Andrä Jörg, Howe Jörg, Garidel Patrick, Rössle Manfred, Richter Walter, Leiva-León José, Moriyon Ignacio, Bartels Rainer, Gutsmann Thomas, Brandenburg Klaus
Forschungszentrum Borstel, Leibniz-Zentrum für Medizin und Biowissenschaften, Biophysik, Parkallee 10, 23845 Borstel, Germany.
Biochem J. 2007 Sep 1;406(2):297-307. doi: 10.1042/BJ20070279.
On the basis of formerly investigated peptides corresponding to the endotoxin-binding domain from LALF [Limulus anti-LPS (lipopolysaccharide) factor], a protein from Limulus polyphemus, we have designed and synthesized peptides of different lengths with the aim of obtaining potential therapeutic agents against septic shock syndrome. For an understanding of the mechanisms of action, we performed a detailed physicochemical and biophysical analysis of the interaction of rough mutant LPS with these peptides by applying FTIR (Fourier-transform infrared) spectroscopy, SAXS (small-angle X-ray scattering), calorimetric techniques [DSC (differential scanning calorimetry) and ITC (isothermal titration calorimetry)] and FFTEM (freeze-fracture transmission electron microscopy). Also, the action of the peptides on bacteria of different origin in microbial assays was investigated. Using FTIR and DSC, our results indicated a strong fluidization of the lipid A acyl chains due to peptide binding, with a decrease in the endothermic melting enthalpy change of the acyl chains down to a complete disappearance in the 1:0.5 to 1:2 [LPS]:[peptide] molar ratio range. Via ITC, it was deduced that the binding is a clearly exothermic process which becomes saturated at a 1:0.5 to 1:2 [LPS]:[peptide] molar ratio range. The results obtained with SAXS indicated a drastic change of the aggregate structures of LPS into a multilamellar stack, which was visualized in electron micrographs as hundreds of lamellar layers. This can be directly correlated with the inhibition of the LPS-induced production of tumour necrosis factor alpha in human mononuclear cells, but not with the action of the peptides on bacteria.
基于之前对来自鲎试剂（鲎抗脂多糖因子）的内毒素结合域相应肽段的研究，鲎试剂是一种来自美洲鲎的蛋白质，我们设计并合成了不同长度的肽段，旨在获得针对脓毒症休克综合征的潜在治疗药物。为了理解其作用机制，我们通过傅里叶变换红外光谱（FTIR）、小角X射线散射（SAXS）、量热技术[差示扫描量热法（DSC）和等温滴定量热法（ITC）]以及冷冻断裂透射电子显微镜（FFTEM），对粗糙突变型脂多糖与这些肽段的相互作用进行了详细的物理化学和生物物理分析。此外，还研究了这些肽段在微生物检测中对不同来源细菌的作用。利用FTIR和DSC，我们的结果表明，由于肽段结合，脂质A酰基链发生了强烈的流化，酰基链的吸热熔化焓变降低，在1:0.5至1:2 [脂多糖]:[肽段]摩尔比范围内完全消失。通过ITC推断，结合是一个明显的放热过程，在1:0.5至1:2 [脂多糖]:[肽段]摩尔比范围内达到饱和。SAXS获得的结果表明，脂多糖的聚集体结构急剧转变为多层堆叠，在电子显微镜照片中可看到数百个层状结构。这可以直接与脂多糖诱导人单核细胞产生肿瘤坏死因子α的抑制作用相关，但与肽段对细菌的作用无关。
