ABSTRACT
Cytokines regulate immune responses by binding to cell surface receptors, including the common subunit beta (ßc), which mediates signaling for GM-CSF, IL-3, and IL-5. Despite known roles in inflammation, the structural basis of IL-5 receptor activation remains unclear. We present the cryo-EM structure of the human IL-5 ternary receptor complex, revealing architectural principles for IL-5, GM-CSF, and IL-3. In mammalian cell culture, single-molecule imaging confirms hexameric IL-5 complex formation on cell surfaces. Engineered chimeric receptors show that IL-5 signaling, as well as IL-3 and GM-CSF, can occur through receptor heterodimerization, obviating the need for higher-order assemblies of ßc dimers. These findings provide insights into IL-5 and ßc receptor family signaling mechanisms, aiding in the development of therapies for diseases involving deranged ßc signaling.
Subject(s)
Cryoelectron Microscopy , Granulocyte-Macrophage Colony-Stimulating Factor , Interleukin-3 , Protein Multimerization , Receptors, Interleukin-5 , Signal Transduction , Humans , Binding Sites , Cytokine Receptor Common beta Subunit/metabolism , Cytokine Receptor Common beta Subunit/genetics , Cytokine Receptor Common beta Subunit/chemistry , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/chemistry , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , HEK293 Cells , Interleukin-3/metabolism , Interleukin-3/chemistry , Interleukin-3/genetics , Interleukin-5/metabolism , Models, Molecular , Protein Binding , Receptors, Interleukin-5/metabolism , Receptors, Interleukin-5/genetics , Receptors, Interleukin-5/chemistry , Single Molecule Imaging , Structure-Activity RelationshipABSTRACT
Protein-protein interactions (PPIs) are attractive targets as they are critical in a variety of biological processes and pathologies. As an illustration, the interleukin 3 (IL-3) and its α subunit receptor (IL-3Rα) are two proteins belonging to the cytokine or receptor ßc family and are involved in several disorders like inflammatory diseases or hematological malignancies. This PPI exhibits a low binding affinity and a complex formed by a mutant form of IL-3 (superkine) and IL-3Rα have emerged from the literature, with an increase of the affinity. Therefore, in this study, we performed molecular dynamics simulations and binding energy calculation in order to evaluate protein dynamics and to characterize the main interactions between IL-3 and IL-3Rα, considering both wild-type and mutant. First, in the case of IL-3Rα/IL-3 wild-type complex, IL-3Rα can adopt three different conformations essentially driven by NTD domain, including the open and closed conformations, previously observed in crystal structures. Additionally, our results reveal a third conformation that has a distinct interaction profile that the others. Interestingly, these conformational changes are attenuated in IL-3Rα/IL-3 mutant complex. Then, we highlighted the contribution of different residues which interact principally with IL-3 or IL-3Rα conserved region. As for the mutated residue at position 135 of IL-3, other residues such as IL-3 E138, IL-3 D40, IL-3Rα Y279, IL-3Rα K235, or IL-3Rα R277 seem important for a low or a high binding affinity. Altogether these findings yield new information that could be exploited in a drug discovery process.
Subject(s)
Interleukin-3 Receptor alpha Subunit , Interleukin-3 , Molecular Dynamics Simulation , Humans , Interleukin-3/chemistry , Molecular Conformation , Protein Binding , Interleukin-3 Receptor alpha Subunit/chemistryABSTRACT
Interleukin-3 (IL-3) is a cytokine belonging to the family of common ß (ßc) and is involved in various biological systems. Its activity is mediated by the interaction with its receptor (IL-3R), a heterodimer composed of two distinct subunits: IL-3Rα and ßc. IL-3 and its receptor, especially IL-3Rα, play a crucial role in pathologies like inflammatory diseases and therefore are interesting therapeutic targets. Here, we have performed an analysis of these proteins and their interaction based on structural and evolutionary information. We highlighted that IL-3 and IL-3Rα structural architectures are conserved across evolution and shared with other proteins belonging to the same ßc family interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The IL-3Rα/IL-3 interaction is mediated by a large interface in which most residues are surprisingly not conserved during evolution and across family members. In spite of this high variability, we suggested small regions constituted by few residues conserved during the evolution in both proteins that could be important for the binding affinity.
