Abstract
Spiders spin high performance threads that have diverse mechanical properties for specific biological applications. To better understand the molecular mechanism by which spiders anchor their threads to a solid support, we solubilized the attachment discs from black widow spiders and performed in-solution tryptic digests followed by MS/MS analysis to identify novel peptides derived from glue silks. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and cDNA library screening, we isolated a novel member of the silk gene family called pysp1 and demonstrate that its protein product is assembled into the attachment disc silks. Alignment of the PySp1 amino acid sequence to other fibroins revealed conservation in the non-repetitive C-terminal region of the silk family. MS/MS analysis also confirmed the presence of MaSp1 and MaSp2, two important components of dragline silks, anchored within the attachment disc materials. Characterization of the ultrastructure of attachment discs using scanning electron microscopy studies support the localization of PySp1 to small diameter fibers embedded in a glue-like cement, which network with large diameter dragline silk threads, producing a strong, adhesive material. Consistent with elevated PySp1 mRNA levels detected in the pyriform gland, MS analysis of the luminal contents extracted from the pyriform gland after tryptic digestion support the assertion that PySp1 represents one of the major constituents manufactured in the pyriform gland. Taken together, our data demonstrate that PySp1 is spun into attachment disc silks to help affix dragline fibers to substrates, a critical function during spider web construction for prey capture and locomotion.
Highlights
All spiders spin silk, but members of the highly diverse suborder Araneomorphae (ϳ37,000 species) spin multiple high performance fibers that enable them to perform a wide range of functions, including prey capture, locomotion, and protection of developing offspring [1, 2]
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(3–5), AcSp1 or AcSp1-like[6,7], TuSp1(8 –11), MiSp1 and MiSp2 or MiSp1-like[12,13], and Flag silk[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 (GAn), (ii) polyalanine blocks (An), (iii) GGX (X ϭ subset of residues, which include Leu, Ile, and Ala), and (iv) GPGXn
Based upon observational and histological data, the pyriform gland has been implicated in the production of attachment disc silks, which function to affix dragline silk to substrates (19 –21)
Summary
“safety line” for the spider has been observed to fuse with attachment disc silk, providing dragline silk with a secure anchor point for locomotor functions to assist prey capture and predator evasion. Given the nature of attachment disc silks, which are spun in a gluey liquid material that dries to facilitate affixing dragline silk, it is uncertain whether the biochemical properties of the attachment disc silk fibroins would be highly divergent from traditional silk family members. Phylogenetic analysis of the C terminus revealed that PySp1 was a member of the spider silk family, and showed that it was a highly divergent fibroin with distinctive internal molecular features. These data suggest the unique chemical properties of PySp1 likely facilitate its ability to be spun into a sticky liquid environment that is well suited to cement dragline silk to solid surfaces. Given the specialized chemical properties of PySp1, these findings have substantial impact for structural biologists and material scientists seeking to engineer synthetic silks that have diverse properties for applications that include body armor, ropes and cords, tissue engineering, and drug delivery
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