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 insolution 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.

Background: Spiders extrude adhesive glues to form connection joints that mediate web construction and prey wrapping. Results: DNA microarray analysis and mass spectrometry reveal new protein glue constituents that comprise connection joints. Conclusion: Spider glue proteins represent a diverse group of polypeptides with distinct molecular architectures. Significance: Learning how spider glues mediate the fusion of fibers is crucial for understanding adhesion mechanisms in biology.

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion. Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands. Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk], tubuliform [synthesizes egg case silk], flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] and pyriform [produces attachment disc silk]. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Spiders spin high performance fibers that have diverse mechanical properties for movement, prey capture and protection of eggs. To investigate the mechanism by which spiders anchor their fibers to solid matrices, we solubilized the attachment discs from black widow spiders (cob weavers) 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 have identified the first spider glue fibroin of the silk gene family called Pyriform Spidroin 1 (PySp1), as well as a closely related fibroin in orb weavers using conventional nucleic acid‐nucleic acid hybridization. Biophysical characterization of black widow spider attachment disc fibers using atomic force microscopy, as well as biochemical analyses of PySp1 using circular dichroism and NMR, demonstrate that PySp1 has distinct molecular features relative to other silk family members. Given the unique biophysical and chemical properties of PySp1, this fibroin could be ideal for use in spinning artificial silks through genetic engineering for different commercial applications, which include body armor, tissue engineering, sutures for the medical industry, and materials for the automobile and aerospace industry. This research was supported by the NSF RUI Grant MCB‐0544087.

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