Subject(s)
Evolution, Molecular , Interleukin-3/chemistry , Receptors, Interleukin-3/chemistry , Amino Acid Sequence , Animals , Humans , Interleukin-3/genetics , Protein Conformation , Receptors, Interleukin-3/genetics , Sequence Homology, Amino AcidABSTRACT
The interleukin-3 (IL-3) receptor is a cell-surface heterodimer that links the haemopoietic, vascular and immune systems and is overexpressed in acute and chronic myeloid leukaemia progenitor cells. It belongs to the type I cytokine receptor family in which the α-subunits consist of two fibronectin III-like domains that bind cytokine, and a third, evolutionarily unrelated and topologically conserved, N-terminal domain (NTD) with unknown function. Here we show by crystallography that, while the NTD of IL3Rα is highly mobile in the presence of IL-3, it becomes surprisingly rigid in the presence of IL-3 K116W. Mutagenesis, biochemical and functional studies show that the NTD of IL3Rα regulates IL-3 binding and signalling and reveal an unexpected role in preventing spontaneous receptor dimerisation. Our work identifies a dual role for the NTD in this cytokine receptor family, protecting against inappropriate signalling and dynamically regulating cytokine receptor binding and function.
Subject(s)
Interleukin-3 Receptor alpha Subunit/chemistry , Interleukin-3 Receptor alpha Subunit/metabolism , Protein Domains , Signal Transduction , Amino Acid Sequence , Animals , Binding Sites/genetics , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Crystallography, X-Ray , HEK293 Cells , Humans , Interleukin-3/chemistry , Interleukin-3/genetics , Interleukin-3/metabolism , Interleukin-3 Receptor alpha Subunit/genetics , Molecular Dynamics Simulation , Mutation , Protein BindingABSTRACT
Human interleukin-3 (hIL-3) is a pleiotropic cytokine that stimulates the differentiation and proliferation of multipotent hematopoietic cells thus making it a therapeutically important molecule. In this study, its poor expression yield was improved by addressing various upstream bottlenecks in E. coli heterologous system. The codon-optimized hIL-3 gene was cloned under various signal sequences and solubility enhancer fusion tags for its hyper-expression under a strong T7 promoter. The optimization of shake flask expression studies resulted in a hIL-3 protein concentration of 225 mg/L in the form of inclusion bodies (IBs). Lowering of inducer concentration and cultivation temperature did not improve its solubility. The hIL-3 protein was refolded from IBs and resulted a protein recovery yield of 53% after optimization of refolding conditions. The refolded protein was subsequently purified using Ni-NTA affinity chromatography and gave â¼95% pure protein. The conformational properties of the refolded hIL-3 protein were studied by CD and fluorescence spectrometry where protein showed 40% α-helix and 12% ß-sheets with a fluorescence emission maxima at 344 nm. The molecular identity was further confirmed by MALDI-TOF/TOF and western blot analysis. The biological activity of refolded protein was confirmed via cell proliferation assay on human erythroleukemia TF-1 cells where commercial hIL-3 was taken as a standard control.
Subject(s)
Gene Expression , Interleukin-3 , Protein Refolding , Cell Line , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Interleukin-3/biosynthesis , Interleukin-3/chemistry , Interleukin-3/genetics , Interleukin-3/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purificationABSTRACT
Human interleukin-3 (hIL-3) is a therapeutically important cytokine involved in the maturation and differentiation of various cells of the immune system. The codon-optimized hIL-3 gene was cloned in fusion with the N-terminus α-mating factor signal peptide of Saccharomyces cerevisiae under an inducible alcohol oxidase 1 (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. A Zeocin concentration up to 2000 mg/L was used to select hyper-producers. The shake flask cultivation studies in the Pichia pastoris GS115 host resulted a maximum recombinant hIL-3 expression level of 145 mg/L in the extracellular medium under the control of AOX1 promoter. The batch fermentation strategy allowed us to attain a fairly pure glycosylated hIL-3 protein in the culture supernatant at a final concentration of 475 mg/L with a high volumetric productivity of 4.39 mg/L/h. The volumetric product concentration achieved at bioreactor level was 3.28 folds greater than the shake flask results. The 6x His-tagged protein was purified using Ni-NTA affinity chromatography and confirmed further by western blot analysis using anti-6x His tag antibody. The glycosylation of recombinant hIL-3 protein was confirmed in a PNGase F deglycosylation reaction where it showed a molecular weight band pattern similar to E. coli produced non-glycosylated hIL-3 protein. The structural properties of recombinant hIL-3 protein were confirmed by CD and fluorescence spectroscopy where protein showed 40 % α-helix, 12 % ß-sheets with an emission maxima at 343 nm. MALDI-TOF-TOF analysis was used to establish the protein identity. The biological activity of purified protein was confirmed by the human erythroleukemia TF-1 cell proliferation assay.
