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Endotoxin binding protein : Novel therapeutic strategy of endotoxin shock.

内毒素结合蛋白：内毒素休克的新治疗策略。

基本信息

批准号：
08670314
负责人：
HIRATA Michimasa
金额：
$ 1.66万
依托单位：
Iwate Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

We purified 18kDa cationic anti-microbial protein (CAP18) with lipopolysaccharide (LPS)-neutralizing activities from rabbit granulocytes. We also cloned a CAP18 family protein from a human bone marrow library. The cloned DNA encoded 140 amino acid residues (CAP181-140). Like the rabbit protein this molecule is comprised of two domains, a highly conserved N-terminal domain of unknown function and a less conserved C-terminal anti-microbial and LPS-neutralizing domain. In the present study, we synthesized new human peptides to identify in more detail the active domain within C-terminal fragment. Synthetic peptide (27 mer, F12-V38) of C-terminal fragment binds to LPS,inhibits LPS-induced activation of Limulus amebocyte lysate, LPS-induced reactive nitrogen release by macrophages and protect mice from LPS lethality. Treatment with the 27 mer peptide also blocked the increase in TNF-alpha levels induced by LPS injection. this peptide also showed antimicrobial activity versus both gram-negative bacteria such as Escerichia coli O157 : H7, salmonella typhimiurium, Klebsiella pneumoniae, Pseudomonas aeruginosa and gram-positive bacteria such as Staphylo-coccus aureus and Streptococcus pneumoniae. Truncation of hydrophobic amino acids from this 27 mer peptide caused a significant decrease in all activities indicating that this 27-mer fragment is the minimal antimicrobial and LPS-neutralizing domain of CAP18.The amphipathic and alpha-helical structure of this peptide may play a major role in the expression of these activities. The importance of C-terminus hydophobic residues in CAP18 was also suggested. CAP18 and derived peptides may act as host defense protein against infectious diseases, and have therapeutic potential for sepsis and endotoxin shock.

我们从兔粒细胞中纯化了具有脂多糖（LPS）中和活性的18 kDa阳离子抗菌蛋白（CAP 18）。我们还从人骨髓文库中克隆了CAP 18家族蛋白。克隆的DNA编码140个氨基酸残基（CAP 181 -140）。与兔蛋白一样，该分子由两个结构域组成，一个高度保守的未知功能的N-末端结构域和一个较不保守的C-末端抗微生物和LPS-中和结构域。在本研究中，我们合成了新的人肽，以更详细地确定C-末端片段内的活性结构域。C端合成肽（27 mer，F12-V38）与LPS结合，抑制LPS诱导的鲎变形细胞裂解物活化和巨噬细胞活性氮释放，保护小鼠免受LPS致死。用27聚体肽处理也阻断了由LPS注射诱导的TNF-α水平的增加。该肽还显示出对革兰氏阴性细菌如大肠杆菌O 157：H7、鼠伤寒沙门氏菌、肺炎克雷伯氏菌、铜绿假单胞菌和革兰氏阳性细菌如金黄色葡萄球菌和肺炎链球菌的抗微生物活性。该27-mer肽段的疏水性氨基酸的截短导致所有活性的显著降低，表明该27-mer片段是CAP 18的最小抗菌和LPS中和结构域。该肽段的两亲性和α-螺旋结构可能在这些活性的表达中起主要作用。还提出了CAP 18中C-末端疏水残基的重要性。CAP 18及其衍生肽可能作为宿主防御蛋白对抗感染性疾病，并对脓毒症和内毒素休克具有治疗潜力。