Expression and secretion of the heterodimeric protein interleukin-12 in plant cell suspension culture

It has been suggested that plant cell culture is the most suitable system for producing small-to-medium quantities of specialized, expensive, and high-purity proteins. Here, we report that a heterodimeric protein, human interleukin-12 (hIL-12), was expressed and secreted into culture medium in a biologically active form. A transgenic plant expressing hIL-12 was constructed by sexual crossing of plants that expressed each subunit of the protein. From a piece of transgenic plant, callus was induced and cell suspension culture was established. The biological activity and amount of hIL-12 secreted into culture medium were analyzed using bioassays and ELISA. Analysis of cellular localization demonstrated that the protein was secreted into the culture medium together with its intrinsic signal peptide.     

Charged surface residues of FKBP12 participate in formation of the FKBP12-FK506-calcineurin complex.

The mechanism of FK506 immunosuppression has been proposed to proceed by formation of a tight-binding complex with the intracellular 12-kDa FK506-binding protein (FKBP12). The FK506-FKBP12 complex then acts as a specific high-affinity inhibitor of the intracellular protein phosphatase PP2B (calcineurin), interrupting downstream dephosphorylation events required for T-cell activation. Site-directed mutagenesis of many of the surface residues of FKBP12 has no significant effect on its affinity for calcineurin. We have identified, however, three FKBP12 surface residues (Asp-37, Arg-42, and His-87) proximal to a solvent-exposed segment of bound FK506 that may be direct contacts in the calcineurin complex. Site-directed mutagenesis of two of these residues decreases the affinity of FKBP12-FK506 for calcineurin (Ki) from 6 nM for wild-type FKBP12 to 3.7 microM for a R42K/H87V double mutant, without affecting the peptidylprolyl isomerase activity or FK506 affinity of the mutant protein. These FKBP12 mutations along with several substitutions on FK506 known to affect calcineurin binding form a roughly 100-A2 region of the FKBP12-FK506 complex surface that is likely to be within the calcineurin binding site.     

Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-12 (cytotoxic lymphocyte maturation factor).

IL-12 is a heterodimeric cytokine that was identified on the basis of its ability to synergize with IL-2 in the induction of cytotoxic effector cells and was originally called cytotoxic lymphocyte maturation factor (CLMF). IL-12 was also found to stimulate the proliferation of PHA-activated lymphoblasts which were greater than 90% CD3+ T cells. In this report we further characterize the effects of IL-12 on lymphocyte proliferation. Studies with purified subpopulations of PHA-activated lymphoblasts and with cloned lines of human T cells indicated that IL-12 caused the proliferation of activated T cells of both the CD4+ and CD8+ subsets. This effect of IL-12 was independent of IL-2 because it was not blocked by antibodies to either IL-2 or IL-2R. The maximum proliferation induced by IL-12 was 31 to 72% of the maximum caused by IL-2; however, IL-12 was active at a lower effective concentration (EC50 = 8.5 +/- 1.3 pM) than IL-2 (EC50 = 52 +/- 8 pM). Combination of suboptimal amounts of IL-12 and IL-2 resulted in additive proliferation, up to the maximum induced by IL-2 alone. IL-12 also caused the proliferation of lymphocytes activated by culture with IL-2 for 6 to 12 days. CD56+ NK cells were among the IL-12-responsive cells in the IL-2-activated lymphocyte population. Unlike IL-2 or IL-7, IL-12 caused little or no proliferation of resting peripheral blood mononuclear cells (PBMC). In this regard, IL-12 was similar to IL-4. However, IL-12 could enhance the proliferation of resting PBMC caused by suboptimal amounts of IL-2, whereas IL-4 inhibited IL-2-induced PBMC proliferation. Thus, IL-12 is a growth factor for activated human T cells and NK cells; however, its spectrum of lymphocyte growth-promoting properties is distinct from that of IL-2, IL-4, or IL-7.     