Subject(s)
Interleukin-3/biosynthesis , Pichia/genetics , Alcohol Oxidoreductases/genetics , Bioreactors , Codon , Fermentation , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Humans , Interleukin-3/chemistry , Interleukin-3/genetics , Pichia/metabolism , Promoter Regions, Genetic , Protein Sorting Signals/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolismABSTRACT
Greatly expanded brain volume is one of the most characteristic traits that distinguish humans from other primates. Recent studies have revealed genes responsible for the dramatically enlarged human brain size (i.e., the microcephaly genes), and it has been well documented that many microcephaly genes have undergone accelerated evolution along the human lineage. In addition to being far larger than other primates, human brain volume is also highly variable in general populations. However, the genetic basis underlying human brain volume variation remains elusive and it is not known whether genes regulating human brain volume variation also have experienced positive selection. We have previously shown that genetic variants (near the IL3 gene) on 5q33 were significantly associated with brain volume in Chinese population. Here, we provide further evidence that support the significant association of genetic variants on 5q33 with brain volume. Bioinformatic analyses suggested that rs31480 is likely to be the causal variant among the studied SNPs. Molecular evolutionary analyses suggested that IL3 might have undergone positive selection in primates and humans. Neutrality tests further revealed signatures of positive selection of IL3 in Han Chinese and Europeans. Finally, extended haplotype homozygosity (EHH) and relative EHH analyses showed that the C allele of SNP rs31480 might have experienced recent positive selection in Han Chinese. Our results suggest that IL3 is an important genetic regulator for human brain volume variation and implied that IL3 might have experienced weak or modest positive selection in the evolutionary history of humans, which may be due to its contribution to human brain volume.
Subject(s)
Adaptation, Physiological , Brain/anatomy & histology , Evolution, Molecular , Interleukin-3/genetics , Amino Acid Sequence , Animals , China , Humans , Interleukin-3/chemistry , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
Interleukin-3 (IL-3) is a cytokine secreted by mast cells and activated T-cells known to be an important regulator of differentiation, survival, proliferation and activation of a range of haemopoietic lineages. The effects of IL-3 on target cells are mediated by a transmembrane receptor system composed of a cytokine-specific α-subunit and a ß-subunit, the principal signalling entity. In the mouse, two ß-subunits have co-evolved: a common ß-subunit (ßc) shared between IL-3 and the related cytokines IL-5 and granulocyte/macrophage colony-stimulating factor (GM-CSF); and an IL-3-specific ß-subunit (ßIL-3). ßIL-3 differs from ßc in its specificity for IL-3 and its capacity to bind IL-3 directly in the absence of an α-subunit, and, in the absence of structural information, the basis for these properties has remained enigmatic. In the present study, we have solved the crystal structure of the ßIL-3 ectodomain at 3.45 Å (1 Å=0.1 nm) resolution. This structure provides the first evidence that ßIL-3 adopts an arch-shaped intertwined homodimer with similar topology to the paralogous ßc structure. In contrast with apo-ßc, however, the ligand-binding interface of ßIL-3 appears to pre-exist in a conformation receptive to IL-3 engagement. Molecular modelling of the IL-3-ßIL-3 interface, in conjunction with previous mutational studies, suggests that divergent evolution of both ßIL-3 and IL-3 underlies their unique capacity for direct interaction and specificity.
