The molecular structures of major ampullate silk proteins of the wasp spider,Argiope bruennichi: A second blueprint for synthesizing de novo silk

The dragline silk of orb-weaving spiders possesses extremely high tensile strength and elasticity. To date, full-length sequences of only two genes encoding major ampullate silk protein (MaSp) in Latrodectus hesperus have been determined. In order to further understand this gene family, we utilized in this study a variety of strategies to isolate full-length MaSp1 and MaSp2 cDNAs in the wasp spider Argiope bruennichi. A. bruennichi MaSp1 and MaSp2 are primarily composed of remarkably homogeneous ensemble repeats containing several complex motifs, and both have highly conserved C-termini and N-termini. Two novel amino acid motifs, GGF and SGR, were found in MaSp1 and MaSp2, respectively. Amino acid composition analysis of silk, luminal contents and predicted sequences indicates that MaSp1 and MaSp2 are two major components of major ampullate glands and that the ratio of MaSp1 to MaSp2 is approximately 3:2 in dragline silk. Furthermore, both the MaSp1:MaSp2 ratio and the conserved termini are closely linked with the production of high quality synthetic fibers. Our results make an important contribution to our understanding of major ampullate silk protein structure and provide a second blueprint for creating new composite silk which mimics natural spider dragline silk.     

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.     

From EST sequence to spider silk spinning: identification and molecular characterisation of Nephila senegalensis major ampullate gland peroxidase NsPox

Spider dragline silk is renowned as one of the toughest materials of its kind. In nature, spider silks are spun out of aqueous solutions under environmental conditions. This is in contrast to production of most synthetic fibres, where hazardous solvents, high temperatures and pressure are used. In order to identify some of the chemical processes involved in spider silk spinning, we have produced a collection of cDNA sequences from specific regions of Nephila senegalensis major ampullate gland. We examined in detail the sequence and expression of a putative Nephila senegalensis peroxidase gene (NsPox) from our EST collection. NsPox encodes a protein with similarity to Drosophila melanogaster and Aedes aegypti peroxidases. Northern analysis and in situ localisation experiments revealed that NsPox is expressed in major and minor ampullate glands of the spider where the main components of the dragline silk are produced. We suggest that NsPox plays a role in dragline silk fibre formation and/or processing.     

NMR assignments of the N-terminal domain of Nephila clavipes spidroin 1

The building blocks of spider dragline silk are two fibrous proteins secreted from the major ampullate gland named spidroins 1 and 2 (MaSp1, MaSp2). These proteins consist of a large central domain composed of approximately 100 tandem copies of a 35–40 amino acid repeat sequence. Non-repetitive N and C-terminal domains, of which the C-terminal domain has been implicated to transition from soluble and insoluble states during spinning, flank the repetitive core. The N-terminal domain until recently has been largely unknown due to difficulties in cloning and expression. Here, we report nearly complete assignment for all ¹H, ¹³C, and ¹⁵N resonances in the 14 kDa N-terminal domain of major ampullate spidroin 1 (MaSp1-N) of the golden orb-web spider Nephila clavipes.     

N-terminal nonrepetitive domain common to dragline, flagelliform, and cylindriform spider silk proteins

Spider silk has been extensively studied for its outstanding mechanical properties. Partial intermediate and C-terminal sequences of different spider silk proteins have been determined, and during the past decade also N-terminal domains have been characterized. However, only some of these N-terminal domains have been reported to contain signal peptides, leaving the mechanism whereby they enter the secretory pathway open to speculation. Here we present the sequence of a 394-residue N-terminal region of the Euprosthenops australis major ampullate spidroin 1 (MaSp1). A close comparison with published sequences from other species revealed the presence of N-terminal signal peptides followed by an approximately 130-residue nonrepetitive domain. From secondary structure predictions, helical wheel analysis, and circular dichroism spectroscopy this domain is concluded to contain five alpha-helices and is a conserved constituent of hitherto analyzed dragline, flagelliform, and cylindriform spider silk proteins.     

