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

Abstract

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 ϳ300-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 glandrestricted 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.The ability to spin multiple task-specific silks is a defining feature of the diverse order Araneae (Ͼ37,000 described species). Araneoid spiders use specialized abdominal glands to manufacture up to seven different protein-based silks/glues that have diverse mechanical properties (1). Spinning high performance fibers with different mechanical properties enable spiders to perform a wide range of functions, including prey capture, locomotion, and protection of developing offspring (2).Amino acid sequences of spider fibroins (spidroins) share a number of distinctive features. Repeats of four fundamental amino acid motifs characterize the majority of sequenced spider silks as follows: (i) alternating glycine and alanine ((GA) n ), (ii) polyalanine (A n ), (iii) GGX (X ϭ subset of residues), and (iv) GPGGX. Biochemical studies indicate that these motifs correspond to distinct structural modules, e.g. A n and (GA) n repeats form crystalline ␤-sheets, whereas ␤-spirals are generated from a series of concatenated ␤-turns from the repeat structure GPGGX (3). It has been proposed that these different structural modules contribute to the mechanics of the fibers. Combinations of these motifs form larger repetitive units termed ensemble repeats, which are organized in tandem copies throug...

Spider Glue Proteins Have Distinct Architectures Compared with Traditional Spidroin Family Members

“…Mass Spectrometric Analysis-In-solution tryptic digests of the solubilized wrapping silk as well as other fiber types were performed as previously described (9,10). The MS map and peptide MS/MS spectra were acquired with a MALDI/TOF/ TOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems, Foster City, CA) operated in reflector mode or MS/MS mode, respectively.…”

“…Wrapping silk was collected as previously described (10). Prior to imaging, the samples were coated to a thickness of ϳ14 -20 nm with gold alloy using a Pelco SC-7 autosputter coater with an FTM-2 film thickness.…”

“…These structural proteins share common protein architectures, including the presence of internal block repeats, and nonrepetitive conserved N and C termini (4 -6). Six of the seven silk-producing glands have been shown to produce fibers with the exception of the aggregate gland (7)(8)(9)(10)(11)(12)(13). Due to the morphology of the aggregate gland, its spinning duct, and the anatomical features of its spigots, it has been debated whether this gland synthesizes structural proteins that self-assemble into fibers or whether it exclusively secretes nonstructural adhesive proteins.…”

Pyriform Spidroin 1, a Novel Member of the Silk Gene Family That Anchors Dragline Silk Fibers in Attachment Discs of the Black Widow Spider, Latrodectus hesperus

“…Over the past 20 years, seven distinct members of the silk gene family have been identified and characterized at the molecular level, which include the silk proteins MaSp1 and MaSp2 (dragline silk) (3)(4)(5), AcSp1 or AcSp1-like (wrapping silk and egg case silk) (6,7), TuSp1 (egg case silk)(8 -11), MiSp1 and MiSp2 or MiSp1-like (temporary spiral capture silk or web reinforcement silk) (12,13), and Flag silk (capture spiral silk in orb weavers) (14). These spider fibroins have revealed that they share a number of distinctive properties, including four fundamental amino acid repeat motifs that characterize the majority of the family: (i) alternating glycine alanine couplets (GA n ), (ii) polyalanine blocks (A n ), (iii) GGX (X ϭ subset of residues, which include Leu, Ile, and Ala), and (iv) GPGX n .…”

Spider Silk Composites and Applications

“…In addition to aciniform silks being constituents of egg cases, these fibers are also used for prey capture (Hayashi et al, 2004;Vasanthavada et al, 2007). Because detailed molecular data are available for dragline and tubuliform silks and these fiber types are available in large quantities, we are attempting to study the mechanical behavior of synthetic composite spider silk threads spun from regenerated dragline and egg sac silk protein mixtures.…”