Silks from araneoid spiders have become an active area of research for material scientists, biochemists, and molecular biologists. Mechanical properties of spider silk such as elasticity, tensile strength, and toughness make the manufacturing of silk for medical sutures, body armor, ropes and other synthetic material applications great possibilities. The difficulties of having a black widow spider farm to harvest silk, due to their cannibalistic nature, make recombinant expression of silk proteins a fundamental goal of spider silk research. In order to express silk fibers, cDNAs encoding the corresponding silk fiber products must first be isolated and identified. One of the first steps in gene identification relies on the identification of the proteins in the silk fibers. No previous study has demonstrated the molecular constituents of gumfooted lines. In the course of this research, the core fibroins in the gumfooted lines were identified to be members of the Major Ampullate Spidroin family (MaSp), using mass spectrometry. This research was the first to identify the core fibroins of the gumfooted lines. Novel peptide fragments from solubilized gumfooted lines were acquired from manual de novo MSIMS sequencing after in-gel tryptic digestion. These peptide fragments showed post-translational modifications consistent with glycosylation, which aligns with the reported chemical properties of glue proteins. Novel peptide sequences were also acquired from the attachment discs as well as novel scanning electron microscopy images and reveal, for the first time, the physical attributes and molecular properties of threads attached to the surface of an immobilized structure. This study was the first to identify the molecular constituents of the attachment discs.

Spider silk from the female black widow spider, Latrodectus hesperus, is made of extraordinary biomolecules of nature. Efforts of the scientific community to commercially synthesize silks have become a collaborating, yet competitive race, to characterize the proteins that contribute to its intriguing biomechanics. Little has been reported on aqueous silk molecules in black widow spider silk, which are quite different from the large water insoluble core fibroins. In this study, a novel, aqueous aggregate gland derived silk factor (AgSF 1) was investigated using proteomics and immunological approaches. Western blot analyses of whole tissue lysates and solublized silk fibers revealed high levels of AgSFl in the aggregate gland, in the web scaffolding junctions, and in wrapping silk. MS/MS analyses of tryptic digest products from solubilized wrapping silk and aggregate gland whole cell lysate also confirmed the presence of AgSFl in these samples. Possible post-translational modifications were also analyzed by two-dimensional gel electrophoresis (2DE) and MS/MS analysis. AgSFl was localized in the web scaffolding junctions and our data supports a role as an adhesive silk protein that serves as a center for connecting scaffolding fibers that functions to reduce the tensile strength of scaffolding fibers, which facilitates capture of aerial insects.

Little is actually known about the transcriptional regulation of spider silk as most studies have focused on the material properties of silks. We isolated and mapped the TuSp1 core promoter from the black widow spider, Latrodectus hesperus. Using a genomic DNA walking strategy, we have isolated an upstream segment (581 bp) of genomic DNA containing the promoter as well as the first exon of the TuSp1 gene. This upstream regulatory element was able to initiate transcription in insect cells when placed upstream the promoterless firefly luciferase reporter gene. Initiation of transcription was orientation dependent, as insertion of this upstream regulatory module in the reverse orientation led to inefficient transcriptional initiation. Only 170 bp of upstream sequence was required for strong transcriptional initiation, showing that core promoter resides within the first 170 bp of upstream 5' -flanking DNA. We also demonstrate the bHLH factor SGSF1 can repress gene transcription of the TuSp1 core promoter, implying SGSF I might participate in the transcriptional regulation of the TuSp1 gene in vivo.