Subject(s)
Cytokine Receptor Common beta Subunit/chemistry , Interleukin-3/chemistry , Animals , Crystallography, X-Ray , Cytokine Receptor Common beta Subunit/metabolism , Evolution, Molecular , Interleukin-3/metabolism , Mice , Models, Molecular , Protein Conformation , Protein Multimerization , Protein Structure, Tertiary , Recombinant Proteins/chemistryABSTRACT
Human interleukin-3 (hIL-3) is a polypeptide growth factor that regulates the proliferation, differentiation, survival and function of hematopoietic progenitors and many mature blood cell lineages. Although recombinant hIL-3 is a widely used laboratory reagent in hematology, standard methods for its preparation, including those employed by commercial suppliers, remain arduous owing to a reliance on refolding insoluble protein expressed in E. coli. In addition, wild-type hIL-3 is a poor substrate for radio-iodination, which has been a long-standing hindrance to its use in receptor binding assays. To overcome these problems, we developed a method for expression of hIL-3 in E. coli as a soluble protein, with typical yields of >3mg of purified hIL-3 per litre of shaking microbial culture. Additionally, we introduced a non-native tyrosine residue into our hIL-3 analog, which allowed radio-iodination to high specific activities for receptor binding studies whilst not compromising bioactivity. The method presented herein provides a cost-effective and convenient route to milligram quantities of a hIL-3 analog with wild-type bioactivity that, unlike wild-type hIL3, can be efficiently radio-iodinated for receptor binding studies.
Subject(s)
Escherichia coli/genetics , Interleukin-3/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Chromatography, Reverse-Phase , DNA Primers , Humans , Interleukin-3/chemistry , Interleukin-3/genetics , Interleukin-3/physiology , Mass Spectrometry , Mice , Molecular Sequence Data , Sequence Homology, Amino Acid , SolubilityABSTRACT
Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are members of a discrete family of cytokines that regulates the growth, differentiation, migration and effector function activities of many hematopoietic cells and immunocytes. These cytokines are involved in normal responses to infectious agents, bridging innate and adaptive immunity. However, in certain cases, the overexpression of these cytokines or their receptors can lead to excessive or aberrant initiation of signaling resulting in pathological conditions, with chronic inflammatory diseases and myeloid leukemias the most notable examples. Recent crystal structures of the GM-CSF receptor ternary complex and the IL-5 binary complex have revealed new paradigms of cytokine receptor activation. Together with a wealth of associated structure-function studies, they have significantly enhanced our understanding of how these receptors recognize cytokines and initiate signals across cell membranes. Importantly, these structures provide opportunities for structure-based approaches for the discovery of novel and disease-specific therapeutics. In addition, recent biochemical evidence has suggested that the GM-CSF/IL-3/IL-5 receptor family is capable of interacting productively with other membrane proteins at the cell surface. Such interactions may afford additional or unique biological activities and might be harnessed for selective modulation of the function of these receptors in disease.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/chemistry , Interleukin-3/chemistry , Interleukin-5/chemistry , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/chemistry , Receptors, Interleukin-3/chemistry , Receptors, Interleukin-5/chemistry , Crystallography, X-Ray , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Inflammation/immunology , Inflammation/metabolism , Inflammation/pathology , Interleukin-3/immunology , Interleukin-3/metabolism , Interleukin-5/immunology , Interleukin-5/metabolism , Leukemia, Myeloid/immunology , Leukemia, Myeloid/metabolism , Leukemia, Myeloid/pathology , Lymphocytes/immunology , Lymphocytes/metabolism , Lymphocytes/pathology , Models, Molecular , Protein Binding , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Receptors, Interleukin-3/immunology , Receptors, Interleukin-3/metabolism , Receptors, Interleukin-5/immunology , Receptors, Interleukin-5/metabolism , Signal Transduction , Structure-Activity RelationshipABSTRACT
Yeast Pichia pastoris has been widely utilized to express heterologous recombinant proteins. P. pastoris expressed recombinant porcine interleukin 3 (IL3) has been used for porcine stem cell mobilization in allo-hematopoietic cell transplantation models and pig-to-primate xeno-hematopoietic cell transplantation models in our lab for many years. Since the yeast glycosylation mechanism is not exactly the same as those of other mammalian cells, P. pastoris expressed high-mannose glycoprotein porcine IL3 has been shown to result in a decreased serum half-life. Previously this was avoided by separation of the non-glycosylated porcine IL3 from the mixture of expressed glycosylated and non-glycosylated porcine IL3. However, this process was very inefficient and lead to a poor yield following purification. To overcome this problem, we engineered a non-N-glycosylated version of porcine IL3 by replacing the four potential N-glycosylation sites with four alanines. The codon-optimized non-N-glycosylated porcine IL3 gene was synthesized and expressed in P. pastoris. The expressed non-N-glycosylated porcine IL3 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50 HQ. In vivo mobilization studies performed in our research facility demonstrated that the non-N-glycosylated porcine IL3 still keeps the original stem cell mobilization function.