A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rbeta1 and a novel cytokine receptor subunit,IL-23R

IL-23 is a heterodimeric cytokine composed of the IL-12p40 "soluble receptor" subunit and a novel cytokine-like subunit related to IL-12p35, termed p19. Human and mouse IL-23 exhibit some activities similar to IL-12, but differ in their capacities to stimulate particular populations of memory T cells. Like IL-12, IL-23 binds to the IL-12R subunit IL-12Rbeta1. However, it does not use IL-12Rbeta2. In this study, we identify a novel member of the hemopoietin receptor family as a subunit of the receptor for IL-23, "IL-23R." IL-23R pairs with IL-12Rbeta1 to confer IL-23 responsiveness on cells expressing both subunits. Human IL-23, but not IL-12, exhibits detectable affinity for human IL-23R. Anti-IL-12Rbeta1 and anti-IL-23R Abs block IL-23 responses of an NK cell line and Ba/F3 cells expressing the two receptor chains. IL-23 activates the same Jak-stat signaling molecules as IL-12: Jak2, Tyk2, and stat1, -3, -4, and -5, but stat4 activation is substantially weaker and different DNA-binding stat complexes form in response to IL-23 compared with IL-12. IL-23R associates constitutively with Jak2 and in a ligand-dependent manner with stat3. The ability of cells to respond to IL-23 or IL-12 correlates with expression of IL-23R or IL-12Rbeta2, respectively. The human IL-23R gene is on human chromosome 1 within 150 kb of IL-12Rbeta2.     

Complementation of buried lysine and surface polar residues in a designed heterodimeric coiled coil

The coiled coil is an attractive target for protein design. The helices of coiled coils are characterized by a heptad repeat of residues denoted a to g. Residues at positions a and d form the interhelical interface and are usually hydrophobic. An established strategy to confer structural uniqueness to two-stranded coiled coils is the use of buried polar Asn residues at position a, which imparts dimerization and conformational specificity at the expense of stability. Here we show that polar interactions involving buried position-a Lys residues that can interact favorably only with surface e' or g' Glu residues also impart structural uniqueness to a designed heterodimeric coiled coil with the nativelike properties of sigmoidal thermal and urea-induced unfolding transitions, slow hydrogen exchange and lack of ANS binding. The position-a Lys residues do not, however, confer a single preference for helix orientation, likely reflecting the ability of Lys at position a to from favorable interactions with g' or e' Glu residues in the parallel and antiparallel orientations, respectively. The Lys-Glu polar interaction is less destabilizing than the Asn-Asn a-->a' interaction, presumably reflecting a higher desolvation penalty associated with the completely buried polar position-a groups. Our results extend the range of approaches for two-stranded coiled-coil design and illustrate the role of complementing polar groups associated with buried and surface positions of proteins in protein folding and design.     

Alanine-scanning mutagenesis of alpha-helix D segment of interleukin-13 reveals new functionally important residues of the cytokine

We documented that alpha-helices A, C, and D in human interleukin-13 (IL13) participate in interaction with its respective receptors. We hypothesized that alpha-helix D is the site II of the cytokine that binds IL13Ralpha1, a component of the normal tissue heterodimeric signaling IL13/4 receptor (IL13/4R), and that alpha-helix D independently binds a monomeric IL13Ralpha2 receptor, which is a non-signaling glioma-restricted receptor for IL13. Therefore, we alanine-scanned mutagenized helix D of IL13 to identify the residues involved in the respective receptors interaction. Recombinant muteins of IL13 were produced in Escherichia coli, and their structural integrity and identity were verified. The alanine mutants were tested in functional cellular assays, in which IL13 interaction with IL13Ralpha2 (glioma cells) or an ability to functionally stimulate IL13/4R (TF-1 cells) were examined, and also in binding assays. We found that residues 105, 106, and 109 of the d-helix of IL13 are responsible for interacting with the glioma-associated receptor. Moreover, glutamic acids at positions 92 and 110, and leucine at position 104 was found to be important for IL13/4R stimulation. Thus, alpha-helix D of IL13 is the primary site responsible for interaction with the IL13 binding proteins. We propose a model that illustrates the binding mode of IL13 with cancer-related IL13Ralpha2 and physiological IL13/4R.     

Charged residues in the M2 region of alpha-hENaC play a role in channel conductance

The epithelial Na⁺channel (ENaC) is a low-conductance channel that is highly selectivefor Na⁺ andLi⁺ overK⁺ and impermeable toanions. The molecular basis underlying these conductionproperties is not well known. Previous studies with the ENaC subunitsdemonstrated that the M2 region of -ENaC is critical to channelfunction. Here we examine the effects of reversing the negative chargesof highly conserved amino acids in -subunit human ENaC (-hENaC)M1 and M2 domains. Whole cell and single-channel currentmeasurements indicated that the M2 mutations E568R, E571R, and D575Rsignificantly decreased channel conductance but did not affectNa⁺:K⁺permeability. We observed no functional perturbations from the M1mutation E108R. Whole cell amiloride-sensitive current recorded fromoocytes injected with the M2 -hENaC mutants along with wild-type (wt) - and -hENaC was low (46-93 nA) compared with the wtchannel (1-3 µA). To determine whether this reduced macroscopiccurrent resulted from a decreased number of mutant channels at theplasma membrane, we coexpressed mutant -hENaC subunits with greenfluorescent protein-tagged - and -subunits. Confocal laserscanning microscopy of oocytes demonstrated that plasma membranelocalization of the mutant channels was the same as that of wt. Theseexperiments demonstrate that acidic residues in the secondtransmembrane domain of -hENaC affect ion permeation and are thuscritical components of the conductive pore of ENaC.

