Kioi Mitomu, Seetharam Saraswathy, Puri Raj K
Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene Therapies, Food and Drug Administration, Center for Biologics Evaluation and Research, Bethesda, MD 20892, USA.
FASEB J. 2006 Nov;20(13):2378-80. doi: 10.1096/fj.06-5995fje. Epub 2006 Oct 3.
A high-affinity receptor for interleukin (IL)-13 (interleukin-13R alpha 2) is over-expressed in disease-related fibroblasts and neoplastic cells and is involved in cancer, allergic, and inflammatory diseases. The extracellular domain of IL-13R alpha2 (ECD alpha2) could be cleaved, which serves as a decoy receptor. We have expressed and purified ECD alpha2 in both Escherichia coli (E. coli) and mammalian systems as a soluble fragment and studied its biological activities. Although both products of ECD alpha2 showed IL-13 inhibitory activities, mammalian cell-derived ECD alpha2 appeared to be superior compared with purified protein from E. coli. When expressed in E. coli, ECD alpha2 appeared to be a monomer of 42 but a 60 kDa protein when purified from mammalian cells due to heavy glycosylation. The purified glycosylated ECD alpha2 efficiently inhibited IL-13-induced STAT6 phosphorylation in immune and Hodgkin's lymphoma cell lines, IL-13 binding, and cytotoxicity of IL-13 cytotoxin in various cancer cell lines. The improved potency of mammalian cell-derived ECD alpha2 was shown over ECD alpha2/Fc fusion protein. The N-linked glycosylation of ECD alpha2 was found to be essential for optimal IL-13 inhibitory activity as deglycosylation by PNGase F showed lower activity. ECD alpha2 did not inhibit IL-4-induced STAT6 phosphorylation, indicating that inhibitory effects of ECD alpha2 are receptor specific. These results indicate that glycosylated ECD alpha2 can serve as a potent inhibitor of IL-13 in a variety of conditions in which IL-13 is a key mediator, e.g., pulmonary, allergic, fibrotic, and neoplastic diseases.
白细胞介素（IL）-13的高亲和力受体（白细胞介素-13Rα2）在疾病相关的成纤维细胞和肿瘤细胞中过度表达，并参与癌症、过敏和炎症性疾病。IL-13Rα2的细胞外结构域（ECDα2）可被切割，作为诱饵受体发挥作用。我们已在大肠杆菌和哺乳动物系统中表达并纯化了作为可溶性片段的ECDα2，并研究了其生物学活性。尽管ECDα2的两种产物均显示出IL-13抑制活性，但与从大肠杆菌纯化的蛋白质相比，哺乳动物细胞来源的ECDα2似乎更具优势。当在大肠杆菌中表达时，ECDα2似乎是42 kDa的单体，但从哺乳动物细胞中纯化时则是60 kDa的蛋白质，这是由于其高度糖基化。纯化的糖基化ECDα2可有效抑制免疫细胞系和霍奇金淋巴瘤细胞系中IL-13诱导的STAT6磷酸化、IL-13结合以及IL-13细胞毒素在各种癌细胞系中的细胞毒性。与ECDα2/Fc融合蛋白相比，哺乳动物细胞来源的ECDα2显示出更高的效力。发现ECDα2的N-连接糖基化对于最佳的IL-13抑制活性至关重要，因为用PNGase F去糖基化后活性降低。ECDα2不抑制IL-4诱导的STAT6磷酸化，表明ECDα2的抑制作用具有受体特异性。这些结果表明，糖基化的ECDα2在IL-13作为关键介质的多种情况下，如肺部、过敏、纤维化和肿瘤疾病中，可作为有效的IL-13抑制剂。
