Isolation, characterization, and cDNA sequence of a carotenoid binding protein from the silk gland of Bombyx mori larvae
Isolation, characterization, and cDNA sequence of a carotenoid binding protein from the silk gland of Bombyx mori larvae
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DOI：
10.1074/jbc.m204507200
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发表时间：
2002-08-30
影响因子：
4.8
通讯作者：
Tsuchida, K
中科院分区：
文献类型：
--
作者：
Tabunoki, H;Sugiyama, H;Tsuchida, K
从家蚕幼虫丝腺中分离到一种类胡萝卜素结合蛋白（CBP）。该蛋白质具有33 kDa的表观分子量，并以1：1的摩尔比结合类胡萝卜素。叶黄素占结合类胡萝卜素的90%，而α-胡萝卜素和β-胡萝卜素是次要成分。免疫学分析表明CBP仅存在于丝腺、中肠、睾丸和卵巢的黄色组织中。B的几种表型。与类胡萝卜素转运相关的桑属突变体已被用于表征CBP。Y（黄色血淋巴）基因控制类胡萝卜素从中肠腔吸收到中肠上皮，而带有+（Y）基因的幼虫缺乏这种特性。免疫印迹分析证实了仅具有显性Y基因的突变体中存在CBP。免疫组化证实CBP定位于中肠上皮的绒毛，表明CBP可能参与类胡萝卜素的吸收。从家蚕丝腺cDNA文库中克隆了编码297个氨基酸的CBP蛋白。推导的氨基酸序列显示，CBP是类固醇生成急性调节（星星）蛋白家族的新成员，具有独特的StAR相关脂质转移结构域的结构特征，已知有助于脂质转移和识别。重组CBP（rCBP）的叶黄素结合能力，通过孵育rCBP与叶黄素，然后使用抗CBP IgG结合蛋白A-Sepharose的免疫沉淀法测定，证明叶黄素-rCBP复合物的形成。结合特异性的序列分析表明CBP是星星蛋白家族的新成员，结合类胡萝卜素而不是胆固醇。
A carotenoid binding protein (CBP) has been isolated from the silk glands of Bombyx mori larvae. The protein has an apparent molecular mass of 33 kDa and binds carotenoids in a 1:1 molar ratio. Lutein accounts for 90% of the bound carotenoids, whereas a-carotene and beta-carotene are minor components. Immunological analysis demonstrated the presence of CBP only in the yellow-colored tissues of the silk gland, midgut, testis, and ovary. Several phenotypes of B. mori mutants linked to carotenoid transport have been utilized to characterize CBP. The Y (yellow hemolymph) gene controls uptake of carotenoids from the midgut lumen into the midgut epithelium, and larvae with the +(Y) gene lack this property. Immunoblotting analysis confirmed the presence of CBP in mutants with the dominant Y gene only. Immunohistochemistry verified the localization of CBP in the villi of the midgut epithelium, indicating that CBP might be involved in absorption of carotenoids. A cDNA clone for CBP encoding a protein of 297 amino acids has been isolated from the R mori silk gland cDNA library. The deduced amino acid sequence revealed that CBP is a novel member of the steroidogenic acute regulatory (StAR) protein family with its unique structural feature of a StAR-related lipid transfer domain, known to aid in lipid transfer and recognition. Lutein-binding capacity of the recombinant CBP (rCBP) determined by incubating rCBP with lutein followed by immunoprecipitation using anti-CBP IgG conjugated to protein A-Sepharose, demonstrated the formation of a lutein-rCBP complex. Sequence analyses coupled with binding specificity suggest that CBP is a new member of the StAR protein family that binds carotenoids rather than cholesterol.