     

Charged residues are major determinants of the transmembrane orientation of a signal-anchor sequence

Uncleaved signal-anchor sequences of membrane proteins inserted into the endoplasmic reticulum initiate the translocation of either the amino-terminal or the carboxyl-terminal polypeptide segment across the bilayer. Which topology is acquired is not determined by the apolar segment of the signal but rather by the hydrophilic sequences flanking it. To study the role of charged residues in determining the membrane topology, the insertion of mutants of the asialoglycoprotein receptor H1, a single-spanning protein with a cytoplasmic amino terminus, was analyzed in transfected COS-7 cells. When the charged amino acids flanking the hydrophobic signal were mutated to residues of opposite charge, half the polypeptides inserted with the inverted orientation. When, in addition, the amino-terminal domain of the mutant protein was truncated, approximately 90% of the polypeptides acquired the inverted topology. The transmembrane orientation appears to be primarily determined by the charges flanking the signal sequence but is modulated by the domains to be translocated.     

Surface topography of histidine residues in lysozymes.

Several avian and mammalian c-type lysozymes were chromatographed on chelated (to iminodiacetate) and immobilized transition metal ions (Co2+, Ni2+, Cu2+ and Zn2+) under a variety of experimental conditions. The varied affinity of evolutionary variants of the lysozyme family for chelated metal ions, IDA-M(II), can be rationalized primarily in terms of the presence, multiplicity and microenvironments of histidine residues. The chromatographic resolution of some of these closely related proteins attests to the analytical power of immobilized metal-ion affinity chromatography.     

Charged residues in the beta2 subunit involved in GABAA receptor activation

Fast synaptic inhibition in the mammalian central nervous system is mediated primarily via activation of the gamma-aminobutyric acid type A receptor (GABAA-R). Upon agonist binding, the receptor undergoes a structural transition from the closed to the open state. This transition, known as gating, is thought to be associated with a sequence of conformational changes originating at the agonist-binding site, ultimately resulting in opening of the channel. Using site-directed mutagenesis and several different GABAA-R agonists, we identified a number of highly conserved charged residues in the GABAA-R beta2 subunit that appear to be involved in receptor activation. We then used charge reversal double mutants and disulfide trapping to investigate the interactions between these flexible loops within the beta2 subunit. The results suggest that interactions between an acidic residue in loop 7 (Asp146) and a basic residue in pre-transmembrane domain-1 (Lys215) are involved in coupling agonist binding to channel gating.     

Antagonistic RNA aptamer specific to a heterodimeric form of human interleukin-17A/F

Interleukin-17 (IL-17) is a pro-inflammatory cytokine produced primarily by a subset of CD4⁺ T cells, called Th17 cells, that is involved in host defense, inflammation and autoimmune disorders. The two most structurally related IL-17 family members, IL-17A and IL-17F, form homodimeric (IL-17A/A, IL-17F/F) and heterodimeric (IL-17A/F) complexes. Although the biological significance of IL-17A and IL-17F have been investigated using respective antibodies or gene knockout mice, the functional study of IL-17A/F heterodimeric form has been hampered by the lack of an inhibitory tool specific to IL-17A/F. In this study, we aimed to develop an RNA aptamer that specifically inhibits IL-17A/F. Aptamers are short single-stranded nucleic acid sequences that are selected in vitro based on their high affinity to a target molecule. One selected aptamer against human IL-17A/F, AptAF42, was isolated by repeated cycles of selection and counterselection against heterodimeric and homodimeric complexes, respectively. Thus, AptAF42 bound IL-17A/F but not IL-17A/A or IL-17F/F. The optimized derivative, AptAF42dope1, blocked the binding of IL-17A/F, but not of IL-17A/A or IL-17F/F, to the IL-17 receptor in the surface plasmon resonance assay in vitro. Consistently, AptAF42dope1 blocked cytokine GRO-α production induced by IL-17A/F, but not by IL-17A/A or IL-17F/F, in human cells. An RNA footprinting assay using ribonucleases against AptAF42dope1 in the presence or absence of IL-17A/F revealed that part of the predicted secondary structure fluctuates between alternate forms and that AptAF42dope1 is globally protected from ribonuclease cleavage by IL-17A/F. These results suggest that the selected aptamer recognizes a global conformation specified by the heterodimeric surface of IL-17A/F.     

