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.

使用质粒 PET-32a 在大肠杆菌细胞中表达由大鼠甘露糖结合凝集素 (MBL) 和蜂毒肽蜂毒肽 (Mel-MBL) 组成的新型蛋白质。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)的蛋白质以产生Mel-MBL。当 Mel-MBL 与红细胞和细菌细胞一起孵育时，观察到溶血和抗菌活性。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 含有溶血凝集素 CEL-III。该蛋白质的氨基酸序列表明，N 端三分之二是碳水化合物结合结构域，C 端三分之一是推定的寡聚化结构域。生产了几种具有部分C末端结构域氨基酸序列的合成肽，并检查了它们的特性。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。人们发现这种活性是由该肽的膜扰动能力引起的，导致细菌内膜的透化作用增加。还对 C. echinata 中的 C 型凝集素 CEL-I 进行了 X 射线晶体学分析，以研究该凝集素的碳水化合物识别机制。从CEL-I及其与N-乙酰半乳糖胺(GalNAc)的复合物的分析可知，CEL-I通过与Ca ^ 2+ 结合来识别GalNAc，并且通过羰基氧与GalNAc之间形成的氢键实现了非常高的特异性。 CEL-I的精氨酸1-15的侧链。这些结果可以促进具有改变的碳水化合物结合特异性的新型凝集素的设计。

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.Biotechnol.Biochem..

T.Hatakeyama et al.: "Antibacterial peptides 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 末端结构域的抗菌肽，CEL-III”肽：未来的浪潮。

T.Hatakeyama et al.: "Crystallization and preliminary crystallographic study of an invertebrate C-type lectin, CEL-I, from a marine invertebrate Cucumaria echinata"Acta Cryst. D58. 143-144 (2002)

T.Hatakeyama 等人：“来自海洋无脊椎动物 Cucumaria echinata 的无脊椎动物 C 型凝集素 CEL-I 的结晶和初步晶体学研究”Acta Cryst。