Protein modeling, molecular network and molecular dynamics study of newly sequenced interleukin-18 (IL-18) gene in Mus musculus.

Interleukin-18 (IL-18) belongs to the superfamily of IL-1 protein and exerts a pleiotropic pro-inflammatory effect on the body. Generally, this protein is significantly involved in immune defense during infection in cells, but sometimes its anomalous activities produce some inflammatory diseases like rheumatoid arthritis and Crohn's disease. In the present study, the IL-18 gene was isolated from mice and was subsequently cloned and sequenced. Further, the network analysis was carried out to explore the functional role of IL-18 protein in animals. The 3D protein structure of the IL-18 protein was generated and docked with appropriate 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate (CPS) ligand. Later the complex structure of the protein was subjected to molecular dynamics simulation (MDS) for 50 ns to determine the effect of ligand on protein. The network analysis explored the correlation of IL-18 protein with others proteins and their involvement in the different significant pathway to defend the cell from various diseases. As confirmed by MDS, the CPS:IL-18 complex was found to be highly stable. Our results further indicated that CPS ligand has the potential to act as a drug molecule, in future, for counteracting IL-18 activity. To date, no structural details were available for animal IL-18. Hence, the finding of this study will be useful in broadening the horizon towards a better understanding of the functional and structural aspects of IL-18 in animals.

Purification, crystallization and preliminary X-ray crystallographic analysis of human IL-18 and its extracellular complexes.

Interleukin-18 (IL-18), a pro-inflammatory cytokine belonging to the interleukin-1 (IL-1) family, is involved in the pathogenesis of autoimmune/autoinflammatory and allergic diseases such as juvenile idiopathic arthritis and bronchial asthma. IL-18 forms a signalling complex with the IL-18 receptor α (IL-18Rα) and ß (IL-18Rß) chains; however, the detailed activation mechanism remains unclear. Here, the IL-18-IL-18Rα binary and IL-18-IL-18Rα-IL-18Rß ternary complexes were purified and crystallized as well as IL-18 alone. An X-ray diffraction data set for IL-18 was collected to 2.33 Šresolution from a crystal belonging to space group P21, with unit-cell parameters a = 68.15, b = 79.51, c = 73.46 Å, ß = 100.97°. Crystals of both the IL-18 binary and ternary complexes belonging to the orthorhombic space groups P21212 and P212121, respectively, diffracted to 3.10 Šresolution. Unit-cell parameters were determined as a = 135.49, b = 174.81, c = 183.40 Šfor the binary complex and a = 72.56, b = 111.56, c = 134.57 Šfor the ternary complex.

Shotgun proteomic analysis of porcine colostrum and mature milk.

The epitheliochorial nature of the porcine placenta prevents the transfer of maternal immunity. Therefore, ingestion of the colostrum immediately after birth is crucial for neonatal piglets to acquire passive immunity from the sow. We performed a shotgun proteomic analysis of porcine milk to reveal in detail the protein composition of porcine milk. On the basis of the Swiss-Prot database, 113 and 118 proteins were identified in the porcine colostrum and mature milk, respectively, and 50 of these proteins were common to both samples. Some immune-related proteins, including interleukin-18 (IL-18), were unique to the colostrum. The IL-18 concentration in the colostrum and mature milk of four sows was measured to validate the proteomic analysis, and IL-18 was only detected in the colostrum (191.0 ± 53.9 pg/mL) and not in mature milk. In addition, some proteins involved in primary defense, such as azurocidin, which has never been detected in any other mammal's milk, were also identified in the colostrum.

Cloning, in vitro expression, and bioactivity of interleukin-18 isolated from a domestic porcine breed found in Henan.

To evaluate the effects of recombinant porcine interleukin-18 (rpIL-18) on the replication of viruses in host cells and proliferation of lymphocytes, porcine IL-18 (pIL-18) isolated from a domestic big-white porcine breed found in the Henan province (HN) was cloned using a reverse transcriptase-PCR. The cloned HN pIL-18 contained an ORF of 579 base pairs encoding a 192-amino-acid precursor protein. The amino acid sequence of HN pIL-18 was compared with all the other pIL-18 amino acid sequences and varied by at least one amino acid to the consensus of all the others available. HN pIL-18 mature protein gene was inserted into a prokaryotic vector pGEX-4T-1 and expressed in Escherichia coli BL21. The expression of glutathione-S-transferase-pIL18 fusion protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. The rpIL-18 induced in vitro proliferation of concanavalin-A-stimulated porcine splenocytes, as revealed by the MTT assay. We studied the antiviral activities of the rpIL-18 on the replication of porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), and porcine parvovirus (PPV) cultured in two homologous cell lines. The results suggested that rpIL-18 can stimulate the proliferation of lymphocytes and inhibit viral pathogens infecting the porcine population.

