Chen Xi, Howe Jörg, Andrä Jörg, Rössle Manfred, Richter Walter, da Silva Ana Paula Galvão, Krensky Alan M, Clayberger Carol, Brandenburg Klaus
Division of Immunology and Transplantation Biology, Department of Pediatrics, CCSR 2105, Stanford University School of Medicine, Stanford, CA 94305, USA.
Biochim Biophys Acta. 2007 Oct;1768(10):2421-31. doi: 10.1016/j.bbamem.2007.05.001. Epub 2007 May 22.
To combat infections by Gram-negative bacteria, it is not only necessary to kill the bacteria but also to neutralize pathogenicity factors such as endotoxin (lipopolysaccharide, LPS). The development of antimicrobial peptides based on mammalian endotoxin-binding proteins is a promising tool in the fight against bacterial infections, and septic shock syndrome. Here, synthetic peptides derived from granulysin (Gra-pep) were investigated in microbiological and biophysical assays to understand their interaction with LPS. We analyzed the influence of the binding of Gra-pep on (1) the acyl chain melting of the hydrophobic moiety of LPS, lipid A, by Fourier-transform spectroscopy, (2) the aggregate structure of LPS by small-angle X-ray scattering and cryo-transmission electron microscopy, and 3) the enthalpy change by isothermal titration calorimetry. In addition, the influence of Gra-pep on the incorporation of LPS and LPS-LBP (lipopolysaccharide-binding protein) complexes into negatively charged liposomes was monitored. Our findings demonstrate a characteristic change in the aggregate structure of LPS into multilamellar stacks in the presence of Gra-pep, but little or no change of acyl chain fluidity. Neutralization of LPS by Gra-pep is not due to a scavenging effect in solution, but rather proceeds after incorporation into target membranes, suggesting a requisite membrane-bound step.
为了对抗革兰氏阴性菌感染，不仅需要杀死细菌，还需要中和诸如内毒素（脂多糖，LPS）等致病因子。基于哺乳动物内毒素结合蛋白开发抗菌肽是对抗细菌感染和败血性休克综合征的一种有前景的工具。在此，对源自颗粒溶素的合成肽（Gra-肽）进行了微生物学和生物物理分析，以了解它们与LPS的相互作用。我们通过傅里叶变换光谱分析了Gra-肽结合对（1）LPS疏水部分脂质A的酰基链熔化的影响，通过小角X射线散射和冷冻透射电子显微镜分析了（2）LPS的聚集体结构，以及通过等温滴定量热法分析了（3）焓变。此外，监测了Gra-肽对LPS和LPS-LBP（脂多糖结合蛋白）复合物掺入带负电荷脂质体的影响。我们的研究结果表明，在存在Gra-肽的情况下，LPS的聚集体结构发生特征性变化，形成多层堆叠，但酰基链流动性几乎没有变化。Gra-肽对LPS的中和作用不是由于溶液中的清除作用，而是在掺入靶膜后发生，这表明需要一个膜结合步骤。
