Vasanthavada Keshav, Hu Xiaoyi, Falick Arnold M, La Mattina Coby, Moore Anne M F, Jones Patrick R, Yee Russell, Reza Ryan, Tuton Tiffany, Vierra Craig
Department of Chemistry, University of the Pacific, Stockton, California 95211, USA.
J Biol Chem. 2007 Nov 30;282(48):35088-97. doi: 10.1074/jbc.M705791200. Epub 2007 Oct 5.
Spiders produce high performance fibers with diverse mechanical properties and biological functions. Molecular and biochemical studies of spider egg case silk have revealed that the main constituent of the large diameter fiber contains the fibroin TuSp1. Here we demonstrate by SDS-PAGE and protein silver staining the presence of a distinct approximately 300-kDa polypeptide that is found in solubilized egg case sacs. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called AcSp1-like and demonstrate that its protein product is assembled into the small diameter fibers of egg case sacs and wrapping silks from the black widow spider, Latrodectus hesperus. BLAST searches of the NCBInr protein data base using the amino acid sequence of AcSp1-like revealed similarity to AcSp1, an inferred protein proposed to be a component of wrapping silk. However, the AcSp1-like protein was found to display more nonuniformity in its internal iterated repeat modules than the putative AcSp1 fibroin. Real time quantitative PCR analysis demonstrates that the AcSp1-like gene displays an aciniform gland-restricted pattern of expression. The amino acid composition of the fibroins extracted from the luminal contents of the aciniform glands was remarkably similar to the predicted amino acid composition of the AcSp1-like protein, which supports the assertion that AcSp1-like protein represents the major constituent stored within the aciniform gland. Collectively, our findings provide the first direct molecular evidence for the involvement of the aciniform gland in the production of a common fibroin that is assembled into the small diameter threads of egg case and wrapping silk of cob weavers.
蜘蛛能产生具有多种机械性能和生物学功能的高性能纤维。对蜘蛛卵囊丝的分子和生化研究表明，大直径纤维的主要成分包含丝心蛋白TuSp1。在此，我们通过SDS-PAGE和蛋白质银染法证明，在溶解的卵囊囊中存在一种明显的约300 kDa的多肽。结合基质辅助激光解吸电离串联飞行时间质谱和反向遗传学，我们分离出一个名为AcSp1-like的新基因，并证明其蛋白质产物组装成卵囊囊的小直径纤维以及来自黑寡妇蜘蛛（Latrodectus hesperus）的包裹丝。使用AcSp1-like的氨基酸序列对NCBInr蛋白质数据库进行BLAST搜索，发现其与AcSp1相似，AcSp1是一种推测为包裹丝成分的蛋白质。然而，发现AcSp1-like蛋白在其内部重复模块中比假定的AcSp1丝心蛋白表现出更多的不均匀性。实时定量PCR分析表明，AcSp1-like基因呈现出一种局限于葡萄状腺的表达模式。从葡萄状腺管腔内容物中提取的丝心蛋白的氨基酸组成与AcSp1-like蛋白的预测氨基酸组成非常相似，这支持了AcSp-like蛋白代表葡萄状腺中储存的主要成分这一论断。总的来说，我们的研究结果为葡萄状腺参与产生一种共同的丝心蛋白提供了首个直接的分子证据，这种丝心蛋白组装成卵囊的小直径丝以及结网蜘蛛的包裹丝。