[High level secretory expression of recombinant human interleukin 18 in Pichia pastoris].

AIM: To construct eukaryotic expression vector and express human interleukin 18 (hIL-18) in Pichia pastoris. METHODS: The gene encoding of hIL-18 was amplification by PCR. The recombinant pPICZaC/hIL-18 was transformed into the Pichia pastoris X-33 strain via electroporation. The high level expression was selected and assayed by the methods of PCR, SDS-PAGE and Western blot. The rhIL-18 was purified by the methods of hydrophobic chromatography and anion exchange chromatography. The bioactivity of it was initially assayed. RESULTS: The rhIL-18 was secreted into the supernatant and the concentration reached to 202 mg/L. The rhIL-18 was further identified by Western blot with specific antibody binding activity. The purity of the rhIl-18 reached about 95%. And rhIL-18 can synergistically induce PBMC to produce IFN-gamma with IL-2. CONCLUSION: A rhIL-18 is successfully constructed and expressed in Pichia pastoris. And this contributes to further study of its function and activity.

Expression and purification of recombinant human interleukin-18 protein using a yeast expression system.

Interleukin-18 (IL-18) has been reported to exert significant immunoregulatory effects on inhibiting tumor growth through stimulating natural killer (NK) cell cytotoxicity and promoting production of several cytokines, including interferon-gamma (IFN-gamma) and granulocyte/macrophage colony-stimulating factor (GM-CSF). Therefore, IL-18 might serve as a potential therapeutic target for cancer treatment. However, the resource of this protein limits its availability for the clinical practice. The purpose of this study was to express and purify recombinant human (h) IL-18 protein using a yeast expression system. We reported here that hIL-18 gene was cloned into pPICZaC vector for expressing a recombinant hIL-18 protein using a yeast expression system. The recombinant hIL-18 protein was purified using centrifugal filter devices, hydrophobic chromatography, and anion exchange chromatography. The yield and purity of the recombinant hIL-18 reached 45.1% and 97.6%, respectively. This recombinant hIL-18 was shown to induce IFN-gamma production by human peripheral blood mononuclear cells (PBMCs) and enhance NK cell cytotoxicity synergistically with IL-2. Furthermore, these recombinant hIL-18-induced effects were the same as those by standard hIL-18. Therefore, the yeast expression system used in this study provides a useful method to produce large-scale of hIL-18 for the clinical application.

[Development of sandwich ELISA for equine interferon-gamma detection].

AIM: To develop a quantitative ELISA by measuring interferon (IFN-gamma) of equine lymphocytes. METHODS: Sandwich ELISA for equine IFN-gamma was developed using mAb A5 as a capture antibody and biotinylated mAb SB10 as a detection antibody. RESULTS: The detection limit of the sandwich ELISA for equine IFN-gamma was 1 microg/L and did not show cross-reactivity with recombinant equine IL-18. Equine IFN-gamma was detected by ELISA in culture medium of the peripheral blood mononuclear cells (PBMCs) stimulated with ConA or PMA/Ionomycin. CONCLUSION: This method can be used to help understand the role of this cytokine in various equine diseases and develop specific cell-mediated immunity assay.

[Separation of interleukin-18 on a weak anion exchange monolithic column].

A weak anion exchange column was prepared and a separation method of interleukin-18 (IL-18) was developed on the column. The chromatographic column with glycidyl methacrylate as functional monomer and ethylene dimethacrylate as cross-linker was prepared by a free radical polymerization within a 64 mm x 4.6 mm i.d. column. The epoxide groups of the column were modified by a reaction with diethylamine that affords ionizable functionalities required for ion exchange chromatography. The influences of the contents of the cross-linker and porogen on column pressure were investigated. The factors influencing the separation, such as stationary phases, buffers, pH values, salts, and contents of organic solvent, were studied. The optimal conditions for separating IL-18 from its related proteins were obtained. The proposed method is simple, sensitive, rapid and reproducible.

