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 18kDa 阳离子抗微生物蛋白 (CAP18)。我们还从人类骨髓库中克隆了 CAP18 家族蛋白。克隆的 DNA 编码 140 个氨基酸残基 (CAP181-140)。与兔蛋白一样，该分子由两个结构域组成，一个功能未知的高度保守的 N 端结构域和一个不太保守的 C 端抗菌和 LPS 中和结构域。在本研究中，我们合成了新的人类肽，以更详细地鉴定 C 端片段内的活性结构域。 C 端片段的合成肽（27 mer，F12-V38）与 LPS 结合，抑制 LPS 诱导的鲎阿米巴细胞裂解物的活化、LPS 诱导的巨噬细胞的活性氮释放，并保护小鼠免受 LPS 致死。 27 mer 肽治疗也阻止了 LPS 注射诱导的 TNF-α 水平的增加。该肽还对革兰氏阴性菌（如大肠杆菌 O157 : H7、鼠伤寒沙门氏菌、肺炎克雷伯菌、铜绿假单胞菌）和革兰氏阳性菌（如金黄色葡萄球菌和肺炎链球菌）表现出抗菌活性。该 27 聚体肽的疏水性氨基酸的截断导致所有活性显着降低，表明该 27 聚体片段是 CAP18 的最小抗菌和 LPS 中和结构域。该肽的两亲性和 α 螺旋结构可能发挥主要作用在这些活动的表达中发挥作用。 CAP18 中 C 末端疏水残基的重要性也被提出。 CAP18 和衍生肽可作为宿主防御感染性疾病的防御蛋白，并具有治疗脓毒症和内毒素休克的潜力。