Novel therapeutic strategy in the treatment of infectious diseases by anti-microbial, endotoxin-neutralizing proteins.

通过抗微生物、内毒素中和蛋白治疗传染病的新治疗策略。

基本信息

批准号：
10670267
负责人：
HIRATA Michimasa
金额：
$ 2.05万
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：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

We have found 18kDa cationic anti-microbial proteins (CAP18) with LPS-neutralizing activity from rabbit and human neutrophils. The 27mer C-terminal fragment of human CAP 18 (27mer ; CAP18_<109-135>) has been identified as the anti-microbial and LPS-neutralizing domain. We investigated the effects of modification and/or substitution of amino acid residues of CAP18 peptides on their activities. Acetylation of N-terminal and amidation of C-terminal residues of the 27mer in-creased LPS-neutralizing and antimicrobial activities. Substitution of E_<119> and K_<128> of 27mer to L (27LL) or F (27FF) increased LPS binding activity (〜30-40 fold) and also blocked LPS-induced lethality in mice. Substitution of E_<119> to L_<119> increased LPS-binding activity (〜8 -fold), however, this peptide (27L) could not protect vs. endotoxin shock. Truncation of 9 amino acids from the N-terminus of 27mer (18mer : CAP18_<118-135>) resulted in a significant decrease in LPS-binding activity even though both terminuses are chemically modified. Increasing the hydrophobic amino acid residues (Leucin=L) in 18mer (18LL) increased LPS-binding activity (〜30-fold) and anti-microbial activity versus gram-positive bacteria such as MRSA (methicillin resistant staphylococcus aureus ; >3 -folds), however, 18LL could not protect vs. endotoxin shock in mice. Further replacement of three positions of 18LL by lysine (K) significantly increased LPS-binding activity (〜120-fold), antimicrobial activity vs. gram negative bacteria such as E.coli O157 : H7 and S.typhimurium, and protect mice from endotoxin shock.From these results, the importance of the stability of alpha-helical structure, and the balance of hydrophobic/ hydrophilic (basic) amino acid residues in CAP18 peptide was suggested.

我们从兔和人中性粒细胞中发现了具有 LPS 中和活性的 18kDa 阳离子抗微生物蛋白 (CAP18)。人 CAP 18 的 27mer C 端片段（27mer；CAP18_<109-135>）已被鉴定为抗微生物蛋白。我们研究了氨基酸残基的修饰和/或取代的影响。 CAP18 肽的活性。27mer 的 N 端乙酰化和 C 端残基的酰胺化增加了 27mer 的 LPS 中和和抗菌活性。 F (27FF) 增加了 LPS 结合活性（约 30-40 倍），并且还阻断了 LPS 诱导的小鼠死亡。 E_<119>替换为L_<119>增加了LPS结合活性（〜8倍），然而，该肽（27L）不能防止27mer N端截短的9个氨基酸。 (18mer：CAP18_<118-135>) 导致 LPS 结合活性显着降低，即使两个末端都经过化学修饰。与革兰氏阳性菌如 MRSA（耐甲氧西林金黄色葡萄球菌；>3 倍）相比，18mer (18LL) 中的疏水性氨基酸残基（亮氨酸=L）增加了 LPS 结合活性（约 30 倍）和抗菌活性然而，18LL不能保护小鼠免受内毒素休克，进一步用赖氨酸(K)替代18LL的三个位置显着增加。 LPS 结合活性（约 120 倍），相对于革兰氏阴性菌（如大肠杆菌 O157：H7 和鼠伤寒沙门氏菌）的抗菌活性，并保护小鼠免受内毒素休克。从这些结果来看，α- 稳定性的重要性提出了 CAP18 肽中螺旋结构和疏水/亲水（碱性）氨基酸残基的平衡。