蜘蛛拖丝蛋白基因的构建及在大肠杆菌中的表达

蜘蛛大壶腹线产生的拖丝是非常优良的纤维蛋白, 具有独特的强度和弹性。基于拖丝蛋白高度重复序列和部分cDNA序列, 合成蜘蛛拖丝蛋白基因单体, 通过头尾相连的构建策略, 得到拖丝蛋白多聚体, 与原核高效表达载体pET30a(+)连接, 转化大肠杆菌BLR(DE3), 用IPTG诱导表达。 表达产物经His.Bind树脂金属螯合亲和层析一步纯化, 纯度达90%以上, 表达量为20mg/L。SDS-PAGE和蛋白质印迹图谱显示表达产物分子量为37kD, 其值与氨基酸组分分析结果与理论推算值基本符合。      

Structural studies of spider silk proteins in the fiber

Although spider silk has been studied for a number of years the structures of the proteins involved have yet to be definitely determined. X-ray diffraction and solid-state nuclear magnetic resonance (NMR) were used to study major ampullate (dragline) silk from Nephila clavipes. The silk was studied in its natural state, in the supercontacted state and in the restretched state following supercontraction. The natural silk structure is dominated by β-sheets aligned parallel to the fiber axis. Supercontraction is characterized by randomizing of the orientation of the β-sheet. When the fiber is restretched alignment is regained. However, the same reorientation was observed for wetting of minor ampullate silk which does not supercontract. Thus, the reorientation of β-sheets alone cannot explain the supercontraction in dragline silk. Cocoon silk showed very little β-sheet orientation in the natural state and there were no changes upon wetting. NMR and X-ray diffraction data are consistent with the β-sheets arising from the poly-alanine sequences known to be present in the proteins of major ampullate silk as has been proposed previously. © 1997 John Wiley & Sons, Ltd.     

Web building and silk properties functionally covary among species of wolf spider

Although phylogenetic studies have shown covariation between the properties of spider major ampullate (MA) silk and web building, both spider webs and silks are highly plastic so we cannot be sure whether these traits functionally covary or just vary across environments that the spiders occupy. As MaSp2‐like proteins provide MA silk with greater extensibility, their presence is considered necessary for spider webs to effectively capture prey. Wolf spiders (Lycosidae) are predominantly non‐web building, but a select few species build webs. We accordingly collected MA silk from two web‐building and six non‐web‐building species found in semirural ecosystems in Uruguay to test whether the presence of MaSp2‐like proteins (indicated by amino acid composition, silk mechanical properties and silk nanostructures) was associated with web building across the group. The web‐building and non‐web‐building species were from disparate subfamilies so we estimated a genetic phylogeny to perform appropriate comparisons. For all of the properties measured, we found differences between web‐building and non‐web‐building species. A phylogenetic regression model confirmed that web building and not phylogenetic inertia influences silk properties. Our study definitively showed an ecological influence over spider silk properties. We expect that the presence of the MaSp2‐like proteins and the subsequent nanostructures improves the mechanical performance of silks within the webs. Our study furthers our understanding of spider web and silk co‐evolution and the ecological implications of spider silk properties.     

Chromosome Mapping of Dragline Silk Genes in the Genomes of Widow Spiders (Araneae,Theridiidae)

With its incredible strength and toughness, spider dragline silk is widely lauded for its impressive material properties. Dragline silk is composed of two structural proteins, MaSp1 and MaSp2, which are encoded by members of the spidroin gene family. While previous studies have characterized the genes that encode the constituent proteins of spider silks, nothing is known about the physical location of these genes. We determined karyotypes and sex chromosome organization for the widow spiders, Latrodectus hesperus and L. geometricus (Araneae, Theridiidae). We then used fluorescence in situ hybridization to map the genomic locations of the genes for the silk proteins that compose the remarkable spider dragline. These genes included three loci for the MaSp1 protein and the single locus for the MaSp2 protein. In addition, we mapped a MaSp1 pseudogene. All the MaSp1 gene copies and pseudogene localized to a single chromosomal region while MaSp2 was located on a different chromosome of L. hesperus. Using probes derived from L. hesperus, we comparatively mapped all three MaSp1 loci to a single region of a L. geometricus chromosome. As with L. hesperus, MaSp2 was found on a separate L. geometricus chromosome, thus again unlinked to the MaSp1 loci. These results indicate orthology of the corresponding chromosomal regions in the two widow genomes. Moreover, the occurrence of multiple MaSp1 loci in a conserved gene cluster across species suggests that MaSp1 proliferated by tandem duplication in a common ancestor of L. geometricus and L. hesperus. Unequal crossover events during recombination could have given rise to the gene copies and could also maintain sequence similarity among gene copies over time. Further comparative mapping with taxa of increasing divergence from Latrodectus will pinpoint when the MaSp1 duplication events occurred and the phylogenetic distribution of silk gene linkage patterns.     

