Biochemical and functional analysis of an IL-15 alternative splice variant
| Autor: | Chih-Hsiu Wang, 王之秀 |
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| Rok vydání: | 2010 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 98 Interleukin 15 (IL-15) is a pleiotropic cytokine which plays an essential role in innate and adaptive immune cell function and homeostasis. Expression of IL-15 protein is known to be tightly controlled at transcriptional and translational levels. Whether alternative pre-mRNA splicing is also involved in regulating IL-15 function is not clear. In this study, the biochemical properties and biological functions of IL-15ΔE7 which is encoded from an alternatively spliced mRNA having partial deletion in the exon 7 of IL-15 gene were assessed in vitro. The full length of IL-15 and IL-15ΔE7 cDNAs encoding the mature protein was cloned individually in an expression plasmid flanked with IL-2 leader peptide at the N-terminus and a FLAG tag at the C-terminus and were expressed in COS-7 cells by transient transfection method. Both IL-15 and IL-15ΔE7 were sensitive to Endo H treatment. However, their banding patterns on SDS-PAGE were different by Western blotting, suggesting the post-translational modifications on these proteins were different. Most of the IL-15ΔE7 protein retained intracellularly and accumulated in the ER as identified by colocalization with ER chaperone calnexin. Very few IL-15ΔE7 protein was secreted and failed to support HT-2 cell proliferation as well as inhibited the biological activity of IL-2 and IL-15 in a dose dependent manner. In addition, flow cytometric analysis showed that the exogenous addition of IL-15ΔE7 reduced the upregulation of surface IL-15 receptor α (IL-15Rα) induced by IL-15. Co-transfection of IL-15ΔE7 and IL-15Rα led to the intracellular accumulation of IL-15Rα which might inhibit the trans-presentation by limiting expression of IL-15Rα on the cell surface. In summary, IL-15ΔE7 was accumulated in the ER without efficient secretion. Once secreted, the extremely low amount of IL-15ΔE7 may have a regulatory role for IL-15 signaling by limiting IL-15Rα surface expression. The functional mechanism as well as the biological significance of this alternative splice variant remain to be clarified. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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