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

家蚕幼虫茧蛋白LCP30是一种成熟时分子量为22k的碱性蛋白。克隆的LCP30 cDNA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普遍存在的细胞粘附基序。本文从家蚕幼虫被膜中纯化了原生形态的LCP30，并对其分子特性和生理功能进行了研究。通过硫酸铵沉淀、离子交换色谱和制备聚丙烯酰胺凝胶电泳等步骤，从5龄幼虫粗被毛的胍-盐酸提取物中纯化出LCP30。通过将LCP30吸附在几丁质纸上，利用LCP30.3特异性抗体对抗原进行免疫检测，定性地证实了LCP30与几丁质结合的活性。利用放射性标记LCP30和粉末状几丁质，定量测定了LCP30的几丁质结合活性。盐浓度和二价阳离子对LCP30与几丁质的结合影响不大。反应混合物中n -乙酰壳聚糖的存在抑制了放射性标记的LCP30与几丁质的结合。放射性标记的LCP30与几丁质的结合被未标记的LCP30.4所取代。用家蚕培养的细胞进行的实验证明，以含有RGD序列的寡肽作为特异性竞争对手的抑制实验证明了细胞的附着性。LCP30介导的细胞附着需要二价阳离子。细菌表达的β -半乳糖苷酶/LCP30融合蛋白具有几丁质结合和细胞附着活性，为未来利用人工诱变的LCP30进行角质层组织的分子机制研究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.