NMR assignments of the human cytokine interleukin-33

We report near complete NMR backbone and side chain assignments of the human cytokine interleukin-33 (IL-33) in solution. IL-33 is the latest addition to the family of interleukin-1 homologous cytokines and was shown to be involved in inflammation and autoimmune diseases.     

Grouper interleukin-12, linked by an ancient disulfide-bond architecture,exhibits cytokine and chemokine activities

Interleukin-12 (IL-12) is a pleiotropic cytokine which bridges innate and adaptive immunity in defense against pathogens. IL-12 proved to be an effective and successful adjuvant to enhance both the innate and adaptive immune responses and could be applicable for a rationale vaccine formulation in fish against pathogen infection. We have cloned the p35 and p40 cDNAs of IL-12 from orange-spotted grouper (Epinephelus coioides). Grouper IL-12 most resembles with sea bass orthologues; moderate to low identity with other teleost and mammalian counterparts. The structural model of grouper IL-12 heterodimer revealed NC¹⁴¹F three amino acid patch of grouper p35, which is present in teleost p35 but absent in mammalian and avian p35, and is spatially nearby the conserved cysteine residue located at A-helix of p35 to form a disulfide bond when the 14aa peptide located at loop 1 of grouper p35 was aligned with human corresponding exon 4, instead of exon 5. The results indicated that the loss of this 3aa patch during evolution was compensated by the duplication of exon 4 in mammalian p35 to gain another cysteine residue to form a disulfide bond, evidenced by chicken p35 which does not contain NCF corresponding 3-aa patch nor exon 4 duplication. Accordingly, the inter-chain disulfide bond of IL-12 heterodimer is conserved from teleost to mammalian IL-12. A single chain grouper IL-12 (scgIL-12) construct linked by (G₄S)₃ was successfully expressed in baculovirus-insect cell system; its identity has been confirmed by LC/MS/MS. In addition, the biological activity of recombinant scgIL-12 (rscgIL-12) are demonstrated for its stimulation of PBL proliferation, chemotactic migration, induction of TNF-α gene expression and a plausible adjuvant effect of prolonged protection against parasite infection in fish. We illustrated the first time in lower vertebrate that grouper IL-12 possesses both cytokine and chemokine activities.     

Enhancement of the antiangiogenic activity of interleukin-12 by peptide targeted delivery of the cytokine to alphavbeta3 integrin

We engineered a fusion protein, mrIL-12vp [mouse recombinant interleukin (IL)-12 linked to vascular peptide], linking the vascular homing peptide CDCRGDCFC (RGD-4C), a ligand for alphavbeta3 integrin, to mrIL-12 to target IL-12 directly to tumor neovasculature. The fusion protein stimulated IFN-gamma production in vitro and in vivo, indicating its biological activity was consistent with mrIL-12. Immunofluorescence techniques showed mrIL-12vp specifically bound to alphavbeta3 integrin-positive cells but not to alphavbeta3 integrin-negative cells. In corneal angiogenesis assays using BALB/c mice treated with either 0.5 microg/mouse/d of mrIL-12vp or mrIL-12 delivered by subcutaneous continuous infusion, mrIL-12vp inhibited corneal neovascularization by 67% compared with only a slight reduction (13%) in angiogenesis in the mrIL-12-treated animals (P = 0.008). IL-12 receptor knockout mice given mrIL-12vp showed a marked decrease in the area of corneal neovascularization compared with mice treated with mrIL-12. These results indicate that mrIL-12vp inhibits angiogenesis through IL-12-dependent and IL-12-independent mechanisms, and its augmented antiangiogenic activity may be due to suppression of endothelial cell signaling pathways by the RGD-4C portion of the fusion protein. Mice injected with NXS2 neuroblastoma cells and treated with mrIL-12vp showed significant suppression of tumor growth compared with mice treated with mrIL-12 (P = 0.03). Mice did not show signs of IL-12 toxicity when treated with mrIL-12vp, although hepatic necrosis was present in mrIL-12-treated mice. Localization of IL-12 to neovasculature significantly enhances the antiangiogenic effect, augments antitumor activity, and decreases toxicity of IL-12, offering a promising strategy for expanding development of IL-12 for treatment of cancer patients.     

Crystal structure of the cytokine interleukin-1 beta.

