Kuzmenko Alexander I, Wu Huixing, McCormack Francis X
Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, Ohio 45267-0564, USA.
Biochemistry. 2006 Feb 28;45(8):2679-85. doi: 10.1021/bi0522652.
We have reported that Gram-negative organisms decorated with rough lipopolysaccharide (LPS) are particularly susceptible to the direct antimicrobial actions of the pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D). In this study, we examined the lipid and LPS components required for the permeabilizing effects of the collectins on model bacterial membranes. Liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), with or without rough Escherichia coli LPS (J5), smooth E. coli LPS (B5), or cholesterol, were loaded with self-quenching probes and exposed to native or oxidatively modified SP-A. Fluorescence that resulted from permeabilization of liposomes and diffusion of dyes was assessed by microscopy or fluorimetry. Human SP-A and melittin increased the permeability of J5 LPS/POPE liposomes, but not B5 LPS/POPE liposomes or control (POPE only) liposomes. At a human SP-A concentration of 100 microg/mL, the permeability of the J5 LPS/POPE membranes increased 4.4-fold (p < 0.02) compared to the control with no added SP-A. Rat SP-A and SP-D also permeabilized the J5-containing liposomes. Incorporation of cholesterol into J5 LPS/POPE liposomes at a POPE:cholesterol molar ratio of 1:0.15 blocked human SP-A or melittin-induced permeability (p < 0.05) compared to cholesterol-free liposomes. Exposure of human SP-A to surfactant lipid peroxidation blocked the permeabilizing activity of the protein. We conclude that SP-A permeabilizes phospholipid membranes in an LPS-dependent and rough LPS-specific manner, that the effect is neither SP-A- nor species-specific, and that oxidative damage to SP-A abolishes its membrane destabilizing properties. Incorporation of cholesterol into the membrane enhances resistance to permeabilization by SP-A, most likely by increasing the packing density and membrane rigidity.
我们曾报道，带有粗糙脂多糖（LPS）的革兰氏阴性菌对肺凝集素、表面活性蛋白A（SP-A）和D（SP-D）的直接抗菌作用尤为敏感。在本研究中，我们检测了凝集素对模型细菌膜通透作用所需的脂质和LPS成分。由1-棕榈酰-2-油酰-sn-甘油-3-磷酸乙醇胺（POPE）组成的脂质体，添加或不添加粗糙型大肠杆菌LPS（J5）、光滑型大肠杆菌LPS（B5）或胆固醇，装载自猝灭探针后，与天然或氧化修饰的SP-A接触。通过显微镜或荧光测定法评估脂质体通透及染料扩散所产生的荧光。人SP-A和蜂毒肽增加了J5 LPS/POPE脂质体的通透性，但未增加B5 LPS/POPE脂质体或对照（仅含POPE）脂质体的通透性。在人SP-A浓度为100μg/mL时，与未添加SP-A的对照相比，J5 LPS/POPE膜的通透性增加了4.4倍（p<0.02）。大鼠SP-A和SP-D也使含J5的脂质体通透。当POPE与胆固醇的摩尔比为1:0.15时，将胆固醇掺入J5 LPS/POPE脂质体中，与不含胆固醇的脂质体相比，可阻断人SP-A或蜂毒肽诱导的通透性（p<0.05）。人SP-A暴露于表面活性剂脂质过氧化反应会阻断该蛋白的通透活性。我们得出结论，SP-A以LPS依赖性和粗糙LPS特异性方式使磷脂膜通透，该效应既非SP-A特异性也非物种特异性，并且SP-A的氧化损伤会消除其膜去稳定特性。将胆固醇掺入膜中可增强对SP-A诱导通透的抗性，这很可能是通过增加堆积密度和膜刚性来实现的。