[Construction and prokaryotic expression of a recombinant immunotoxin fused with mouse interleukin 18 and truncated Pseudomonas exotoxin].

OBJECTIVE: To construct a new recombinant immunotoxin expression vector by fusion of mouse interleukin 18 (mIL-18) gene and a truncated form of A (PE38) gene, and examine the expression of IL-18-PE38 fusion protein in Escherichia coli. METHODS: mIL-18 cDNA was cloned from mouse liver tissue through reverse transcription-polymerase chain reaction (RT-PCR). The mIL-18 cDNA was subcloned into a PE38 gene inserted fusion protein expression plasmid. The recombinant vector was identified by restriction endonucleases digestion, PCR and DNA sequencing. After transformed into E. coli BL21 and induced by IPTG, the expressed product was obtained and detected the molecular weight and specificity by SDS-PAGE and Western-blotting. RESULTS: The new recombinant immunotoxin expression vector was constructed successfully. The IL-18-PE38 fusion protein was expressed in E. coli BL21, and the molecular weight of the expression product was identical to the expected value. In addition, the protein expressed could react with the specific antibody against mIL-18. CONCLUSION: IL-18-PE38 recombinant immunotoxin expression vector will provide the basis for study on the targeted cytotoxic activity to Th1 cell and may have some potential value in the treatment of autoimmune disease and T cell leukemias.

Method for purifying porcine mature interleukin-18 from silkworm haemolymph.

Recombinant porcine mature interleukin-18 (rPomIL-18) has been purified from the haemolymph of silkworms. After co-infection of two recombinant baculoviruses (BmAcpVL1392-IL-18-His and BmAcpVL1392-casp-1) into the silkworm, rPomIL-18 was produced and secreted into the haemolymph. Polyethylene glycol (PEG) 6000 was added to the haemolymph at 8% (v/w) to precipitate storage proteins which would otherwise bind non-specifically to the metal chelating column and the supernatant then was applied to Sepharose bonded with Ni2+. rPomIL-18 was eluted from the column using 100 mM imidazole buffer at pH 8 with a purity of 93.6%. Approximately 5.3 mg purified rPomIL-18 was obtained from 22 ml haemolymph. It could induce interferon-gamma formation from porcine peripheral blood mononuclear cells.

Systematic optimization of active protein expression using GFP as a folding reporter.

Many recombinant proteins have been used as drugs; however, human proteins expressed using heterologous hosts are often insoluble. To obtain correctly folded active proteins, many optimizations of expression have been attempted but usually are found to be applicable only for specific targets. Interleukin-18 (IL-18) has a key role in many severe disorders including autoimmune diseases, and therapeutic approaches using IL-18 have been reported. However, production of IL-18 in Escherichia coli resulted in extensive inclusion body formation and previous conventional screenings of expression conditions could obtain only a condition with a low yield. To address the problem, we applied a folding reporter system using green fluorescent protein (GFP) for screening of the expression conditions for hIL-18. The established system efficiently screened many conditions, and optimized conditions for the expression of hIL-18 significantly enhanced the final yield of the active protein. Systematic screening using a GFP reporter system could be applied for the production of other proteins and in other organisms.

Identification of metastasis-associated proteins by proteomic analysis and functional exploration of interleukin-18 in metastasis.

