Potential Antimicrobial Strategy Using Genetically Engineered Homo & Hetero-Dimer of alpha-Lactalbumin and Lysozyme

使用基因工程人的潜在抗菌策略

• 批准号: 13660129
• 负责人: IBRAHIM Hisham r.
• 金额: $ 2.18万
• 依托单位: Kagoshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660129/
• 关键词: Lysozyme; α-Lactalbumin; Antimicrobial; Interaction; Hetero-Dimer; Pichia Expression System; Mammary Epithelial Cells; New Drug Design; 抗菌性; ホモダイマー; カルシウム結合; カルシウム

Mammalian milk is characterized by a complex host defense system that prevents the growth of microbial pathogens that may pervade the infant. However, little is known of the fate of the antimicrobial components and precisely how they interact to protect the recipient. Alpha-lactalbumin (α-LA) is one of the major proteins in milk of all species and has the unusual property of acting to modify the action of galactosyltransferase to a lactose synthase. On the other hand, lysozyme (LZ), a homologous protein to α-LA, is a ubiquitous antimicrobial in a variety of tissues and secretions. These two proteins uniquely co-exist in mammalian milk but the significance of their distinct presence in milk is completely unknown. In this study, LZ and α-LA were investigated to elucidate the nature of their interaction and antibacterial synergy. A complex of LZ and α-LA exhibited greatly enhanced antibacterial activity against the Gram-positive and -negative bacteria. Under physiological pH, α-LA associa … More ted with LZ, whereas the complex existed mainly as LZ-αLA heterodimer with LZ homodimer. The results provided the first demonstration of association of α-LA with LZ leading to the formation of a distinct complex with significantly enhanced bactericidal activity. Our results strongly suggest the important role of LZ and α-LA in the host defense system of the newborn against gastric microbial infections. Furthermore, a parallel genetic study was initiated with the objectives of cloning the cDNAs of human LZ (hLZ) and human LA (hLA) from human mammary epithelial cells, establish high-level expression system in Pichia under the native signal peptides of the two human proteins, and to design a stable in-frame fusion constructs between hLZ and hLA., Human LZ, α-LA, and their LZ-α-LA fusion construct were successfully produced in Pichia methanolica with expression level exceeding 20 mg/L and thus offer a great opportunity for the design of potential antimicrobial drug in the treatment of infectious diseases. Less

哺乳动物奶的特点是有一个复杂的宿主防御系统，可以防止可能渗透到婴儿体内的微生物病原体的生长。然而，人们对抗菌成分的命运以及它们确切地如何相互作用来保护接受者知之甚少。α-乳清蛋白(α-LA)是所有种类的牛奶中的主要蛋白质之一，具有将半乳糖基转移酶修饰为乳糖合成酶的特殊性质。另一方面，溶菌酶(LZ)是一种与α-LA同源的蛋白质，是一种在各种组织和分泌物中普遍存在的抗菌剂。这两种蛋白质在哺乳动物的乳汁中独一无二地共存，但它们在乳汁中不同存在的意义完全不清楚。本研究以LZ和α-LA为研究对象，探讨其相互作用和抗菌协同作用的性质。LZ与α-LA形成的复合体对革兰氏阳性菌和革兰氏阴性菌均表现出较强的抗菌活性。在生理pH条件下，α-LA Associa…与LZ结合较多，而LZ-αLA与LZ均二聚体形成的络合物主要以LZ-LZ均二聚体形式存在。这一结果首次证明了α-LA与LZ结合形成了具有显著增强杀菌活性的独特的复合体。我们的结果有力地表明LZ和α-LA在新生儿抵抗胃微生物感染的宿主防御系统中起着重要作用。此外，我们还开展了一项平行的遗传学研究，目的是从人乳腺上皮细胞中克隆人LZ(HLZ)和人LA(HL A)的基因，在毕赤酵母中建立这两种蛋白天然信号肽作用下的高效表达系统，并设计稳定的hLZ和HL A之间的框内融合结构。人L Z、α-LA及其LZ-α-LA融合载体在甲醇毕赤酵母中的表达水平超过20 mg/L，为设计潜在的治疗感染性疾病的抗菌药物提供了良好的机会。较少

项目成果

H.R.IBRAHIM, U.Thomas, A.Pellegrini: "A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action"Journal of Biological Chemistry. 276(47). 43767-43774 (2001)

H.R.IBRAHIM、U.Thomas、A.Pellegrini：“溶菌酶活性位点裂缝上唇处的螺旋-环-螺旋肽赋予有效的抗菌活性和膜透化作用”《生物化学杂志》。

R.IBRAHIM, T.Aoki: "New Promises of Lysozyme as Immune Modulator and Antimicrobial Agent for Nutraceuticals"Foods & Food Ingredients Journal. 208(5). 361-374 (2003)

R.IBRAHIM、T.Aoki：“溶菌酶作为营养保健品的免疫调节剂和抗菌剂的新前景”

H.R.IBRAHIM, T.Matsuzaki, T.Aoki: "Genetic evidence that antibacterial activity of lysozyme is independent of its catalytic function."FEBS Letters. 506. 27-32 (2001)

H.R.IBRAHIM、T.Matsuzaki、T.Aoki：“溶菌酶的抗菌活性与其催化功能无关的遗传证据。”FEBS 快报。

H.R.IBRAHIM, T.Aoki: "New Promises of Lysozyme as Immune Modulator and Antimicrobial Agent for Nutraceuticals"Journal of Foods & Food Ingredients. (In Press).

H.R.IBRAHIM、T.Aoki：“溶菌酶作为营养保健品免疫调节剂和抗菌剂的新前景”食品杂志

H.R.IBRAHIM, U.Thomas, A.Pellegrini: "A helix-loop-helix peptide at the upper lip of the active site cleft of Lysozyme confers potent antimicrobial activity with membrane permeabilization action."Journal of Biological Chemistry. 276(47). 43767-43774 (2001

H.R.IBRAHIM、U.Thomas、A.Pellegrini：“溶菌酶活性位点裂缝上唇处的螺旋-环-螺旋肽赋予了有效的抗菌活性和膜透化作用。”生物化学杂志。