia ni -derived (Tn5) cells using the baculovirus Autografha californica nuclear polyhedorosis virus (AcNPV) as a vector. Tn5 cells infected with recombinant virus containing a whole IL-18 protein coding region sequence secreted porcine precursor IL-18 into the culture medium. On the other hand, Tn5 cells infected with recombinant virus containing a mature IL-18 protein coding region sequence expressed several proteins in the cell lysates, but did not secrete mature protein into the culture medium efficiently. Immunoblotting analysis of recombinant protein showed cross-reactivity with anti-human IL-18 polyclonal antibody. The mature form of porcine IL-18 protein induced IFN-gamma production in suboptimal doses of anti-CD3 antibody and concanavalin A- (ConA) stimulated porcine peripheral blood mononuclear cells (PBMC), but the precursor form had little effect.     

Effects of site-specific mutations on biologic activities of recombinant human IL-6

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.     

Expression, renaturation and purification of recombinant human interleukin 4 from Escherichia coli

The lymphokine human interleukin 4 (IL-4) has been expressed from a plasmid in the cytoplasm of Escherichia coli. Advantage has been taken of insolubility of the human IL-4 in E. coli for rapid purification of this protein in only a few steps. We describe extraction and renaturation procedures which solubilize human IL-4 yielding biologically active protein. The protein was purified to homogeneity by one passage over a gel-filtration column. The refolded human IL-4 was characterized by N-terminal sequence analysis, amino acid analysis and bioassays. The refolded E. coli-derived human IL-4 has biological activity on T and B cells and binds to the human IL-4 receptor, comparable to mammalian expressed human IL-4, indicating that the protein is folded correctly.     

Molecular cloning and expression of the murine interleukin-5 receptor

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.     

Cloning and characterization of IL-1HY2, a novel interleukin-1 family member

The interleukin-1 (IL-1) family members play an important role in the process of inflammation and host defense. We describe here the identification and characterization of a novel member of the IL-1 family, IL-1HY2. The human IL-1HY2 protein shares significant amino acid sequence similarity (37%) with the IL-1 receptor antagonist and has a predicted three-dimensional structure similar to that of the IL-1 receptor antagonist. The IL-1HY2 gene is located in close proximity to other IL-1 family genes on human chromosome 2, and the genomic organization of the IL-1HY2 gene is highly conserved with other IL-1 family members. IL-1HY2 protein is secreted from mammalian cells, and the purified recombinant IL-1HY2 protein binds soluble IL-1 receptor type I. IL-1HY2 is expressed in human skin, spleen, and tonsil. Immunohistochemical analysis showed that the IL-1HY2 protein is expressed in the basal epithelia of skin and in proliferating B cells of the tonsil. These data suggest that IL-1HY2 is a novel IL-1 family member and that it may participate in a network of IL-1 family members to regulate adapted and innate immune responses.     

Characterization of murine IL-1 beta. Isolation, expression, and purification

One cDNA clone encoding a truncated murine IL-1 beta (M IL-1 beta) sequence was isolated from a murine macrophage cDNA library. We reconstituted the coding sequence of the 152-residue mature protein and expressed it in Escherichia coli. rM IL-1 beta was purified to homogeneity and characterized by oligonucleotide and NH2-terminal sequence analysis. Purified rM IL-1 beta exhibited biologic activity equivalent to 7.8 x 10(7) units/mg in the murine thymocyte proliferation assay and 9.9 x 10(3) units/mg in the human gingival fibroblast PGE2 production assay, indicative of species specificity. The isoelectric point of rM IL-1 was found to be 8.85. The circular dichroism spectrum revealed that the secondary structure of M IL-1 is indistinguishable from that of the human protein. Receptor binding studies indicated the rM IL-1 bound to murine EL-4.1 thymoma cells in a specific and dose-dependent fashion with an affinity of 32 pM. Competition binding data suggested that murine and human IL-1 compete for a single class of receptor. Antisera were generated in rabbits against both murine and human IL-1. Results of ELISA binding and antisera neutralization assays indicated that there are common antigenic sites between the two IL-1 beta molecules. These domains are of functional importance because they are capable of mediating the neutralization of biologic activity.     

