Construction of the novel functional proteins composed of calcium-dependent lectin and bioactive peptides

钙依赖性凝集素和生物活性肽组成的新型功能蛋白的构建

基本信息

批准号：
12660082
负责人：
HATAKEYAMA Tomomitsu
金额：
$ 2.05万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

A novel protein composed of rat mannose-binding lectin (MBL) and bee venom peptide melittin (Mel-MBL), was expressed in Escherichia coll cells using the plasmid PET-32a. The protein expressed as a fusion protein with thioredoxin (Trx-Mel-MBL) was then cleaved with enterokinase to produce Mel-MBL. When Mel-MBL was incubated with erythrocytes and bacterial cells, hemolytic as well as antibacterial activities were observed. These activities were found to be dependent on the carbohydrate-binding ability ofMBL. The marine invertebrate Cucumaria echinata contains the hemolytic lectin CEL-III. The amino acid sequence of this protein suggested that N-terminal two-thirds are carbohydrate binding domains and C-terminal one-third is a putative oligomerization domain. Several synthetic peptides having the parts of the amino acid sequence of the C-terminal domain were produced, and their properties were examined. As a result, P332 corresponding to the sequence beginning at position 332 exhibited strong antibacterial activity for Gram-positive bacteria, especially Staphylococcus aureus. This activity was found to result from the membrane perturbing ability of this peptide, leading to the increase in permeabilization of the inner membrane of the bacteria. X-ray crystallographic analysis of the C type lectin CEL-I in C. echinata was also performed in order to investigate the carbohydrate-recognition mechanism of this lectin. From the analysis of CEL-I and its complex with N-acetylgalactosamine (GalNAc), it was revealed that CEL I recognizes GalNAc through binding with Ca^<2+>, and very high specificity was enabled by hydrogen bonds formed between carbonyl oxygen and the side chain ofarginine 1 15 of CEL-I. These results could facilitate the designing of novel lectins with altered carbohydrate-binding specificity.

一种由大鼠甘露糖结合凝集素（MBL）和蜜蜂毒液肽蜂胶（MEL-MBL）组成的新型蛋白质，使用质粒PET-32A在大肠菌细胞中表达。然后用硫氧还蛋白（TRX-MEL-MBL）用肠球菌裂解以硫氧还蛋白（TRX-MEL-MBL）表示的蛋白质，以产生MEL-MBL。当Mel-MBL与红细胞和细菌细胞孵育时，观察到溶血和抗菌活性。发现这些活动取决于MBL的碳水化合物结合能力。海洋无脊椎动物黄瓜echinata包含溶血凝集素Cel-III。该蛋白的氨基酸序列表明，N末端三分之二是碳水化合物结合域，而C末端三分之一是推定的寡聚结构域。产生了几种具有C-末端结构域氨基酸序列部分的合成肽，并检查了其性质。结果，对应于位置332的序列的p332表现出革兰氏阳性细菌，尤其是金黄色葡萄球菌的强抗菌活性。发现这种活性是由于该肽的膜扰动能力引起的，导致细菌内膜的透化增加。为了研究该凝集素的碳水化合物识别机制，还进行了C. echinata中C型凝集素Cel-I的X射线晶体学分析。从对Cel-I及其与N-乙酰基乳二胺（GALNAC）的复合物的分析中，可以发现CEL I通过与Ca^<2+>结合来识别GalNAC，并且通过羰基氧和侧链链链链链球氨酸1 15的CEL-I的氢键启用了非常高的特异性。这些结果可以促进具有改变碳水化合物结合特异性的新型凝集素的设计。