The crystal structure of human recombinant interleukin-1 beta has been determined at 3.0 A resolution by the isomorphous replacement method in conjunction with solvent flattening techniques. The model prior to refinement has a crystallographic R-factor of 42.3%. The structure is composed of 12 beta-strands forming a complex network of hydrogen bonds. The core of the structure can best be described as a tetrahedron whose edges are each formed by two antiparallel beta-strands. The interior of this structure is filled with hydrophobic side chains. There is a 3-fold repeat in the folding of the polypeptide chain. Although this folding pattern suggests gene triplication, no strong internal sequence homology between topologically corresponding residues exists. The folding topology of interleukin-1 beta is very similar to that described by McLachlan (1979) J. Mol. Biol., 133, 557-563, for soybean trypsin inhibitor.     

Assignment of genes encoding a unique cytokine (IL12) composed of two unrelated subunits to chromosomes 3 and 5.

IL12 (formerly NKSF or CLMF) is a unique cytokine composed of two unrelated disulfide-linked subunits. The larger 40-kDa subunit (p40) is a member of the cytokine receptor family, and the smaller 35-kDa subunit (p35) is related to IL6 and GCSF. The chromosomal localization of these two subunits was determined by PCR analysis of DNA from rodent-human hybrids. More refined mapping was obtained by PCR analysis of hybrids containing translocation chromosomes and for p40, by analysis of radiation hybrids. The subunits map to different chromosomes: p40 (IL12B) to 5q31-q33 and p35 (IL12A) to 3p12-3q13.2.     

Interactions of interleukin-12A and interleukin-12B polymorphisms on the risk of intracranial aneurysm

Several lines of evidence indicate that inflammatory processes play pivotal role in the development of intracranial aneurysm (IA). Recently, polymorphisms in the interleukin-12 (IL-12) gene were shown to be associated with immune-mediated inflammatory disease. The aim of this study was to investigate the interactions of IL-12A and IL-12B polymorphisms on the risk of IA in a Chinese population. A total of 422 individuals (including 164 patients with IA and 258 controls) were involved in the study. The polymorphisms (i.e., rs2243115 and rs568408 in IL-12A and rs3212227 in IL-12B) were genotyped by polymerase chain reaction–restriction fragment length polymorphism assay and DNA sequencing. We found an association of the AC/CC genotypes and C allele of IL-12B rs3212227 with an increased risk of IA, compared with the AA genotype and A allele (AC/CC vs. AA: OR?=?2.09, 95?% CI: 1.29–3.38; C vs. A: OR?=?1.45, 95?% CI: 1.10–1.91). Moreover, a significant gene interaction of IL-12A and IL-12B was evident on the risk of IA, and subjects carrying variant genotypes of IL-12B rs3212227 had an increased risk of IA. In the stratified analysis by gender, the IL-12B rs3212227 AC/CC genotypes had an increased risk of IA compared with the AA genotype in male patients (AC/CC vs. AA: OR?=?4.63, 95?% CI: 1.92–11.16). These findings suggest that the IL-12A and IL-12B independently and jointly be involved in the susceptibility to IA.     

Gene-gene interactions between interleukin-12A and interleukin-12B with the risk of brain tumor

Emerging evidence from preclinical and clinical studies has shown that interleukin-12 (IL-12) has some effectiveness against endogenously arising brain tumor. The aim of this study was to investigate interactions of IL-12A and IL-12B polymorphisms on the risk of brain tumor. We analyzed IL-12A rs2243115 and IL-12B rs3212227 polymorphisms in 170 patients with brain tumor and 222 healthy controls in a Chinese population using a polymerase chain reaction-restriction fragment length polymorphism assay and DNA sequencing method. Individuals carrying a G allele of IL-12A rs2243115 had a significantly higher risk of developing brain tumor compared with those carrying a T allele (odds ratio [OR]=2.01, 95% confidence interval [CI], 1.17-3.45). After stratification analysis according to tumor types, a similarly higher risk was detected in patients with glioma (OR=2.56, 95% CI, 1.25-5.21). When gene-gene interactions were examined, carriers at both loci rs2243115 TG/GG and rs3212227 AC/CC had a 2.62-fold increased risk of glioma compared with those with rs2243115 TT and rs3212227 AC/CC genotypes (OR=2.62, 95% CI, 1.05-6.50). This study provides evidence that IL-12A rs2243115 may be associated with the risk of brain tumor. Additionally, gene-gene interactions of IL-12A rs2243115 and IL-12B rs3212227 may contribute to brain tumor susceptibility.