Environmentally induced post‐spin property changes in spider silks: influences of web type,spidroin composition and ecology

Many spiders use silk to construct webs that must function for days at a time, whereas many other species renew their webs daily. The mechanical properties of spider silk can change after spinning under environmental stress, which could influence web function. We hypothesize that spiders spinning longer‐lasting webs produce silks composed of proteins that are more resistant to environmental stresses. The major ampullate (MA) silks of orb web spiders are principally composed of a combination of two proteins (spidroins) called MaSp1 and MaSp2. We expected spider MA silks dominated by MaSp1 to have the greatest resistance to post‐spin property change because they have high concentrations of stable crystalline β‐sheets. Some orb web spiders that spin three‐dimensional orb webs, such as Cyrtophora, have MA silks that are predominantly composed of MaSp1. Hence, we expected that the construction of three‐dimensional orb webs might also coincide with MA silk resistance to post‐spin property change. Alternatively, the degree of post‐spin mechanical property changes in different spider silks may be explained by factors within the spider's ecosystem, such as exposure to solar radiation. We exposed the MA silks of ten spider species from five genera (Nephila, Cyclosa, Leucauge, Cyrtophora, and Argiope) to ecologically high temperatures and low humidity for 4 weeks, and compared the mechanical properties of these silks with unexposed silks. Using species pairs enabled us to assess the influence of web dimensionality and MaSp composition both with and without phylogenetic influences being accounted for. We found neither the MaSp composition nor the three‐dimensionality of the orb web to be associated with the degree of post‐spin mechanical property changes in MA silk. The MA silks in Leucauge spp. are dominated by MaSp2, which we found to have the least resistance to post‐spin property change. The MA silk in Argiope spp. is also dominated by MaSp2, but has high resistance to post‐spin property change. The ancestry of Argiope is unresolved, but it is largely a tropical genus inhabiting hot, open regions that present similar stressors to silk as those of our experiment. Ecological factors thus appear to influence the vulnerability of orb web spider MA silks to post‐spin property change. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 580–588.     

Proline and processing of spider silks

Major ampullate (MAA) silks from a variety of spider species were collected by artificial silking that adjusted the samples to have similar breaking strains. Those silks are highly comparable in post-yield mechanical properties, but their supercontraction behaviors and initial moduli vary in large ranges and both correlate with the content of one amino acid, proline. These relationships, in combination with protein sequence data, support the hypothesis that the proline-related motif, that is, GPGXX, may play a key role in silk. This also explains the interspecific variability of spider dragline silk. Moreover, MAA silks from three representative species were prepared in a range of processing conditions and their mechanical properties were compared. Our results indicate how chemical compositions, coupled with processing conditions, shape the mechanical properties of the spider silk.     

Multiple recombining loci encode MaSp1, the primary constituent of dragline silk, in widow spiders (Latrodectus: Theridiidae)

Spiders spin a functionally diverse array of silk fibers, each composed of one or more unique proteins. Most of these proteins, in turn, are encoded by members of a single gene family thought to have arisen through duplication and divergence of an ancestral silk gene. Because of its remarkable mechanical properties, orb weaver dragline silk, a composite of 2 proteins (MaSp1 and MaSp2), is the best studied. Here, we demonstrate that multiple loci encode MaSp1 in widow spiders (Latrodectus). Because these copies may be the result of more recent duplication events than those leading to the currently recognized silk gene paralogs, they offer insight into the early evolutionary fate of silk gene duplicates. In addition to 3 presumed functional MaSp1 loci in Latrodectus hesperus (Western black widow) and Latrodectus geometricus (brown widow) genomes, we find a MaSp1 pseudogene in L. hesperus, demonstrating the potential for unrecognized extinction of silk gene paralogs. We also document recombination events among L. hesperus MaSp1 loci and between Latrodectus MaSp1 loci and MaSp2. This result supports the hypothesis that concerted evolution occurs not only within an individual silk gene but also among silk gene paralogs. One of the L. geometricus MaSp1 copies encodes a protein that has diverged in amino acid composition and potentially converged on the secondary structure of MaSp2. Based on the presence of multiple MaSp1 loci and the phylogenetic distribution of MaSp1 versus MaSp2, we propose that MaSp2 is derived from an ancestral MaSp1 duplicate. Finally, divergence has occurred in the upstream flanking sequences of the L. hesperus MaSp1 loci, the region most likely to contain regulatory motifs, providing ample opportunity for differential expression. However, the benefits associated with increased protein production may be the primary mechanism maintaining multiple functional MaSp1 copies in widow genomes.     