Very little is currently known about mechanisms underlying cancer metastasis. In the present study, metastasis-associated proteomes were separated and identified by comparative proteomic analysis, and the metastasis-related function of candidate protein interleukin-18 (IL-18) was further elucidated. First, a pair of highly and poorly metastatic sublines (termed PLA801D and PLA801C, respectively), originating from the same parental PLA801 cell line, was identified by spontaneous tumorigenicity and metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach was used to compare the protein expression profiles between PLA801C and PLA801D sublines. Eleven proteins were identified and further verified by one-dimensional Western blotting, Northern blot and/or semiquantitative reverse transciptase polymerase chain reaction analysis. Compared with those in poorly metastatic PLA801C subline, cytokeratin 18, tissue transglutaminase, Rho GDP-dissociation inhibitor 1, tropomyosin, fibroblast type, IL-18 and annexin I were significantly up-regulated, while protein disulfide isomerase, heat shock protein 60, peroxiredoxin 1, chlorine intracellular channel protein 1 (CLI1) and creatine kinase, B chain were significantly down-regulated in the highly metastatic PLA801D subline. Intriguingly, all the identified candidate proteins except for CLI1 have been shown to be somehow associated with distinct aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. Considering that IL-18 was present in highly metastatic PLA801D but absent in poorly metastatic PLA801C, the association of IL-18 with metastasis was further elucidated by introducing IL-18 sense/IL-18 antisense into PLA801C/PLA801D sublines simultaneously. The results demonstrated that ectopically expressed IL-18 promoted cell motility in vitro and down-regulated E-cadherin expression of PLA801C transfectants, while IL-18 antisense remarkably decreased cell invasion potency in vitro and notably increased E-cadherin expression of PLA801D transfectants, indicating that IL-18 might play a role in metastasis by inhibiting E-cadherin expression.

High level expression and purification of bioactive bovine interleukin-18 using a baculovirus system.

Bioactive recombinant bovine interleukin-18 (rboIL-18) was expressed using a baculovirus system. Normally, IL-18 is translated as a precursor form of a 24kDa polypeptide and processed by IL-1beta converting enzyme (ICE) to a mature bioactive form of 18kDa protein. Hence, to express active form IL-18, we constructed two recombinant baculoviruses containing boIL-18 and human ICE (hICE) genes, respectively, and superinfected these viruses into insect cells. Superinfection of both recombinant viruses into the cells resulted in the expression of a 24kDa precursor form and an 18kDa mature form detectable in the supernatant by immunoblotting using anti-porcine IL-18 antibody. Culture supernatant from the superinfected cells showed a synergistic effect with recombinant boIL-12 for production of interferon-gamma (IFN-gamma) in bovine peripheral mononuclear cells. By addition of histidine hexamer at the C-terminal of boIL-18, the mature IL-18 was purified. Bioactivity remained after purification.

An alternative form of IL-18 in human blood plasma: complex formation with IgM defined by monoclonal antibodies.

Monoclonal Abs 21 and 132 were raised against human functionally inactive rIL-18, and plasma IL-18 levels were determined by the sandwich ELISA established with these mABS: Plasma IL-18, designated type 2, was detected by this ELISA, and the levels found were not consistent with those obtained with the commercially available kit for determination of functionally active IL-18 (type 1). Type 1 was detected in all volunteers, whereas type 2 was detected in approximately 30% of healthy subjects, and the levels of type 2 in their blood plasma were high (25-100 ng/ml) compared with those of type 1 (0.05-0.3 ng/ml). We purified IL-18 type 2 from blood plasma of volunteers with high IL-18 type 2 concentrations, and its M(r) was determined to be 800 kDa by SDS-PAGE and molecular sieve HPLC. The purified 800-kDa protein, either caspase-1-treated or untreated, expressed no or marginal IL-18 function in terms of potentiation of NK-mediated cytolysis and IFN-gamma induction, and it barely bound IL-18R-positive cells. N-terminal amino acid analysis indicated that the purified protein was IgM containing a minimal amount of IL-18 proform and its fragment. Again, the purified IgM from IL-18 type2-positive volunteers exhibited cross-reaction with mAb 21 against IL-18. This band was not detected with 125-2H, an mAb against functionally active IL-18. Hence, human IgM carries functionally inactive IL-18 forming a disulfide-bridged complex, and this IL-18 moiety is from 10- to 100-fold higher than the conventional type 1 IL-18 in blood circulation in approximately 30% normal subjects.

[Expression and purification of murine interleukin 18 in Escherichia coli and its antitumor effects].

Total RNA was extracted from murine hepatocytes, and the cDNA of interleukin 18(IL-18) was amplified by RT-PCR. The cDNA was introduced into the expression vector pJW2 and sequenced. Under heat induction, the recombinant murine IL-18(rmIL-18) was expressed in inclusion bodies in E. coli with the yield accounting for 18% of total bacteria proteins. The inclusion bodies were dissolved with 5 mol/L urea, and rmIL-18 was purified using Sephadex G-100 column chromatography. In the presence of 0.5 mg/L Con A, the purified rmIL-18 showed dose-dependent IFN-gamma-inducing activity in murine splenocytes. The purified rmIL-18 exhibited significant antitumor effects in Kunming mice challenged intraperitoneally (i.p.) with H22 hepatocarcinoma when administered 10 micrograms rmIL-18 i.p. on days 1, 4 after challenge, and the mice survived resisted the rechallenged with H22 cells.

