Egg case protein-1 -: A new class of silk proteins with fibroin-like properties from the spider Latrodectus hesperus
Egg case protein-1 -: A new class of silk proteins with fibroin-like properties from the spider Latrodectus hesperus
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DOI：
10.1074/jbc.m412316200
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发表时间：
2005-06-03
影响因子：
4.8
通讯作者：
Vierra, C
中科院分区：
文献类型：
--
作者：
Hu, XY;Kohler, K;Vierra, C
蜘蛛能产生多种类型的丝，这些丝表现出不同的机械特性和生物功能。大多数关于蜘蛛丝的分子研究都集中在从拖丝和捕获丝中提取的丝蛋白上，这两种重要的丝类型与蜘蛛的生存有关。在我们的研究中，我们重点研究了卵壳丝的特性，这是黑寡妇蜘蛛（Latrodectus hesperus）产生的第三种丝纤维。利用扫描电子显微镜对卵壳丝的物理结构进行分析，发现存在大小直径的纤维。用强蛋白质变性剂8 M胍盐酸盐对纤维进行增溶，通过SDS-PAGE和蛋白银染色证实卵壳丝中含有丰富的100 kda蛋白双偶体成分。结合基质辅助激光解吸电离串联飞行时间质谱法和反向遗传学，我们分离了一个名为ep -1的新基因，该基因编码100 kda物种的一种蛋白质成分。利用ECP-1的初级序列对NCBInr蛋白数据库进行BLAST搜索，发现其与蜘蛛和家蚕的丝蛋白相似，在ECP-1中有两个不同的区域。这些区域包含保守的重复丝蛋白基序poly（Ala）和poly(Gly-Ala)，但令人惊讶的是，在epc -1的初级序列中没有发现更大的集合重复序列。与丝素蛋白的丝腺限制性表达模式一致，证明了ECP-1主要在管状腺中产生，在大壶状腺和小壶状腺中检测到较低的水平。研究还显示，通过独特的富含半胱氨酸的n端区域形成二硫键，epc -1单体单元可以组装成更高的聚集结构。总的来说，我们的发现为卵丝的成分提供了新的见解，并确定了一类新的丝蛋白，它们与蜘蛛丝基因家族的传统成员相比具有独特的分子特征。
Spiders produce multiple types of silk that exhibit diverse mechanical properties and biological functions. Most molecular studies of spider silk have focused on fibroins from dragline silk and capture silk, two important silk types involved in the survival of the spider. In our studies we have focused on the characterization of egg case silk, a third silk fiber produced by the black widow spider, Latrodectus hesperus. Analysis of the physical structure of egg case silk using scanning electron microscopy demonstrates the presence of small and large diameter fibers. By using the strong protein denaturant 8 M guanidine hydrochloride to solubilize the fibers, we demonstrated by SDS-PAGE and protein silver staining that an abundant component of egg case silk is a 100-kDa protein doublet. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called ecp-1, which encodes for one of the protein components of the 100-kDa species. BLAST searches of the NCBInr protein data base using the primary sequence of ECP-1 revealed similarity to fibroins from spiders and silkworms, which mapped to two distinct regions within the ECP-1. These regions contained the conserved repetitive fibroin motifs poly( Ala) and poly(Gly-Ala), but surprisingly, no larger ensemble repeats could be identified within the primary sequence of ECP-1. Consistent with silk gland-restricted patterns of expression for fibroins, ECP-1 was demonstrated to be predominantly produced in the tubuliform gland, with lower levels detected in the major and minor ampullate glands. ECP-1 monomeric units were also shown to assemble into higher aggregate structures through the formation of disulfide bonds via a unique cysteine-rich N-terminal region. Collectively, our findings provide new insight into the components of egg case silk and identify a new class of silk proteins with distinctive molecular features relative to traditional members of the spider silk gene family.