Department of Bioscience and Biotechnology, BMIC, Konkuk University, Seoul 143-701.
Laboratory of Cytokine Immunology, Institute of Biomedical Science and Technology, College of Medicine, Konkuk University, Seoul 143-701.
J Biol Chem. 2011 Dec 2;286(48):41296-41311. doi: 10.1074/jbc.M111.269225. Epub 2011 Sep 29.
Papiliocin is a novel 37-residue cecropin-like peptide isolated recently from the swallowtail butterfly, Papilio xuthus. With the aim of identifying a potent antimicrobial peptide, we tested papiliocin in a variety of biological and biophysical assays, demonstrating that the peptide possesses very low cytotoxicity against mammalian cells and high bacterial cell selectivity, particularly against Gram-negative bacteria as well as high anti-inflammatory activity. Using LPS-stimulated macrophage RAW264.7 cells, we found that papiliocin exerted its anti-inflammatory activities by inhibiting nitric oxide (NO) production and secretion of tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-2, producing effects comparable with those of the antimicrobial peptide LL-37. We also showed that the innate defense response mechanisms engaged by papiliocin involve Toll-like receptor pathways that culminate in the nuclear translocation of NF-κB. Fluorescent dye leakage experiments showed that papiliocin targets the bacterial cell membrane. To understand structure-activity relationships, we determined the three-dimensional structure of papiliocin in 300 mm dodecylphosphocholine micelles by NMR spectroscopy, showing that papiliocin has an α-helical structure from Lys(3) to Lys(21) and from Ala(25) to Val(36), linked by a hinge region. Interactions between the papiliocin and LPS studied using tryptophan blue-shift data, and saturation transfer difference-NMR experiments revealed that Trp(2) and Phe(5) at the N-terminal helix play an important role in attracting papiliocin to the cell membrane of Gram-negative bacteria. In conclusion, we have demonstrated that papiliocin is a potent peptide antibiotic with both anti-inflammatory and antibacterial activities, and we have laid the groundwork for future studies of its mechanism of action.
蜂毒素是一种新型的 37 个残基的抗菌肽，最近从燕尾蝶 Papilio xuthus 中分离出来。为了鉴定一种有效的抗菌肽，我们在各种生物学和生物物理测定中测试了蜂毒素，结果表明该肽对哺乳动物细胞的细胞毒性非常低，对细菌细胞具有高度选择性，特别是对革兰氏阴性菌以及具有高抗炎活性。使用 LPS 刺激的巨噬细胞 RAW264.7 细胞，我们发现蜂毒素通过抑制一氧化氮（NO）的产生和肿瘤坏死因子（TNF）-α和巨噬细胞炎症蛋白（MIP）-2的分泌来发挥其抗炎活性，其效果可与抗菌肽 LL-37 相媲美。我们还表明，蜂毒素所涉及的先天防御反应机制涉及 Toll 样受体途径，最终导致 NF-κB 的核转位。荧光染料渗漏实验表明蜂毒素靶向细菌细胞膜。为了了解结构-活性关系，我们通过 NMR 光谱法在 300mm 十二烷基磷酸胆碱胶束中确定了蜂毒素的三维结构，结果表明蜂毒素具有从 Lys(3)到 Lys(21)和从 Ala(25)到 Val(36)的α-螺旋结构，由铰链区连接。使用色氨酸蓝移数据和饱和转移差异-NMR 实验研究了蜂毒素与 LPS 之间的相互作用，结果表明 N 端螺旋上的 Trp(2)和 Phe(5)在吸引蜂毒素到革兰氏阴性菌细胞膜中起着重要作用。总之，我们已经证明蜂毒素是一种具有抗炎和抗菌活性的强效肽抗生素，并为其作用机制的进一步研究奠定了基础。
