tailieunhanh - Báo cáo Y học: Engineering and use of 32P-labeled human recombinant interleukin-11 for receptor binding studies

Human interleukin-11 (hIL-11) is a pleiotropic cytokine that is involved in numerous biological activities such as hematopoiesis, osteoclastogenesis, neurogenesis and female fertility. IL-11 is obviously a key reagent to study the IL-11 receptors. However, conventional radio-iodination techni-ques lead to a loss of IL-11 bioactivity. Here, we report the construction and the production of a new recombinant human IL-11 (FPDIL-11). In this molecule, a speci®c phosphorylation site (RRASVA) has been introduced at the N-terminus of rhIL-11. . | Eur. J. Biochem. 269 61-68 2002 FEBS 2002 Engineering and use of 32P-labeled human recombinant interleukin-11 for receptor binding studies Xiao-Ming Wang1 Jean-Marc Wilkin1 Olivier Boisteau2 Dimitri Harmegnies1 Chrystel Blanc3 Paul Vandenbussche1 Felix A. Montero-Julian3 Yannick Jacques2 and Jean Content1 1Institut Pasteur de Bruxelles Belgium 2Groupe Recherche CytokinesịRécepteurs Unite Institut de Biologie Nantes France 3Immunotech A Beckman-Coulter Company Marseille France Human interleukin-11 hIL-11 is a pleiotropic cytokine that is involved in numerous biological activities such as hematopoiesis osteoclastogenesis neurogenesis and female fertility. IL-11 is obviously a key reagent to study the IL-11 receptors. However conventional radio-iodination techniques lead to a loss of IL-11 bioactivity. Here we report the construction and the production of a new recombinant human IL-11 FPAIL-11 . In this molecule a specific phosphorylation site RRASVA has been introduced at the N-terminus of rhIL-11. It can be specifically phosphorylated by bovine heart protein kinase and accordingly easily radiolabeled with 32P. A high radiological specific activity 250 000 of protein was obtained with the retention of full biological activity of the protein. The binding of 32P-labeled FPAIL-11 to Ba F3 cells stably transfected with plasmids encoding human IL-11 receptors a and b chains IL-11Ra and gp130 was specific and saturable with a high affinity as determined from Scatchard plot analysis. Availability of this new ligand should prompt further studies on IL-11R structure expression and regulation. Keywords interleukin-11 cytokines phosphorylation radiolabeling receptor binding. Interleukin-11 IL-11 is a pleiotropic cytokine that has been shown to exhibit multiple effects on hematopoietic and nonhematopoietic systems including the liver gastrointestinal tract lung heart central nervous system bone joint and immune system 1 . IL-11 has hematopoietic and .