Egg Case Protein 3: A Constituent of Black Widow Spider Tubuliform Silk.

Spider silk has outstanding mechanical properties, rivaling some of the best materials on the planet. Biochemical analyses of tubuliform silk have led to the identification of TuSp1, egg case protein 1, and egg case protein 2. TuSp1 belongs to the spidroin superfamily, containing a non-repetitive N- and C-terminal domain and internal block repeats. ECP1 and ECP2, which lack internal block repeats and sequence similarities to the highly conserved N- and C-terminal domains of spidroins, have cysteine-rich N-terminal domains. In this study, we performed an in-depth proteomic analysis of tubuliform glands, spinning dope, and egg sacs, which led to the identification of a novel molecular constituent of black widow tubuliform silk, referred to as egg case protein 3 or ECP3. Analysis of the translated ECP3 cDNA predicts a low molecular weight protein of 11.8 kDa. Real-time reverse transcription-quantitative PCR analysis performed with different silk-producing glands revealed ECP3 mRNA is predominantly expressed within tubuliform glands of spiders. Taken together, these findings reveal a novel protein that is secreted into black widow spider tubuliform silk.

Comprehensive Proteomic Analysis of Spider Dragline Silk from Black Widows: A Recipe to Build Synthetic Silk Fibers.

The outstanding material properties of spider dragline silk fibers have been attributed to two spidroins, major ampullate spidroins 1 and 2 (MaSp1 and MaSp2). Although dragline silk fibers have been treated with different chemical solvents to elucidate the relationship between protein structure and fiber mechanics, there has not been a comprehensive proteomic analysis of the major ampullate (MA) gland, its spinning dope, and dragline silk using a wide range of chaotropic agents, inorganic salts, and fluorinated alcohols to elucidate their complete molecular constituents. In these studies, we perform in-solution tryptic digestions of solubilized MA glands, spinning dope and dragline silk fibers using five different solvents, followed by nano liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis with an Orbitrap Fusion™ Tribrid™. To improve protein identification, we employed three different tryptic peptide fragmentation modes, which included collision-induced dissociation (CID), electron transfer dissociation (ETD), and high energy collision dissociation (HCD) to discover proteins involved in the silk assembly pathway and silk fiber. In addition to MaSp1 and MaSp2, we confirmed the presence of a third spidroin, aciniform spidroin 1 (AcSp1), widely recognized as the major constituent of wrapping silk, as a product of dragline silk. Our findings also reveal that MA glands, spinning dope, and dragline silk contain at least seven common proteins: three members of the Cysteine-Rich Protein Family (CRP1, CRP2 and CRP4), cysteine-rich secretory protein 3 (CRISP3), fasciclin and two uncharacterized proteins. In summary, this study provides a proteomic blueprint to construct synthetic silk fibers that most closely mimic natural fibers.

Analysis of aqueous glue coating proteins on the silk fibers of the cob weaver, Latrodectus hesperus.

Elucidation of the molecular composition and physical properties of spider glue is necessary to understand its function in the mechanics of the web and prey capture. Previous reports have indicated that components of the adhesive coating contain inorganic molecules, phosphorylated glycoproteins, lipids, and organic low-molecular mass (LMM) compounds. Using a proteomic strategy, we have investigated the viscid, aqueous components that coat different silk fiber types from the black widow spider, Latrodectus hesperus. After in-solution tryptic digestion of the aqueous protein material extracted from egg case sacs, gumfooted lines, and the web scaffolding connection joints, followed by peptide analysis using MALDI tandem TOF mass spectrometry, we demonstrate that these fibers are coated with common peptides. Utilizing a reverse genetics approach, we have isolated the cDNAs encoding two distinct fiber coating products, which we have named spider coating peptide 1 and 2 (SCP-1 and SCP-2). Secreted forms of SCP-1 and SCP-2 contain 36 and 19 amino acids, respectively, and their primary sequences display no significant similarities to ensemble repeat units from traditional fibroins. Quantitative real-time reverse transcription PCR analyses show that these mRNAs are chiefly produced by the aggregate gland. Biochemical studies also demonstrate that the SCP-1 peptide has intrinsic metal binding properties, suggesting a role of peptide-metal ion interactions with the fiber constituents to enhance thread performance. Collectively, these investigations are the first to reveal a novel role for the aggregate gland in the production of peptides that coat spider silk threads.

Characterization of a novel class II bHLH transcription factor from the black widow spider, Latrodectus hesperus, with silk-gland restricted patterns of expression.

Members of the basic helix-loop-helix (bHLH) family are required for a number of different developmental pathways, including lymphopoiesis, myogenesis, neurogenesis, and sex determination. Screening a cDNA library prepared from silk-producing glands of the black widow spider, we have identified a new bHLH transcription factor named SGSF. Within the bHLH region, SGSF showed considerable conservation with other HLH proteins, including Drosophila melanogaster achaete and scute, as well as three HLH proteins identified by gene prediction programs. The expression pattern of SGSF was restricted to a subset of silk-producing glands, which include the tubuliform and major ampullate glands. SGSF was capable of binding an E-box element as a heterodimer with the E protein, E47, but was unable to bind this motif as a homodimer. SGSF was demonstrated to be a nuclear transcription factor capable of attenuating the transactivation of E47 homodimers in mammalian cells. SGSF represents the first example of a silk gland-restricted bHLH protein, and its expression pattern suggests that SGSF plays a role in regulating differentiation of cells in the spider that control silk gland formation or egg case silk gene expression.

Molecular and mechanical properties of major ampullate silk of the black widow spider, Latrodectus hesperus.

Molecular and material properties of major ampullate silk were studied for the cobweb-building black widow spider Latrodectus hesperus. Material properties were measured by stretching the silk to breaking. The strength was 1.0 +/- 0.2 GPa, and the extensibility was 34 +/- 8%. The secondary structure of the major ampullate silk protein was studied using carbon-13 NMR spectroscopy. Alanine undergoes a transition from a coiled structure in pre-spun silk to a beta sheet structure in post-spun silk. We have also isolated two distinct cDNAs (both about 500 bp) which encode proteins similar to major ampullate spidroin 1 and 2 (MaSp1 and MaSp2). The MaSp1-like silk contains polyalanine runs of 5-10 residues as well as GA and GGX motifs. The MaSp2-like silk contains polyalanine runs of varying length as well as GPG(X)(n) motifs. L. hesperus major ampullate silk is more like major ampullate silk from other species than other L. hesperus silks.