Alternative Forms of IL-4 Gene
Development of Technique for Diagnosis and Monitoring IL-4delta2, Being a Product of Natural Alternative Splicing of Human IL-4 Gene, in Norm and Pathology: Laboratory and Clinical Studies
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
8 Project completed
Senior Project Manager
Weaver L M
Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany
- Institute of Immunology, Russia, Moscow
- Immunology Research Institute of New England, USA, MA, Fitchburg
Project summaryInterleukin-4 (IL-4) is a 15-kDa protein produced by activated T cells , mast cells, and basophils. It regulates a wide spectrum of cellular functions in hematopoietic and non-hematopoietic cells, therefore it is not surprising that the regulation of IL-4 activity is pivotal in determining the outcome of certain diseases - leshmaniasis, leprosy, schistosomiasis and allergic responses. Recently, a new mechanism for potential regulation of IL-4 activity was described, in which a second mRNA is alternatively spliced from the IL-4 gene. A naturally occurring splice variant of human IL-4, called IL-4delta2, was cloned, expressed, and its functional effects were tested. In IL-4delta2, the second exon (residues 22-37) of IL-4 is omitted by alternative splicing, with exons 1, 3, and 4 joined in an open reading frame. It was found that IL-4delta2 RNA is expressed in much higher levels than IL-4 RNA in thymocytes and bronchoalveolar lavage cells, suggesting tissue specificity of expression. IL-4delta2 cDNA was expressed in yeast. Recombinant human (rh) IL-4delta2 was partially purified and found to be glycosylated, with a protein core of 13 to 15 kDa. Unlike rhIL-4, rhIL-4delta2 did not act as a co-stimulator for T cell proliferation. However, rhIL-4delta2 inhibited the ability of rhIL-4 to act as a T cell co-stimulator. Iodinated IL-4delta2 was found to bind specifically to human PBMC and tumor lines known to express IL-4 receptors. Excess unlabelled IL-4 inhibited cellular binding of labeled IL-4delta2. Thus, hIL-4delta2 is a naturally occurring splice variant of IL-4 that is preferentially expressed in the thymus and airways and inhibits function of complete IL-4. The three-dimensional model of the hIL-4delta2 was developed taking into account that the second exon codes the main part of the long loop AB connected the helices A and B in parallel superposition. Therefore the hydrophobic core and the native fold of the rest part of IL-4delta2 molecule could be preserved without any significant changes only in the case of revolution of the helix A relatively to other helices. In the result, the dominated a left-handed four-helix bundle structure of IL-4 with an up-up-down-down structural patterns is transformed to the IL-4delta2 structure with a down-up-down-down structural patterns.
The project is based on the knowledge of (i) a new mechanism for potential regulation of IL-4 activity, in which a second mRNA is alternatively spliced from the IL-4 gene; (ii) predicted three-dimensional structure of the hIL-4delta2; (iii) importance of the switch from the IL-4 to IL-4delta2 expression at a recovery from bronchial asthma.
The aim of the Project
Development of the technique of rhIL-4delta2 diagnosis, its monitoring in patients with atopical bronchial asthma and pollinosis and the studies on correlation between clinico-immunological indexes of allergic patients and the level of rhIL-4 and rhIL-4delta2 including: clinical manifestation of disease, cutaneous tests with provocative allergens, free histamine and luecotrien tests, analysis of correlation of T-helpers and T-suppressers