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Studies on physicochemical and cytochemical analyzes of major cuticle proteins of silkworm, Bombyx mori

家蚕主要角质层蛋白的理化和细胞化学分析研究

基本信息

批准号：
05640773
负责人：
IZUMI Susumu
金额：
$ 1.22万
依托单位：
Tokyo Metropolitan University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05640773/
关键词：
Insect Silkworm Bombyx Chitin Expression vector Fusion protein Cell adhension Cuticle N-アセチルグルコサアミン

项目摘要

A major larval cuitcle protein of Bombyx mori, termed "LCP30" is a basic protein with molecular weight 22k in mature form. The sequence analysis of the cloned LCP30 cDNA has shown the presence of an arginine-glycine-aspartic acid (RGD) tripeptide sequence, which is a consensus cell-adhesive motif occurring in many extracellular matrix proteins.In the present work, native form of LCP30 was purified from the larval integument of B.mori and its molecular properties and physiological functions were studied.1. LCP30 was purified to homogeneity from the guanidine-HCl extract of crude integuments of the fifth instar larvae through the steps of ammounium sulfate precipitation, ion exchange chromatography and preparative polyacrylamide gel elecctrophoresis.2. Chitin-binding activity of LCP30 was qualitatively demonstrated by adsorption of LCP30 on chitin paper followed by immuno-detection of antigen by use of specific antibody against LCP30.3. Chitin-binding activity of LCP30 was quantitatively determined by the assay method using the radiolabeled LCP30 and powdered chitin. Binding of LCP30 to chitin was little influenced by salt concentrations and divalent cations. Binding of the radiolabeled LCP30 to chitin was inhibited by the presence of N-acetvlchitobiose in the reaction mixture . Binding of the radiolabeled LCP30 to chitin was competed by the unlabeled LCP30.4. The experiments employing the cultured cells of B.mori proved the cell attachment was demonstrated by the inhibition experiments using oligopeptide bearing RGD sequence as specific competitor. Cell attachment mediated by LCP30 required divalent cations.5. Bacterially expressed beta-galactosidase/LCP30 fusion protein exhibited chitin-binding as well as cell-attachment activity, proiding promising results for future studies on the molecular mechanism of cuticle organization by use of artificially mutated LCPs.

桑尺蠖幼虫的一种主要的角质层蛋白，称为“LCP 30”，是一种成熟形式的碱性蛋白，分子量为22 k。对克隆的LCP 30 cDNA进行序列分析，发现其含有一段精氨酸-甘氨酸-天冬氨酸（RGD）三肽序列，这是一种存在于多种细胞外基质蛋白中的共有细胞粘附基序，本工作从家蚕B.mori幼虫体壁中纯化了天然形式的LCP 30，并对其分子特性和生理功能进行了研究.通过硫酸铵沉淀、离子交换层析和制备型聚丙烯酰胺凝胶电泳等步骤，从五龄幼虫体壁盐酸胍粗提液中纯化得到均一的LCP 30.通过将LCP 30吸附在几丁质纸上，然后通过使用针对LCP 30的特异性抗体进行抗原的免疫检测来定性地证明LCP 30的几丁质结合活性。通过使用放射性标记的LCP 30和粉末状甲壳素的测定方法定量测定LCP 30的甲壳素结合活性。LCP 30与几丁质的结合受盐浓度和二价阳离子的影响很小。在反应混合物中存在N-乙酰壳二糖可抑制放射性标记的LCP 30与几丁质的结合。未标记的LCP 30.4竞争放射性标记的LCP 30与几丁质的结合。以家蚕B培养细胞为实验材料，以含有RGD序列的寡肽为特异性竞争物，通过抑制实验证明了该寡肽对家蚕细胞的粘附作用。LCP 30介导的细胞粘附需要二价阳离子.细菌表达的β-半乳糖苷酶/LCP 30融合蛋白具有几丁质结合和细胞附着活性，为进一步研究利用人工突变的LCPs进行角质层组织化的分子机制提供了有希望的结果。