Mechanisms of alpha-defensin bactericidal action: comparative membrane disruption by Cryptdin-4 and its disulfide-null analogue.
Mechanisms of alpha-defensin bactericidal action: comparative membrane disruption by Cryptdin-4 and its disulfide-null analogue.
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DOI：
10.1021/bi800335e
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发表时间：
2008-11-25
期刊：
影响因子：
2.9
通讯作者：
Gizeli, Electra
中科院分区：
文献类型：
--
作者：
Hadjicharalambous, Chrystalleni;Sheynis, Tania;Jelinek, Raz;Shanahan, Michael T.;Ouellette, Andre J.;Gizeli, Electra
哺乳动物α-防御素都具有保守的三链β-折叠结构，该结构受到不变的三二硫键阵列的限制，并且这些肽通过透化靶细胞包膜来发挥杀菌作用。奇怪的是，小鼠α-防御素cryptdin-4（Crp 4）的无序、无二硫键变体（称为（6C/A）-Crp 4）具有与天然肽相同或更大的杀菌活性，这为比较肽与确定组成的磷脂囊泡相互作用和破坏磷脂囊泡的机制提供了理论基础。两个活的E。coliML 35细胞和模型膜，无序的（6C/A）-Crp 4诱导类似于Crp 4的渗漏，但总体膜透化活性较低。Crp 4诱导荧光团从负电性脂质体泄漏强烈依赖于囊泡脂质电荷和组合物，并且将心磷脂掺入低负电性电荷的脂质体中以模拟细菌膜组合物赋予对Crp 4和（6C/A）-Crp 4介导的囊泡裂解的敏感性。使用仿生脂质/聚丁二炔囊泡的膜扰动研究表明，Crp 4在低而不是高负电荷脂质体中具有比（6C/A）-Crp 4更明显的双层表面相互作用，与诱导泄漏的测量直接相关。荧光共振能量转移实验提供的证据表明，在与膜透化偶联的过程中，Crp 4跨高电荷或含心磷脂的膜易位，但（6C/A）-Crp 4不跨脂质双层易位，并始终显示膜表面缔合。因此，尽管（6C/A）-Crp 4的体外杀菌活性更大，但含有Crp 4的天然β-折叠通过移位和形成瞬时膜缺陷比二硫化物无效的Crp 4更有效地诱导膜透化。(6C另一方面，A）-Crp 4似乎诱导更大的膜崩解。
Mammalian α-defensins all have a conserved triple-stranded β-sheet structure that is constrained by an invariant tridisulfide array, and the peptides exert bactericidal effects by permeabilizing the target cell envelope. Curiously, the disordered, disulfide-null variant of mouse α-defensin cryptdin-4 (Crp4), termed (6C/A)-Crp4, has equal or greater bactericidal activity than the native peptide, providing rationale for comparing the mechanisms by which the peptides interact with and disrupt phospholipid vesicles of defined composition. For both live E. coli ML35 cells and model membranes, disordered (6C/A)-Crp4 induced leakage similar to Crp4 but had less overall membrane-permeabilizing activity. Crp4 induction of fluorophore leakage from electronegative liposomes was strongly dependent on vesicle lipid charge and composition, and the incorporation of cardiolipin into liposomes of low electronegative charge to mimic bacterial membrane composition conferred sensitivity to Crp4- and (6C/A)-Crp4-mediated vesicle lysis. Membrane perturbation studies using biomimetic lipid/polydiacetylene vesicles showed that Crp4 had more pronounced bilayer surface interactions than (6C/A)-Crp4 in low rather than high negatively charged liposomes, correlating directly with measurements of induced leakage. Fluorescence resonance energy transfer experiments provided evidence that Crp4 translocates across highly charged or cardiolipin-containing membranes, in a process coupled with membrane permeabilization, but (6C/A)-Crp4 did not translocate across lipid bilayers and consistently displayed membrane surface association. Thus, despite the greater in vitro bactericidal activity of (6C/A)-Crp4, native, β-sheet containing Crp4 induces membrane permeabilization more effectively than disulfide-null Crp4 by translocating and forming transient membrane defects. (6C/A)-Crp4, on the other hand, appears to induce greater membrane disintegration.
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