Egg Case Protein 3: A Constituent of Black Widow Spider Tubuliform Silk.
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ABSTRACT:
SUBMITTER: Shanafelt M
PROVIDER: S-EPMC8399404 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Spider silk research has largely focused on spidroins, proteins that are the primary components of spider silk fibers. Although a number of spidroins have been characterized, other types of proteins associated with silk synthesis are virtually unknown. Previous analyses of tissue-specific RNA-seq libraries identified 647 predicted genes that were differentially expressed in silk glands of the Western black widow, Latrodectus hesperus. Only ∼5% of these silk-gland specific transcripts (SSTs) encode spidroins; although the remaining predicted genes presumably encode other proteins associated with silk production, this is mostly unverified. Here, we used proteomic analysis of multiple silk glands and dragline silk fiber to investigate the translation of the differentially expressed genes. We find 48 proteins encoded by the differentially expressed transcripts in L. hesperus major ampullate, minor ampullate, and tubuliform silk glands and detect 17 SST encoded proteins in major ampullate silk fibers. The observed proteins include known silk-related proteins, but most are uncharacterized, with no annotation. These unannotated proteins likely include novel silk-associated proteins. Major and minor ampullate glands have the highest overlap of identified proteins, consistent with their shared, distinctive ampullate shape and the overlapping functions of major and minor ampullate silks. Our study substantiates and prioritizes predictions from differential expression analysis of spider silk gland transcriptomes.
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Project description:Spider dragline silk represents a biomaterial with outstanding mechanical properties, possessing high-tensile strength and toughness. In black widows at least eight different proteins have been identified as constituents of dragline silk. These represent major ampullate spidroins MaSp1, MaSp2, MaSp', and several low-molecular weight cysteine-rich protein (CRP) family members, including CRP1, CRP2, and CRP4. Molecular modeling predicts that CRPs contain a cystine slipknot motif, but experimental evidence to support this assertion remains to be reported. To advance scientific knowledge regarding CRP function, we recombinantly expressed and purified CRP1 and CRP4 from bacteria and investigated their secondary structure using circular dichroism (CD) under different chemical and physical conditions. We demonstrate by far-UV CD spectroscopy that these proteins contain similar secondary structure, having substantial amounts of random coil conformation, followed by lower levels of beta sheet, alpha helical and beta turn structures. CRPs are thermally and pH stable; however, treatment with reagents that disrupt disulfide bonds impact their structural conformations. Cross-linking mass spectrometry (XL-MS) data also support computational models of CRP1. Taken together, the chemical and thermal stability of CRPs, the cross-linking data, coupled with the structural sensitivity to reducing agents, are experimentally consistent with the supposition CRPs are cystine slipknot proteins.
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Project description:Latrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (α-latrotoxin (α-LTX)), five specialized on insects (α, β, γ, δ, ε- latroinsectotoxins (LITs), and one on crustaceans (α-latrocrustatoxin (α-LCT)). LaTXs bind to specific receptors on the surface of neuronal cells, inducing the release of neurotransmitters either by directly stimulating exocytosis or by forming Ca2+-conductive tetrameric pores in the membrane. Despite extensive studies in the past decades, a high-resolution structure of a LaTX is not yet available and the precise mechanism of LaTX action remains unclear. Here, we report cryoEM structures of the α-LCT monomer and the δ-LIT dimer. The structures reveal that LaTXs are organized in four domains. A C-terminal domain of ankyrin-like repeats shields a central membrane insertion domain of six parallel α-helices. Both domains are flexibly linked via an N-terminal α-helical domain and a small β-sheet domain. A comparison between the structures suggests that oligomerization involves major conformational changes in LaTXs with longer C-terminal domains. Based on our data we propose a cyclic mechanism of oligomerization, taking place prior membrane insertion. Both recombinant α-LCT and δ-LIT form channels in artificial membrane bilayers, that are stabilized by Ca2+ ions and allow calcium flux at negative membrane potentials. Our comparative analysis between α-LCT and δ-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family.
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Project description:Spider silk proteins (fibroins) are renowned for their extraordinary mechanical properties and biomimetic potential. Despite extensive evolutionary, ecological, and industrial interest in these fibroins, only a fraction of the known silk types have been characterized at the molecular level. Here we report cDNA and genomic sequences of the fibroin TuSp1, which appears to be the major component of tubuliform gland silk, a fiber exclusively synthesized by female spiders for egg case construction. We obtained TuSp1 sequences from 12 spider species that represent the extremes of phylogenetic diversity within the Orbicularia (orb-weaver superfamilies, Araneoidea and Deinopoidea) and finer scale sampling within genera. TuSp1 encodes tandem arrays of an approximately 200-aa-long repeat unit and individual repeats are readily aligned, even among species that diverged >125 million years ago. Analyses of these repeats across species reveal the strong influence of concerted evolution, resulting in intragenic homogenization. However, deinopoid TuSp1 repeats also contain insertions of coding, minisatellite-like sequences, an apparent result of replication slippage and nonreciprocal recombination. Phylogenetic analyses of 37 spider fibroin sequences support the monophyly of TuSp1 within the spider fibroin gene family, consistent with a single origin of this ortholog group. The diversity of taxa and silks examined here confirms that repetitive architecture is a general feature of this gene family. Moreover, we show that TuSp1 provides a clear example of modular evolution across a range of phylogenetic levels.
