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

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660082/
• 关键词: lectin; carbohydrate-binding prtein; calcium; invertebrate; self-defense; hemolysin; toxin; X-ray crystallography; カルシウムイオン; 両親媒性ペプチド; 溶血活性; 抗菌活性; クローニング; タンパク質コンジュゲート

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.

以大鼠甘露糖结合凝集素(MBL)和蜂毒肽Melittin(MEL-MBL)为载体，利用表达载体pET-32a，在大肠杆菌中表达了一种新的蛋白质。以硫氧还蛋白融合蛋白(Trx-Mel-MBL)的形式表达，然后用肠激酶酶切制得Mel-MBL。当MEL-MBL与红细胞和细菌细胞孵育时，观察到了溶血和抗菌活性。这些活性依赖于MBL的碳水化合物结合能力。海洋无脊椎动物刺参含有溶血凝集素CEL-III。氨基酸序列分析表明，N端2/3为糖结合区，C端1/3为寡聚区。合成了几种具有C-末端结构域部分氨基酸序列的合成肽，并对它们的性质进行了检测。结果表明，与332位序列对应的P332对革兰氏阳性菌表现出较强的抗菌活性，尤其是对金黄色葡萄球菌的抗菌活性。这种活性是由于这种多肽的膜扰动能力，导致细菌内膜通透性的增加。对棘球藻中C型凝集素CEL-I进行了X射线结晶学分析，以探讨该凝集素的碳水化合物识别机制。通过对CEL-I及其与N-乙酰半乳糖胺(GalNAc)的络合物的分析，发现CEL-I通过与Ca~(2+)和Gt~(2+)的结合识别GalNAc，并且CEL-I的精氨酸115侧链与羰基氧形成氢键使其具有很高的特异性。这些结果有助于设计具有改变的碳水化合物结合专一性的新型凝集素。

项目成果

Tomomitsu Hatakeyama et al.: "Amino acid sequence and carbohydrate-binding analysis of the N-acetyl-D-galactosamine-specific C-type lectin, CEL-I, from the Holothuroidea, Cucumaria echinata"Biosci.Biotechnol.Biochem.. 66. 157-163 (2002)

Tomomitsu Hatakeyama 等人：“来自海参总科、Cucumaria echinata 的 N-乙酰基-D-半乳糖胺特异性 C 型凝集素 CEL-I 的氨基酸序列和碳水化合物结合分析”Biosci.Biotechnol.Biochem.. 66

H.Kuwahara et al.: "Effects of chemical modification of carboxyl groups in the hemolytic lectin CEL-III on its hemolytic and carbohydrate-binding activities"Biosci.Biotechnol.Biochem.. 64. 1278-1281 (2000)

H.Kuwahara 等：“溶血凝集素 CEL-III 中羧基的化学修饰对其溶血和碳水化合物结合活性的影响”Biosci.Biotechnol.Biochem.. 64. 1278-1281 (2000)

T.Hatakeyama, et al.: "Amino acid sequence and carbohydrate-binding analysis of the N-acetyl-D-galactosamine-specific C-type lectin, CEL-I, from the Holothuroidea, Cucumaria echinata"Biosci. Biotechnol. Biochem.. 66. 157-163 (2002)

T.Hatakeyama 等人：“来自海参总科、Cucumaria echinata 的 N-乙酰基-D-半乳糖胺特异性 C 型凝集素 CEL-I 的氨基酸序列和碳水化合物结合分析”Biosci。

T. Hatakeyama et al: "Antibacterial reptides derived from the C-terminal domain of the hemolytic lectin, CEL-III"Peptides : The Wave of the Future. 760-761 (2001)

T. Hatakeyama 等人：“源自溶血凝集素 C 末端结构域的抗菌爬行动物，CEL-III”肽：未来的浪潮。

T. Suenaga et al: "Antibacterial peptides derived from the C-terminal domain of the hemolytic lectin, CEL-III"Peptide Science 2001. 199-202 (2002)

T. Suenaga 等：“源自溶血凝集素 C 末端结构域的抗菌肽，CEL-III”Peptide Science 2001. 199-202 (2002)