Subject(s)
Interleukin-3/genetics , Interleukin-3/isolation & purification , Pichia/genetics , Swine/immunology , Amino Acid Sequence , Animals , Base Sequence , Gene Expression , Glycosylation , Interleukin-3/chemistry , Molecular Sequence Data , Plasmids/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Swine/geneticsABSTRACT
Full-length cDNA (435 bp) of the interleukin-3(IL-3) gene of the Indian water buffalo was amplified by reverse transcriptase-polymerase chain reaction and sequenced. This sequence had 96% nucleotide identity and 92% amino acid identity with bovine IL-3. There are 10 amino acid substitutions in buffalo compared with that of bovine. The amino acid sequence of buffalo IL-3 also showed very high identity with that of other ruminants, indicating functional cross-reactivity. Structural homology modelling of buffalo IL-3 protein with human IL-3 showed the presence of five helical structures.
Subject(s)
Buffaloes/genetics , DNA, Complementary/genetics , Interleukin-3/genetics , Amino Acid Sequence , Animals , Cattle , Cloning, Molecular , DNA, Complementary/chemistry , Humans , India , Interleukin-3/chemistry , Interleukin-3/classification , Molecular Sequence Data , Phylogeny , Protein Structure, Secondary , Ruminants/genetics , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
AIM: to construct a eukaryolic expression plasmid of Ppic9k-IL3-Linker-PE38KDE. METHODS: the IL3 and PE38KDEL gene were amplified by polymerase chain reaction (PCR) and cloned into the eukaryolic expression plasmid Ppic9k-Linker constructed after being sequenced.The recombinant vector confirmed by restriction endonucleases digestion, coenobium PCR and DNA sequence analysis showed that the prokaryotic expression vector Ppic9k-IL3-Linker-PE38KDEL was constructed successfully. CONCLUSION: the fusion gene IL3-PE38KDEL is successfully constructed, which laid a solid foundation for the further research.
Subject(s)
Genetic Vectors/chemical synthesis , Immunotoxins/chemistry , Interleukin-3/genetics , Pichia/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Biomarkers, Tumor/chemistry , Biomarkers, Tumor/genetics , Cloning, Molecular/methods , Genetic Vectors/chemistry , Genetic Vectors/genetics , Humans , Immunotoxins/immunology , Interleukin-3/biosynthesis , Interleukin-3/chemistry , Leukemia/pathology , Leukemia/therapy , Pichia/metabolism , Polymerase Chain Reaction/methods , Sequence Analysis, DNA/methodsABSTRACT
Interleukin-3 (IL-3), a cytokine produced primarily by activated T-cells during immune responses, is a crucial regulator of allergic inflammation. The three-dimensional structure of murine IL-3 (mIL-3) has remained elusive owing to its poor solubility and strong tendency toward aggregation under solution conditions typically used for structural studies. Here we describe the solution properties and structure of mIL-3 determined by NMR using an engineered construct of mIL-3 (mIL-3(33-156)). mIL-3 adopts a four-helical bundle fold, typical of proteins belonging to the short-chain cytokine family, and features a core of highly conserved hydrophobic residues. While significant line broadening and peak disappearance were observed in NMR spectra at higher temperatures, there was no evidence for temperature-dependent changes of the oligomeric state of mIL-3(33-156). Further analysis of the temperature dependence of amide (1)H chemical shifts and backbone (15)N relaxation parameters, including (15)N relaxation dispersion, revealed the presence of significant conformational exchange and local conformational heterogeneity. Residues recently shown by mutagenesis to play key roles in ß(IL-3) receptor recognition and activation, which are located within the α(A) and α(C) helices and aligned on one face of the mIL-3(33-156) structure, are relatively rigid. In contrast, pronounced conformational heterogeneity was observed for a cluster of residues located on the opposite side of mIL-3, which corresponds spatially to sites in the related cytokines human IL-3, IL-5, and GM-CSF that are known to mediate interactions with their respective α-receptor subunits. Such conformational heterogeneity may facilitate the interaction of mIL-3 with each of two naturally occurring mIL-3Rα isoforms, leading to structurally distinct high-affinity complexes.