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Project description:BackgroundThe black widow spider has both extraordinarily neurotoxic venom and three-dimensional cobwebs composed of diverse types of silk. However, a high-quality reference genome for the black widow spider was still unavailable, which hindered deep understanding and application of the valuable biomass.FindingsWe assembled the Latrodectus elegans genome, including a genome size of 1.57 Gb with contig N50 of 4.34 Mb and scaffold N50 of 114.31 Mb. Hi-C scaffolding assigned 98.08% of the genome to 14 pseudo-chromosomes, and with BUSCO, completeness analysis revealed that 98.4% of the core eukaryotic genes were completely present in this genome. Annotation of this genome identified that repetitive sequences account for 506.09 Mb (32.30%) and 20,167 protein-coding genes, and specifically, we identified 55 toxin genes and 26 spidroins and provide preliminary analysis of their composition and evolution.ConclusionsWe present the first chromosome-level genome assembly of a black widow spider and provide substantial toxin and spidroin gene resources. These high-qualified genomic data add valuable resources from a representative spider group and contribute to deep exploration of spider genome evolution, especially in terms of the important issues on the diversification of venom and web-weaving pattern. The sequence data are also firsthand templates for further application of the spider biomass.
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Project description:Spiders (Order Araneae) are essential predators in every terrestrial ecosystem largely because they have evolved potent arsenals of silk and venom. Spider silks are high performance materials made almost entirely of proteins, and thus represent an ideal system for investigating genome level evolution of novel protein functions. However, genomic level resources remain limited for spiders.We de novo assembled a transcriptome for the Western black widow (Latrodectus hesperus) from deeply sequenced cDNAs of three tissue types. Our multi-tissue assembly contained ~100,000 unique transcripts, of which >?27,000 were annotated by homology. Comparing transcript abundance among the different tissues, we identified 647 silk gland-specific transcripts, including the few known silk fiber components (e.g. six spider fibroins, spidroins). Silk gland specific transcripts are enriched compared to the entire transcriptome in several functions, including protein degradation, inhibition of protein degradation, and oxidation-reduction. Phylogenetic analyses of 37 gene families containing silk gland specific transcripts demonstrated novel gene expansions within silk glands, and multiple co-options of silk specific expression from paralogs expressed in other tissues.We propose a transcriptional program for the silk glands that involves regulating gland specific synthesis of silk fiber and glue components followed by protecting and processing these components into functional fibers and glues. Our black widow silk gland gene repertoire provides extensive expansion of resources for biomimetic applications of silk in industry and medicine. Furthermore, our multi-tissue transcriptome facilitates evolutionary analysis of arachnid genomes and adaptive protein systems.
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Project description: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.
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Project description:This study provides a detailed secondary structural characterization of major ampullate dragline silk from Latrodectus hesperus (black widow) spiders. X-ray diffraction results show that the structure of black widow major ampullate silk fibers is comprised of stacked ?-sheet nanocrystallites oriented parallel to the fiber axis and an amorphous region with oriented (anisotropic) and isotropic components. The combination of two-dimensional (2D) (13)C-(13)C through-space and through-bond solid-state NMR experiments provide chemical shifts that are used to determine detailed information about the amino acid motif secondary structure in black widow spider dragline silk. Individual amino acids are incorporated into different repetitive motifs that make up the majority of this protein-based biopolymer. From the solid-state NMR measurements, we assign distinct secondary conformations to each repetitive amino acid motif and, hence, to the amino acids that make up the motifs. Specifically, alanine is incorporated in ?-sheet (poly(Alan) and poly(Gly-Ala)), 3(1)-helix (poly(Gly-Gly-Xaa), and ?-helix (poly(Gln-Gln-Ala-Tyr)) components. Glycine is determined to be in ?-sheet (poly(Gly-Ala)) and 3(1)-helical (poly(Gly-Gly-X(aa))) regions, while serine is present in ?-sheet (poly(Gly-Ala-Ser)), 3(1)-helix (poly(Gly-Gly-Ser)), and ?-turn (poly(Gly-Pro-Ser)) structures. These various motif-specific secondary structural elements are quantitatively correlated to the primary amino acid sequence of major ampullate spidroin 1 and 2 (MaSp1 and MaSp2) and are shown to form a self-consistent model for black widow dragline silk.
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Project description:Due to their abundance and ability to invade diverse environments, many arthropods have become pests of economic and health concern, especially in urban areas. Transcriptomic analyses of arthropod ovaries have provided insight into life history variation and fecundity, yet there are few studies in spiders despite their diversity within arthropods. Here, we generated a de novo ovarian transcriptome from 10 individuals of the western black widow spider (Latrodectus hesperus), a human health pest of high abundance in urban areas, to conduct comparative ovarian transcriptomic analyses. Biological processes enriched for metabolism-specifically purine, and thiamine metabolic pathways linked to oocyte development-were significantly abundant in L. hesperus. Functional and pathway annotations revealed overlap among diverse arachnid ovarian transcriptomes for highly-conserved genes and those linked to fecundity, such as oocyte maturation in vitellogenin and vitelline membrane outer layer proteins, hormones, and hormone receptors required for ovary development, and regulation of fertility-related genes. Comparative studies across arachnids are greatly needed to understand the evolutionary similarities of the spider ovary, and here, the identification of ovarian proteins in L. hesperus provides potential for understanding how increased fecundity is linked to the success of this urban pest.
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