Institute of Immunology, University of Regensburg, Regensburg D-93042, Germany.
J Biol Chem. 2010 Mar 5;285(10):7028-34. doi: 10.1074/jbc.M109.091090. Epub 2010 Jan 12.
Beta-defensins are small antimicrobial polypeptides that are mainly expressed by epithelial cells and play an important role in the antimicrobial innate immune response. In addition to the direct microbicidal effects of these polypeptides, members of the beta-defensin super family have the capacity to promote local innate inflammatory and systemic adaptive immune responses, which are in part mediated by the CC-chemokine receptor CCR6. Here we report the expression of recombinant mBD4 and its human orthologue hBD2 fused to the constant domain of human IgG(1) to obtain correct folding and to increase stability and solubility using the Drosophila S2 expression system. Purified recombinant mBD4:Ig and hBD2:Ig fusion proteins retained potent antimicrobial activity against Gram-negative and Gram-positive bacteria. Furthermore, these beta-defensin fusion proteins showed specific binding to CCR6-expressing cells as revealed by flow cytometry. Interestingly, although hBD2:Ig bound to both human and mouse CCR6-expressing cells, mBD4:Ig did only bind to mCCR6-expressing cells but not to hCCR6-expressing cells. Both beta-defensin fusion proteins demonstrated chemotactic activity for cells expressing the mouse CC-chemokine receptor CCR6. The chemokine ligand CCL20 competed with the beta-defensin fusion proteins for specific binding to CCR6 as analyzed by fluorescence-activated cell sorter analysis. Both beta-defensin fusion proteins demonstrated chemotactic activity for cells expressing the mouse CCR6 receptor, but mBD4:Ig did not induce chemotactic activity of cells expressing human CCR6. This result supports our finding that mBD4 does not interact with human CCR6-expressing cells. Further evidence for specific interaction of the beta-defensin fusion proteins with CCR6-expressing cells is demonstrated by the observation that CCL20 and beta-defensin fusion proteins desensitize each other in inducing chemotactic activity. In addition both mBD4:Ig and hBD2:Ig demonstrated CCR6-independent chemotaxis of freshly isolated mouse resident peritoneal cells and human peripheral blood mononuclear cells, indicating the interaction with another chemotaxis-inducing receptor. Thus, the beta-defensin fusion proteins used in this study retained their biological activity and are a feasible tool to identify and analyze specific beta-defensin receptor interactions.
β-防御素是主要由上皮细胞表达的小抗菌多肽，在抗菌先天免疫反应中发挥重要作用。除了这些多肽的直接杀菌作用外，β-防御素超家族的成员还具有促进局部先天炎症和全身适应性免疫反应的能力，部分由 CC 趋化因子受体 CCR6 介导。在这里，我们报告了使用果蝇 S2 表达系统表达重组 mBD4 及其与人 IgG1 恒定区融合的人同源物 hBD2，以获得正确折叠并增加稳定性和溶解度。纯化的重组 mBD4：Ig 和 hBD2：Ig 融合蛋白保留了针对革兰氏阴性和革兰氏阳性细菌的强大抗菌活性。此外，这些β-防御素融合蛋白通过流式细胞术显示出与表达 CCR6 的细胞的特异性结合。有趣的是，尽管 hBD2：Ig 结合人源和鼠源表达 CCR6 的细胞，但 mBD4：Ig 仅结合鼠源 CCR6 表达细胞而不结合人源 CCR6 表达细胞。两种β-防御素融合蛋白均表现出对表达鼠源 CCR6 的细胞的趋化活性。用荧光激活细胞分选分析表明，趋化因子配体 CCL20 与β-防御素融合蛋白竞争与 CCR6 的特异性结合。两种β-防御素融合蛋白均表现出对表达鼠源 CCR6 受体的细胞的趋化活性，但 mBD4：Ig 不诱导表达人源 CCR6 的细胞的趋化活性。这一结果支持我们的发现，即 mBD4 不与表达人源 CCR6 的细胞相互作用。β-防御素融合蛋白与表达 CCR6 的细胞的特异性相互作用的进一步证据是观察到 CCL20 和β-防御素融合蛋白在诱导趋化活性方面相互脱敏。此外，mBD4：Ig 和 hBD2：Ig 均表现出对新鲜分离的鼠源固有腹膜细胞和人外周血单核细胞的 CCR6 非依赖性趋化作用，表明与另一种趋化诱导受体的相互作用。因此，本研究中使用的β-防御素融合蛋白保留了它们的生物学活性，是识别和分析特定β-防御素受体相互作用的可行工具。
