Wu Zhibin, Hoover David M, Yang De, Boulègue Cyril, Santamaria Fanny, Oppenheim Joost J, Lubkowski Jacek, Lu Wuyuan
Institute of Human Virology, University of Maryland Biotechnology Institute, Baltimore, MD 21201, USA.
Proc Natl Acad Sci U S A. 2003 Jul 22;100(15):8880-5. doi: 10.1073/pnas.1533186100. Epub 2003 Jul 2.
Human defensins form a family of small, cationic, and Cys-rich antimicrobial proteins that play important roles in innate immunity against invading microbes. They also function as effective immune modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells. On the basis of sequence homology and the connectivity of six conserved Cys residues, human defensins are classified into alpha and beta families. Structures of several beta-defensins have recently been characterized, confirming the disulfide connectivity conserved within the family, i.e., Cys1-Cys5, Cys2-Cys4, and Cys3-Cys6. We found that human beta-defensin 3 (hBD3), a recently described member of the growing beta family, did not fold preferentially into a native conformation in vitro under various oxidative conditions. Using the orthogonal protection of Cys1-Cys5 and of Cys1-Cys6, we chemically synthesized six topological analogs of hBD3 with predefined disulfide connectivities, including the (presumably) native beta pairing. Unexpectedly, all differently folded hBD3 species exhibited similar antimicrobial activity against Escherichia coli, whereas a wide range of chemotactic activities was observed with these analogs for monocytes and cells transfected by the chemokine receptor CCR6. Furthermore, whereas substitution of all Cys residues by alpha-aminobutyric acid completely abolished the chemotactic activity of hBD3, the bactericidal activity remained unaffected in the absence of any disulfide bridge. Our findings demonstrate that disulfide bonding in hBD3, although required for binding and activation of receptors for chemotaxis, is fully dispensable for its antimicrobial function, thus shedding light on the mechanisms of action for human beta-defensins and the design of novel peptide antibiotics.
人防御素构成了一类小的、阳离子的、富含半胱氨酸的抗菌蛋白家族，在针对入侵微生物的固有免疫中发挥重要作用。它们还通过选择性地趋化T淋巴细胞和未成熟树突状细胞，在适应性免疫中作为有效的免疫调节剂发挥作用。基于序列同源性和六个保守半胱氨酸残基的连接方式，人防御素被分为α和β家族。最近已对几种β-防御素的结构进行了表征，证实了该家族内保守的二硫键连接方式，即Cys1-Cys5、Cys2-Cys4和Cys3-Cys6。我们发现，人β-防御素3（hBD3）是不断增加的β家族中最近描述的成员，在各种氧化条件下，它在体外不会优先折叠成天然构象。利用Cys1-Cys5和Cys1-Cys6的正交保护，我们化学合成了六种具有预定义二硫键连接方式的hBD3拓扑类似物，包括（推测的）天然β配对。出乎意料的是，所有不同折叠形式的hBD3物种对大肠杆菌均表现出相似的抗菌活性，而这些类似物对单核细胞和通过趋化因子受体CCR6转染的细胞则表现出广泛的趋化活性。此外，虽然用α-氨基丁酸取代所有半胱氨酸残基完全消除了hBD3的趋化活性，但在没有任何二硫键桥的情况下，杀菌活性仍然不受影响。我们的研究结果表明，hBD3中的二硫键结合虽然是趋化受体结合和激活所必需的，但其抗菌功能完全不需要，从而揭示了人β-防御素的作用机制以及新型肽类抗生素的设计方法。
