Endotoxin binding protein : Novel therapeutic strategy of endotoxin shock.

我们从兔粒细胞中纯化了具有脂多糖中和活性的18 kDa阳离子抗微生物蛋白(CAP18)。我们还从人骨髓库中克隆了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-末端结构域，以及一个不太保守的C-末端抗微生物和内毒素中和结构域。在本研究中，我们合成了新的人源多肽，以更详细地鉴定C末端片段中的活性区域。合成肽(27mer，F12-V38)可与内毒素结合，抑制内毒素诱导的凝胶裂解产物的活化，抑制内毒素诱导的巨噬细胞释放反应性氮，保护小鼠免受内毒素的致死。27肽的治疗也阻断了由内毒素注射引起的肿瘤坏死因子-α水平的升高。该多肽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也显示了抗菌活性。截短该27肽的疏水氨基酸后，所有活性均显着降低，表明该27肽片段是CAP18的最小抑菌和中和域，其两亲性和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CAP18中C末端疏水残基的重要性。CAP18及其衍生多肽可能作为宿主防御蛋白对抗感染性疾病，对脓毒症和内毒素休克具有治疗潜力。

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.