Subject(s)
Interleukin-3/chemistry , Peptide Fragments/chemistry , Animals , Hydrogen-Ion Concentration , Interleukin-3/genetics , Mice , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/genetics , Protein Conformation , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Solubility , TemperatureABSTRACT
The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit overlapping activities in the regulation of hematopoietic cells. In humans, the common beta (betac) receptor is shared by the three cytokines and functions together with cytokine-specific alpha subunits in signaling. A widely accepted hypothesis is that receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in human betac (hbetac) and unidentified cysteine residues in the N-terminal domains of the alpha receptors. Since the development of this hypothesis, new data have been obtained showing that domain 1 of hbetac is part of the cytokine binding epitope of this receptor and that an IL-3Ralpha isoform lacking the N-terminal Ig-like domain (the "SP2" isoform) is competent for signaling. We therefore investigated whether distortion of the domain 1-domain 4 ligand-binding epitope in hbetac and the related mouse receptor, beta(IL-3), could account for the loss of receptor signaling when the domain 1 D-E loop disulfide is disrupted. Indeed, mutation of the disulfide in hbetac led to both a complete loss of high affinity binding with the human IL-3Ralpha SP2 isoform and of downstream signaling. Mutation of the orthologous residues in the mouse IL-3-specific receptor, beta(IL-3), not only precluded direct binding of mouse IL-3 but also resulted in complete loss of high affinity binding and signaling with the mouse IL-3Ralpha SP2 isoform. Our data are most consistent with a role for the domain 1 D-E loop disulfide of hbetac and beta(IL-3) in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signaling.
Subject(s)
Interleukin-3/chemistry , Receptors, Interleukin-3/physiology , Amino Acid Sequence , Animals , COS Cells , Cell Proliferation , Chlorocebus aethiops , Disulfides/chemistry , Humans , Mice , Molecular Sequence Data , Mutation , Protein Isoforms , Protein Structure, Tertiary , Receptors, Interleukin-3/chemistry , Signal TransductionABSTRACT
The cytokine interleukin-3 (IL-3) is a critical regulator of inflammation and immune responses in mammals. IL-3 exerts its effects on target cells via receptors comprising an IL-3-specific alpha-subunit and common beta-subunit (beta c; shared with IL-5 and granulocyte-macrophage colony-stimulating factor) or a beta-subunit that specifically binds IL-3 (beta(IL-3); present in mice but not humans). We recently identified two splice variants of the alpha-subunit of the IL-3 receptor (IL-3R alpha) that are relevant to hematopoietic progenitor cell differentiation or proliferation: the full length ("SP1" isoform) and a novel isoform (denoted "SP2") lacking the N-terminal Ig-like domain. Although our studies demonstrated that each mouse IL-3 (mIL-3) R alpha isoform can direct mIL-3 binding to two distinct sites on the beta(IL-3) subunit, it has remained unclear which residues in mIL-3 itself are critical to the two modes of beta(IL-3) recognition and whether the human IL-3R alpha SP1 and SP2 orthologs similarly instruct human IL-3 binding to two distinct sites on the human beta c subunit. Herein, we describe the identification of residues clustering around the highly conserved A-helix residue, Glu(23), in the mIL-3 A- and C-helices as critical for receptor binding and growth stimulation via the beta(IL-3) and mIL-3R alpha SP2 subunits, whereas an overlapping cluster was required for binding and activation of beta(IL-3) in the presence of mIL-3R alpha SP1. Similarly, our studies of human IL-3 indicate that two different modes of beta c binding are utilized in the presence of the hIL-3R alpha SP1 or SP2 isoforms, suggesting a possible conserved mechanism by which the relative orientations of receptor subunits are modulated to achieve distinct signaling outcomes.