一种新的乙型肝炎病毒表面抗原结合蛋白的筛选、表达及其生物学活性的初步研究

为了研究乙肝病毒侵染肝细胞过程中的功能蛋白 ,通过印迹免疫分析技术从人肝cDNA噬菌体表达库中筛选出一株编码乙肝表面抗原结合蛋白 (hepatitisBsurfaceantigenbindingprotein ,HBsAg BP)的cDNA克隆 .基因测序结果表明 ,该cDNA具有独立的开放阅读框架 ,编码 1个由 344个氨基酸残基构成的可溶性蛋白分子 ,属于免疫球蛋白超家族成员 .将该基因克隆到原核表达载体pTriplEx后 ,在E .coliXL1 Blue菌株中获得 4 4kD的重组蛋白 .重组蛋白经Western印迹和ELISA实验证明具有与乙肝表面抗原特异性结合的能力 .进一步经流式细胞仪实验显示 ,在纯化的重组蛋白存在的情况下 ,天然的HBsAg与肝细胞株HepG2的亲和力显著增高 .结果显示 ,该乙肝表面抗原结合蛋白可能是介导乙肝病毒对肝细胞亲和侵染的可溶性辅助受体 .     

Identification of a lactoferrin-derived peptide possessing binding activity to hepatitis C virus E2 envelope protein

Bovine and human lactoferrins (LF) prevent hepatitis C virus (HCV) infection in cultured human hepatocytes; the preventive mechanism is thought to be the direct interaction between LF and HCV. To clarify this hypothesis, we have characterized the binding activity of LF to HCV E2 envelope protein and have endeavored to determine which region(s) of LF are important for this binding activity. Several regions of human LF have been expressed and purified as thioredoxin-fused proteins in Escherichia coli. Far-Western blot analysis using these LF fragments and the E2 protein, expressed in Chinese hamster ovary cells, revealed that the 93 carboxyl amino acids of LF specifically bound to the E2 protein. The 93 carboxyl amino acids of LFs derived from bovine and horse cells also possessed similar binding activity to the E2 protein. In addition, the amino acid sequences of these carboxyl regions appeared to show partial homology to CD81, a candidate receptor for HCV, and the binding activity of these carboxyl regions was also comparable with that of CD81. Further deletion analysis identified 33 amino acid residues as the minimum binding site in the carboxyl region of LF, and the binding specificity of these 33 amino acids was also confirmed by using 33 maltose-binding protein-fused amino acids. Furthermore, we demonstrated that the 33 maltose-binding protein-fused amino acids prevented HCV infection in cultured human hepatocytes. In addition, the site-directed mutagenesis to an Ala residue in both terminal residues of the 33 amino acids revealed that Cys at amino acid 628 was determined to be critical for binding to the E2 protein. These results led us to consider the development of an effective anti-HCV peptide. This is the first identification of a natural protein-derived peptide that specifically binds to HCV E2 protein and prevents HCV infection.     

人白介素6受体基因在痘苗病毒载体系统中的表达

人ＩＬ－６受体是一个在各种细胞上广泛表达的跨膜糖蛋白分子,是ＩＬ－６发挥细胞效应所必需的。本文通过将ＩＬ－６ＲｃＤＮＡ重组到痘苗病毒的ＴＫ基因中构建成重组痘苗病毒ＶＩＬ６Ｒ。细胞原位杂交和ＡＰＡＡＰ染色结果表明,感染ＶＩＬ６Ｒ后的Ｖｅｒｏ细胞中,ＩＬ－６Ｒ在ｍＲＮＡ和蛋白水平上均呈现较强的表达。Ｗｅｓｔｅｒｎｂｌｏｔ分析所表达的分子量为８０ｋＤ,表明所表达的产物是糖基化的。ＩＬ－６结合试验表明,表达的膜ＩＬ－６Ｒ能够结合ｒＩＬ－６,说明它是有功能的。利用ＶＩＬ６Ｒ免疫小鼠后,能够刺激较强的抗体产生。从而为进一步研究ＩＬ－６Ｒ的信号传导和构效关系提供了基础。     

Role of disulfide bonds in biologic activity of human interleukin-6.

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.