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

家蚕的主要幼虫胞壁蛋白，称为“LCP30”，是成熟形式的分子量为22k的碱性蛋白。克隆的LCP30 cDNA的序列分析表明存在精氨酸-甘氨酸-天冬氨酸(RGD)三肽序列，这是许多细胞外基质蛋白中存在的共有细胞粘附基序。本工作从家蚕幼虫体皮中纯化了天然形式的LCP30，并研究了其分子特性和生理功能。从五龄幼虫粗珠被的盐酸胍提取物中，经硫酸铵沉淀、离子交换层析和制备型聚丙烯酰胺凝胶电泳，将LCP30纯化至均质。 2．通过将LCP30吸附在几丁质纸上，然后使用针对LCP30.3的特异性抗体对抗原进行免疫检测来定性证明LCP30的几丁质结合活性。 LCP30的几丁质结合活性通过使用放射性标记的LCP30和粉末几丁质的测定方法进行定量。 LCP30 与几丁质的结合几乎不受盐浓度和二价阳离子的影响。反应混合物中存在的 N-乙酰壳二糖抑制了放射性标记的 LCP30 与几丁质的结合。放射性标记的LCP30与几丁质的结合由未标记的LCP30.4竞争。使用家蚕培养细胞的实验证明，通过使用带有RGD序列的寡肽作为特定竞争剂的抑制实验证明了细胞附着。 LCP30介导的细胞附着需要二价阳离子。5．细菌表达的β-半乳糖苷酶/LCP30融合蛋白表现出几丁质结合以及细胞附着活性，为未来利用人工突变的LCP研究角质层组织的分子机制提供了有希望的结果。

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.