Subject(s)
Cytokine Receptor Common beta Subunit/metabolism , Epitopes/metabolism , Interleukin-3/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cell Line , Cell Proliferation , Cytokine Receptor Common beta Subunit/chemistry , DNA Mutational Analysis , Enzyme Activation , Glutamic Acid/metabolism , Humans , Interleukin-3/chemistry , Interleukin-3 Receptor alpha Subunit/metabolism , Janus Kinase 2/metabolism , MAP Kinase Signaling System , Mice , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation/genetics , Protein Isoforms/chemistry , Protein Isoforms/metabolismABSTRACT
Interleukin-3 (IL-3) is a cytokine that acts as a critical mediator of inflammation and immune responses to infections. IL-3, like interleukin-5 (IL-5) and granulocyte-macrophage colony stimulating factor (GM-CSF), exerts its effects on target cells via receptors composed of cytokine-specific alpha-subunits and a common beta-subunit (betac-subunit, shared with IL-5 and GM-CSF). In contrast to humans, mice also possess an additional beta-receptor, beta(IL-3), that can specifically bind IL-3. Except for a study carried out on an analogue of human IL-3 that contains 14 mutations, structure-related studies of IL-3 have been very limited, largely because of its poor solution behaviour. Here we report (1)H, (13)C, and (15)N chemical shift assignments of murine IL-3 comprising residues 33-156 (SWISS-PROT accession number: P01586), in which the only mutation is an alanine substitution of Cys105. The mIL-3 construct used in the present study was engineered by eliminating residues 27-32 of the N-terminus (the first 26 residues of the primary sequence of mIL-3 are cleaved in vivo during secretion), the C-terminal 10 residues (157-166), and a disulfide bond between Cys105 and Cys166 that is poorly conserved in orthologue sequences. The new construct vastly improves the solubility of murine IL-3 while maintaining its wild-type biological activity.
Subject(s)
Interleukin-3/chemistry , Amino Acid Sequence , Animals , Carbon Isotopes/chemistry , Humans , Hydrogen/chemistry , Interleukin-3/analogs & derivatives , Interleukin-3/genetics , Mice , Mutation, Missense , Nitrogen Isotopes/chemistry , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Secondary , Software , Solubility , Structural Homology, ProteinABSTRACT
Mouse interleukin-3 (mIL-3) is a critical cytokine regulator of myeloid cell differentiation, survival and activation, and consequently this cytokine has become a key reagent for hematological studies in the laboratory. Although bacterial expression has been used for the preparation of recombinant mIL-3 for more than 20 years, the resultant cytokine is known to exhibit poor solubility, be prone to aggregation, and may contain mispaired disulfide bonds. As a result, little structural characterization of mIL-3 has been possible to date. In the present work, we describe a convenient, inexpensive, and scalable protocol for preparing an mIL-3 analog with wild-type bioactivity from Escherichia coli via a simple purification scheme. This analog is typically expressed at >1 mg/l of shaking Super broth culture and, owing to solubility >5 mg/ml, structural studies in solution by nuclear magnetic resonance spectroscopy are feasible for mIL-3 for the first time.
