Expression of the human soluble interleukin 4 receptor in methylotropic yeast / 生物工程学报

Interleukin-4 (IL-4) is a pleiotropic cytokine which plays a pivotal role in shaping immune responses. IL-4 mediates important proinflammatory functions in asthma, including the IgE isotype switch. IL-4 exerts its biological effects through binding to its receptor (IL-4R) complex, with the alpha chain as the high affinity binding subunit. Soluble IL-4R lacks the transmemberane and cytoplasmic domains, so it cannot induce cellular activation. By acting as a decoy to circulating IL-4 and neutralize its activity, its high specificity and affinity make it ideal as an IL-4 antagonist. Some companies have embarked the clinical research for asthma treatment with the sIL-4R and the result revealed well therapeutic effect. With RNA extracted from human monocyte as the template, The sIL-4R cDNA encoding the extracellular domain of IL-4R a chain was obtained by RT-PCR. Compared with the sIL-4R encoding sequence in GenBank, the nucleotide sequencing analysis indicated that there was a A-->G mutation at 148bp and the mutation caused Ile-->Val at 50th amino acid. According to the references, numerous polymorphisms have been identified in the IL-4R gene and the Ile50Val was the only known extracellular variant of human IL-4R. Then the recombinant vector pPIC9K/sIL-4R was constructed, linearized and introduced into Pichia pastoris GS115 by electroporation. The recombinant sIL-4R was identified by SDS-PAGE, Western blot and Ligand binding blot. The SDS-PAGE and Western blot analysis showed that the apparent molecular weight of expressed sIL-4R was about 30kD. And the Ligand binding blot analysis indicated the expressed sIL-4R had the biological activity. The sIL-4R, which had the biological activity, was successfully secretorily expressed in the Pichia pastoris (GS 115).

Essential roles for ID-1 motif of interleukin-4 receptor alpha chain in interleukin-4 signaling / 천식및알레르기

BACKGROUND: Interleukin (IL)-4 is a pleiotropic cytokine that plays an important role in the pathogenesis of the allergic inflammation and asthma. Upon IL-4 receptor (IL-4R) engagement, a variety of signaling mediators, such as JAK kinases and STAT-6 are activated, leading to induction of IL-4 target gene expression including CD23 and germline C epsilon transcription. The function of a membrane-proximal domain of IL-4Ra, termed ID-1, remains to be characterized to date. OBJECTIVE: To assess whether the ID-1 domain mediates the induction of IL-4 target gene expression in a STAT-6-dependent manner. METHODS: The intracellular region of IL-4Ralpha was translationally fused to the extracellular region of IL-2Rbeta to provide ligand specificity to IL-2. Acidic amino acids and serine residues in the ID-1 domain of the chimeric receptor were substituted by site-directed mutagenesis. These receptor cDNAs were stably transfected to M12.4.1 murine B lymphoma cells. Following IL-2 stimulation, wild type and mutant clones for the ID-1 motif were subjected to FACS. RNA blotting and elecroporetic mobility shift assays to address the levels of CD23, germline C epsilon and STAT-6 inductions, respectively. RESULTS: ID-1 mutant clones were defective in gene induction of CD23 and germline C epsilon in response to IL-2 stimulation, as compared with wildtype clones. Moreover, IL-2-mediated STAT-6 activation was abolished in ID-1 mutant clones. CONCLUSION: These results demonstrate that the ID-1 domain of IL-4Ra is essential to induce IL-4 target gene expression through a STAT-6-dependent pathway.