Egg Case Protein-1

Abstract

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.

Aciniform Spidroin, a Constituent of Egg Case Sacs and Wrapping Silk Fibers from the Black Widow Spider Latrodectus hesperus

“…Previous studies have demonstrated that two of the abundant proteins represent egg case protein gene products (Fig. 1, lane 2) (14,16). The ϳ300-kDa protein does not represent the major constituent of the large diameter fiber, TuSp1, which is not readily dissolved under these conditions and is missing from detection (Fig.…”

“…Egg sacs must be designed to resist damage from predator/parasitoid invasion as well as temperature and humidity fluctuations (11)(12)(13). Analyses of the physical structure of egg sacs collected from the cob weaver Latrodectus hesperus, commonly referred to as the black widow spider, have demonstrated that their egg case sacs consist of two different diameter fibers (14,15). MS/MS 4 analyses of black widow peptides generated from solubilized egg case materials digested with trypsin have shown that the large diameter core fibers contain the fibroin TuSp1, as well as the egg case proteins ECP-1 and ECP-2 (egg case protein 1 or 2), which form a trimeric complex in cob weavers (14,16,17).…”

“…Analyses of the physical structure of egg sacs collected from the cob weaver Latrodectus hesperus, commonly referred to as the black widow spider, have demonstrated that their egg case sacs consist of two different diameter fibers (14,15). MS/MS 4 analyses of black widow peptides generated from solubilized egg case materials digested with trypsin have shown that the large diameter core fibers contain the fibroin TuSp1, as well as the egg case proteins ECP-1 and ECP-2 (egg case protein 1 or 2), which form a trimeric complex in cob weavers (14,16,17). Real time quantitative PCR and Northern blot analyses have demonstrated tubuliform-restricted patterns of expression for these gene products (14,(17)(18)(19).…”

“…MS/MS 4 analyses of black widow peptides generated from solubilized egg case materials digested with trypsin have shown that the large diameter core fibers contain the fibroin TuSp1, as well as the egg case proteins ECP-1 and ECP-2 (egg case protein 1 or 2), which form a trimeric complex in cob weavers (14,16,17). Real time quantitative PCR and Northern blot analyses have demonstrated tubuliform-restricted patterns of expression for these gene products (14,(17)(18)(19). Although it has been widely accepted by the silk community that the aciniform glands produce egg case silk constituents and wrapping silk, no molecular evidence has been reported to demonstrate that aciniform-specific fibroins are directly assembled into these diverse materials (1).…”

Modular evolution of egg case silk genes across orb-weaving spider superfamilies

“…Examination of ADF-2, in comparison with additional spidroins, subsequently identified it as a member of the major ampullate spidroin 1 (MaSp1) silk ortholog group (7), used in dragline fibers (13,14). Recently, Hu et al (15) reported another putative component of egg case silk (ECP-1) expressed in silk glands of the black widow spider, Latrodectus hesperus. However, the translated sequence of neither ADF-2 nor ECP-1 could account for the amino acid composition of tubuliform silk fibers or the gland's contents (4,11,15,16,17), indicating that the dominant silk expressed by tubuliform glands must be a different, unidentified protein (4).…”