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The Role of Endotoxin-neutralizing and Antimicrobial Proteins in Innate Immunity

内毒素中和和抗菌蛋白在先天免疫中的作用

基本信息

批准号：
06670300
负责人：
HIRATA Michimasa
金额：
$ 1.34万
依托单位：
Iwate Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

CAP18 (cationic antimicrobial protein, 18kDa) is a 142 amino acid protein originally isolated from rabbit granulocytes using agglutination of LPS-coated erythrocytes as an assay. CAP18 is composed of an N-terminal domain of unknown function (CAP18_<1-105>) and a C-terminal LPS-binding domain (CAP18_<106-142>). Synthetic CAP18_<106-142> and CAP18_<106-137>, a 32-amino acid peptide resulting from the truncation of 5 amino acids from the C-terminus of CAP18_<106-142>, inhibited LPS-induced tissue factor generation, nitric oxide production and TNF release by macrophages. Mice treated with CAP18_<106-142> or CAP18_<106-137> were significantly protected from LPS lethality. Although CAP18_<106-142> and CAP18_<106-137> were highly active, other fragments of CAP18_<106-142>, including CAP18_<110-142> with a truncated N-terminus, did not exhibit LPS-binding and LPS-neutralizing activities. Both peptides had broad antimicrobial activity against both gram-negative bacteria such as Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Pseudomonas aeruginosa (IC_<50> ; 40-100 nM) and gram-positive bacteria such as Staphylococcus aureus (Methicillin sensitive and resistant strains) and Streptococcus pneumoniae (IC_<50> ; 100-200nM).We cloned a CAP-18 family protein from human granulocytes. The cloned cDNA encoded 140 amino acid residues. Human CAP18 (CAP18_<1-140>) was highly homologous to that of rabbit. A32-amino-acid C-terminal fragment (CAP18_<104-135>) was shown to bind LPS,inhibit LPS-induced tissue factor generation by murine macrophages, and protect mice from LPS lethality. This peptide exhibited antimicrobial activity against both gram-negative and gram-positive bacteria. We hypothesize that CAP18 and the derived peptides bind to LPS and alter the capacity of LPS to initiate disseminated intravascular coagulation.In this regard, CAP may act as host defense protein against infectious diseases, and have therapeutic potential for sepsis and endotoxin shock.

CAP 18（阳离子抗菌蛋白，18 kDa）是最初使用LPS包被的红细胞凝集作为测定从兔粒细胞中分离的142个氨基酸的蛋白。CAP 18由N端未知功能结构域（CAP 18_<1-105>）和C端LPS结合结构域（CAP 18_<106-142>）组成。合成的CAP 18_<106-142>和CAP 18_<106-137>（从CAP 18_的C-末端截短5个氨基酸产生的32个氨基酸的肽<106-142>）抑制LPS诱导的组织因子产生、一氧化氮产生和巨噬细胞的TNF释放。用CAP 18_<106-142>或CAP 18_<106-137>处理的小鼠被显著保护免于LPS致死。虽然CAP 18_<106-142>和CAP 18_<106-137>具有高活性，但CAP 18_的其他片段<106-142>，包括<110-142>具有截短的N-末端的CAP 18_，不表现出LPS结合和LPS中和活性。这两种肽对革兰氏阴性菌如大肠杆菌、鼠伤寒沙门氏菌、肺炎克雷伯氏菌、铜绿假单胞菌（IC_<50>; 40-100 nM）和革兰氏阳性菌如金黄色葡萄球菌（甲氧西林敏感和耐药菌株）和肺炎链球菌（IC_<50>; 100- 200 nM）都具有广泛的抗菌活性。克隆的cDNA编码140个氨基酸残基。人CAP 18（CAP 18_<1-140>）与兔的CAP 18高度同源。32-氨基酸C-末端片段（CAP 18_<104-135>）显示结合LPS，抑制LPS诱导的小鼠巨噬细胞产生组织因子，并保护小鼠免受LPS致死。该肽对革兰氏阴性菌和革兰氏阳性菌均表现出抗微生物活性。我们推测CAP 18及其衍生肽与LPS结合，改变LPS启动弥散性血管内凝血的能力，可能作为宿主抗感染的防御蛋白，对脓毒症和内毒素休克具有治疗潜力。