Role of Endotoxin-binding proteins in Non-specific Protection to Infection

内毒素结合蛋白在非特异性感染保护中的作用

• 批准号: 04670250
• 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: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04670250/
• 关键词: Lipopolysaccharide(LPS); LPS-binding protein; Tissue factor; Disseminated intravascular coagulation; Anticoagulant; Antibacteria; LPS-neutralization; リポ多種(LPS); LPS中和; 酸化窒素; 腫瘍壊死因子; エンドトキシンショック

Endotoxin(lipopolysaccharide=LPS), cell wall component of gram-negative bacteria, activates monocytes and macrophages to release cytokines, reactive nitrogen intermediates (RNI), and to generate tissue factor(TF) which initiate coagulation. We have purified 7kDa and 18kDa cationic antibacterial proteins (CAP-7 and CAP-18) with LPS-binding and LPS-neutralizing activities from rabbit granulocytes using as an assay the agglutination of erythrocytes coated with Re-LPS.From protein sequencing, CAP-7 was identified as the C-terminal 37 amino acid fragment of CAP-18. Synthetic peptide #197(identical sequence to CAP-7, Gly1-Try37) and #36-1 (a truncation of CAP consisting of 32 amino acid residues, Gly1-Ala32) showed LPS-binding activity. Each peptide inhibited LPS-induced tissue factor(TF) generation by murine peritoneal macrophages, even added 1 hour after stimulation of cells with LPS.C57BL/6 mice treated with #197 were significantly protected from lethal LPS challenge. Peptide #36 also blo … More cked the LPS-induced lethality. These peptides had antibacterial activity to gram-negative bacteria, such as E.coli, S.typhimurium, K.pneumonia, Ps.aeruginosa and also to gram-positive S.aureus(Methicllin sensitive and resistant strains). Both peptides inhibited TF-and Xa-induced plasma clotting. Using synthetic chromogenic substrates, both CAP7 peptides blocked the coagulation cascade at two sites, activation of factor X to Xa and conversion of Factor II(prothrombin) to factor IIa(prothrombin). In vivo treatment of peptide #197 prevented acute lethality in mice injected with tissue factor (rabbit brain thromboplastin). Two other peptides, #32(Gly1-Phe9) and #50(lle13-Typ37) failed to demonstrate LPS-binding, LPS-neutralizing, antibacterial and anticoagulant activities. The active peptides but not the inactive peptide maintain a putative heparin binding domain at their N-termini. This heparin binding domain is participate in the LPS-binding, LPS neutralizing, antibacterial and anticoaThese active peptides may have a therapeutic potential for treatment for DIC due to sepsis and endotoxin shock. Less

内毒素（脂多糖=LPS），革兰氏阴性菌的细胞壁组分，激活单核细胞和巨噬细胞以释放细胞因子、活性氮中间体（RNI），并产生启动凝血的组织因子（TF）。本研究从兔粒细胞中分离纯化了具有LPS结合和中和活性的7 kDa和18 kDa阳离子抗菌蛋白（CAP-7和CAP-18），经蛋白质序列测定，CAP-7为CAP-18 C端37个氨基酸的片段。合成肽#197（与CAP-7相同的序列，Gly 1-Try 37）和#36-1（由32个氨基酸残基组成的CAP截短物，Gly 1-Ala 32）显示LPS结合活性。每种肽抑制LPS诱导的小鼠腹腔巨噬细胞产生的组织因子（TF），甚至在用LPS刺激细胞后1小时加入。用#197处理的C57 BL/6小鼠显著保护免于致死性LPS攻击。肽#36也是blo ...更多信息 检测LPS诱导的致死率。这些肽对革兰氏阴性细菌如大肠杆菌、鼠伤寒沙门氏菌、肺炎克雷伯菌、铜绿假单胞菌以及革兰氏阳性金黄色葡萄球菌（甲氧西林敏感和耐药菌株）具有抗菌活性。两种肽均抑制TF和Xa诱导的血浆凝血。使用合成的显色底物，两种CAP 7肽在两个位点阻断凝血级联反应，即因子X活化为Xa和因子II（凝血酶原）转化为因子IIa（凝血酶原）。肽#197的体内处理防止了注射组织因子（兔脑促凝血酶原激酶）的小鼠的急性致死。另外两种肽，#32（Gly 1-Phe 9）和#50（Ile 13-Typ 37）未能证明LPS结合、LPS中和、抗菌和抗凝活性。活性肽而非活性肽在其N-末端保持推定的肝素结合结构域。这些活性肽可能对脓毒症和内毒素休克引起的DIC具有潜在的治疗作用。少

项目成果

Hirata M.,Shimomura Y et al: "Endotoxin-neutralizing,antibacterial and anticoagulant activities rabbit CAP18-derived peptides." Gram Negative Sepsis:Basic Science to clinical Investigation. (in press). (1994)

Hirata M.、Shimomura Y 等人：“兔 CAP18 衍生肽具有中和、抗菌和抗凝血活性。”

Hirata, M., Shimomura, Y., Yoshida, M., Morgan, J.G., Palings, I., Wilson D., Yen, M.H. : and Larrick, J.W.: "Characterization of a rabbit cationic protein (CAP-18) with lipopolysaccharide-inhibitory activity" Infect.Immun. (in press). (1994)

平田 M.、下村 Y.、吉田 M.、摩根 J.G.、帕林斯 I.、威尔逊 D.、日元 M.H.

Larrick.J.W,Hirata,Metsl: "A novelgranalocyte-derived peptide with LPS neutralizing activity" J.Immunol. 152. 231-240 (1994)

Larrick.J.W、Hirata、Metsl：“一种具有 LPS 中和活性的新型粒细胞衍生肽”J.Immunol。

平田 陸正: "内毒素結合蛋白質の構造と活性" 第39回毒素シンポジウム予稿集. 80-84 (1992)

Rikumasa Hirata：“内毒素结合蛋白的结构和活性”第 39 届毒素研讨会论文集 80-84（1992 年）。

Larrick,J.W.,Hirata,M et al: "A Novel granulocyte-derived peptide with LPS neutralizing activity" J Immunol. 152. 231-240 (1994)

Larrick,J.W.,Hirata,M 等人：“一种具有 LPS 中和活性的新型粒细胞衍生肽”JImmunol。