Tissue-specific expression of rat IL-18 gene and response to adrenocorticotropic hormone treatment.

IL-18 is a pleiotropic cytokine also proposed to have a role in modulating immune function during stress. Initially found in immune cells, IL-18 mRNA is detectable in several tissues including the cells of the zona reticularis and the zona fasciculata of the adrenal cortex, where its levels are elevated by acute stress or adrenocorticotropic hormone treatment. In the present study, we compared the expression of IL-18 in the adrenal cortex with that of spleen and duodenum, two other IL-18-positive tissues. In situ hybridization showed that, in contrast to the adrenal cortex, in spleen and duodenum IL-18 is primarily localized to immune cells. In duodenum, IL-18 mRNA was also detectable in epithelial cells. Northern blot demonstrated that while the adrenal gland synthesized IL-18 mRNA of 1.1 kb, spleen and duodenum produced a 0.9-kb transcript. RT-PCR, sequencing, Western blot, primer extension, and rapid amplification of cDNA end analysis demonstrated that the three tissues synthesize IL-18 mRNAs containing the same coding region and produce the same IL-18 peptide, but differ in the length of their 5'-untranslated region, indicating tissue-specific usage of the promoter region. Finally, in contrast to the adrenal gland, adrenocorticotropic hormone treatment did not increase the levels of IL-18 mRNA in spleen and duodenum. These results demonstrate tissue-specific expression and promoter usage of IL-18 gene and suggest that the adrenal cortex and not immune cells may be the source of IL-18 during stress.

Production of monoclonal antibodies to porcine interleukin-18 and their use for immunoaffinity purification of recombinant porcine interleukin-18.

We have recently reported the cloning and expression of porcine interleukin-18 (IL-18). In this study, we describe the production of anti-porcine IL-18 monoclonal antibodies (mAb) and their use in the purification of a large amount of recombinant porcine IL-18 by immunoaffinity column chromatography. Five monoclonal antibodies (2-2-B, 2-5-B, 2-13-C, 3-1-C and 5-3-B) were established and characterized. Three (2-2-B, 3-1-C and 5-3-B) of them were of IgG1 subclass, and the other two were IgMs. Epitope analysis of the three IgG1 mAbs showed that they recognized the same epitope. All five mAbs demonstrated reactivity with baculovirus generated porcine IL-18 by immunoblot analysis. Biologically active porcine IL-18 was obtained by immunoaffinity chromatography using anti-porcine IL-18 mAb at more than 85% purity from culture supernatants of Trichoplusia ni (Tn5) derived cells infected with recombinant baculovirus containing the coding sequence of porcine mature IL-18. These results suggest that the anti-porcine IL-18 mAbs established in this study are useful for one-step purification of porcine mature IL-18 as well as the detection of porcine IL-18 by immunoblotting.

Limitations for purification of murine interleukin-18 when expressed as a fusion protein containing the FLAG peptide.

As a strategy to purify recombinant murine Interleukin (IL)-18, we cloned the mature coding region of this protein into the pFLAG-1 expression system. The intent was to use the FLAG peptide "tag" as an amino terminal addition to IL-18 so that purification of this fusion protein (FLAG-IL-18) on anti-FLAG antibody affinity columns could be performed. While significant amounts of recombinant IL-18 were present in E. coli lysates, only a small portion of this material could be recovered on immunoaffinity columns conjugated with an anti-FLAG antibody. Surprisingly, the majority of recombinant IL-18 present in E. coli (strain JM83) bacterial lysates did not contain the FLAG peptide and therefore did not bind to immunoaffinity columns conjugated with an anti-FLAG antibody. However, we found that the BL21 strain of E. coli, which has reduced endogenous protease activity, could express the majority of recombinant IL-18 as the fusion protein, FLAG-IL-18. Taken together, these studies show that it is necessary to consider whether protease sites formed at the FLAG-protein junction can be easily cleaved by the bacterial strain used to express the fusion protein.