Subject(s)
Escherichia coli/metabolism , Interleukin-3 , Protein Engineering/methods , Animals , Base Sequence , Cytokines/genetics , Cytokines/metabolism , Escherichia coli/genetics , Escherichia coli/growth & development , Interleukin-3/chemistry , Interleukin-3/genetics , Interleukin-3/isolation & purification , Interleukin-3/metabolism , Magnetic Resonance Spectroscopy , Mice , Mutagenesis, Site-Directed , SolubilityABSTRACT
We have produced clinical grade of DTIL3K116W, a variant diphtheria toxin-interleukin-3 fusion protein, for treatment of acute myeloid leukemia. The product was filter sterilized, aseptically vialed, and stored at -80 degrees C. It was characterized by Coomassie-stained SDS-PAGE, endotoxin assay, cytotoxicity assay, sterility, mass spectroscopy, receptor binding affinity, ADP-ribosylation, inhibition of normal human CFU-GM, disulfide bond analysis, immunoblots, stability, size exclusion chromatography-HPLC, sequencing, and immunohistochemistry. Vialed product was sterile in 0.25 M NaCl/5 mM Tris, pH 7.9, and had a protein concentration of 1.08 mg/ml. Purity by SDS-PAGE was >99%. Aggregates by HPLC were <1%. Endotoxin levels were 0.296EU/mg. Peptide mapping and mass spectroscopy confirmed its composition and molecular weight. The vialed drug kept reactivity with anti-IL3 and DT antibodies. Potency study revealed a 48-h EC(50) of 0.5 pM on TF1/H-ras cell. Its binding properties were confirmed by competitive experiments showing IC(50) of 1.4 nM. ADP-ribosylation activity was equivalent to DTGM-CSF. Drug did not react with tested frozen human tissue sections by immunohistochemistry. There was no evidence of loss of solubility, proteolysis aggregation, or loss of potency over 6 months at -80 degrees C. Further, the drug was stable at 4 and 25 degrees C in the plastic syringe and administration tubing for 48 h.
Subject(s)
Clinical Trials, Phase I as Topic/methods , Diphtheria Toxin/pharmacology , Interleukin-3/pharmacology , Recombinant Fusion Proteins/pharmacology , Amino Acid Substitution , Bone Marrow Cells/drug effects , Bone Marrow Cells/pathology , Cells, Cultured , Diphtheria Toxin/adverse effects , Diphtheria Toxin/chemistry , Diphtheria Toxin/genetics , Drug Compounding/methods , Drug Contamination/prevention & control , Drug Evaluation, Preclinical , Drug Stability , Genetic Variation/physiology , Humans , Interleukin-3/adverse effects , Interleukin-3/chemistry , Interleukin-3/genetics , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/pathology , Lysine/genetics , Recombinant Fusion Proteins/adverse effects , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Sterilization , Toxicity Tests , Tryptophan/geneticsABSTRACT
The objective was to determine the effects of adding progesterone, 17beta-estradiol (17beta-E2), and FSH during in vitro fertilization on development and sex ratio of murine embryos. Progesterone (33-330 pg/mL), 17beta-estradiol (17beta-E2); 10-70 pg/mL), and FSH (0.01-0.05 IU/mL), were added to human tubal fluid (HTF); this medium (with or without hormones) was used to pre-incubate sperm (2h) and to co-incubate sperm and oocytes (6h). Thereafter, the ova were washed and incubated in mM16 medium and embryo sex was determined (by PCR) on Day 4 (insemination=Day 0). There was no effect (P>0.05) of hormone treatments on rates of cleavage (6 h after cessation of co-incubation with sperm). The only significant effects of added hormones on development were a decrease in the rate of development to at least the morula stage in 165 pg/mL progesterone (0.46+/-0.03 vs. 0.54+/-0.05 in the control, mean+/-S.D.; P<0.05) and a decrease in the blastocyst rate in 0.03 IU/mL FSH (0.34+/-0.00 vs. 0.42+/-0.04 in the control, P<0.05). However, the ratio of male to female embryos was 1.61 and 2.90 following the addition of 99 pg/mL progesterone and 70 pg/mL 17beta-E2, respectively; both of these ratios were different (P<0.01) than in the control group (1.20). In contrast, the addition of FSH to the medium had no significant effect on this ratio (range, 0.78-1.02). We concluded that the addition of progesterone and estradiol to the media during in vitro fertilization did not enhance embryonic development, but significantly increased the proportion of male murine embryos.