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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.

利用质粒PET-32 a在大肠杆菌科尔l细胞中表达了一种由大鼠甘露糖结合凝集素（MBL）和蜂毒肽蜂毒肽（Mel-MBL）组成的新蛋白。然后用肠激酶切割表达为与硫氧还蛋白的融合蛋白（Trx-Mel-MBL）的蛋白以产生Mel-MBL。当Mel-MBL与红细胞和细菌细胞孵育时，观察到溶血以及抗菌活性。发现这些活性依赖于MBL的碳水化合物结合能力。海洋无脊椎动物Cucumaria echinata含有溶血性凝集素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结构域和C-末端三分之一是一个推定的寡聚化结构域。生产了几种具有C-末端结构域的氨基酸序列部分的合成肽，并检查了它们的性质。结果，对应于从位置332开始的序列的P332对革兰氏阳性菌，特别是金黄色葡萄球菌表现出强的抗菌活性。发现这种活性是由于这种肽的膜扰动能力，导致细菌内膜透化的增加。C型凝集素CEL-I的X射线晶体学分析。为了研究这种凝集素的碳水化合物识别机制，还进行了海胆凝集素实验。对CEL-I及其与N-乙酰半乳糖胺（GalNAc）复合物的分析表明，CEL-I通过与Ca ~（2+）结合识别GalNAc，并通过与CEL-I的精氨酸115侧链形成的氢键实现了很高的特异性。这些结果有助于设计具有改变的碳水化合物结合特异性的新型凝集素。