A MaSp2-like gene found in the Amazon mygalomorph spider Avicularia juruensis

Two unique spidroins are present in the silk of the Amazon mygalomorph spider — Avicularia juruensis (Theraphosidae), and for the first time the presence and expression of a major ampullate spidroin 2-like in Mygalomorphae are demonstrated. Molecular analysis showed the presence of (GA)_n, poly-A and GPGXX motifs in the amino acid sequence of Spidroin 2, the last being a motif described so far only in MaSp2 and Flag spidroins. Phylogenetic analysis confirmed the previously known orthologous silk gene clusters, and placed this gene firmly within the orbicularian MaSp2 clade. Gene tree–species tree reconciliations show a pattern of multiple gene duplication throughout spider silk evolution, and pinpoint the oldest speciation in which MaSps must have been present in spiders on the mygalomorph–araneomorph split, 240 MYA. Therefore, while not refuting orb weaver monophyly, MaSp2s, and major ampullate silks in general cannot be classified as orbicularian synapomorphies, but have to be considered plesiomorphic for Opisthothelae. The evidence presented here challenges the simplified notion that mygalomorphs spin only one kind of silk, and adds to the suite of information suggesting a pattern of early niche diversification between Araneomorphae and Mygalomorphae. Additionally, mygalomorph MaSp2-like might accommodate mechanical demands arising from the arboreal habitat preference of Avicularia.     

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring ^1,2. 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 ³. 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 ⁴. 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] ^5,6, tubuliform [synthesizes egg case silk] ^7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] ⁹ and pyriform [produces attachment disc silk] ¹⁰. 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.     

Construction of the synthetic genes for protein analogs of spider silk carcass spidroin 1 and their expression in tobacco plants

To obtain transgenic tobacco plants expressing recombinant analogs of spider dragline silk spidroin 1, artificial 1f5 and 1f9 coding for spidroin 1 analogs were 3'-fused in-frame with the reporter lichenase gene. The Tr2' weak constitutive promoter of Agrobacterium tumefaciens T-DNA and the strong constitutive promoter of the cauliflower mosaic virus 35S RNA gene were used as regulatory elements. The expression cassettes were used to transform agrobacteria and then introduced in tobacco leaf disks. On evidence of Southern hybridization, transgenic plants each carried a single copy of a hybrid gene, which corresponded in size to the constructed one. Zymography and Western blotting revealed full-length hybrid proteins in leaf extracts of transgenic plants. The results testified that plants can maintain and express synthetic genes for spider silks and, consequently, may be used as a convenient producer of recombinant silk analogs.     

Araneoid egg case silk: a fibroin with novel ensemble repeat units from the black widow spider, Latrodectus hesperus

Araneoid spiders use specialized abdominal glands to manufacture up to seven different protein-based silks/glues that have diverse physical properties. The fibroin sequences that encode egg case fibers (cover silk for the egg case sac) and the secondary structure of these threads have not been previously determined. In this study, MALDI tandem TOF mass spectrometry (MS/MS) and reverse genetics were used to isolate the first egg case fibroin, named tubuliform spidroin 1 (TuSp1), from the black widow spider, Latrodectus hesperus. Real-time quantitative PCR analysis demonstrates TuSp1 is selectively expressed in the tubuliform gland. Analysis of the amino acid composition of raw egg case silk closely aligns with the predicted amino acid composition from the primary sequence of TuSp1, which supports the assertion that TuSp1 represents a major component of egg case fibers. TuSp1 is composed of highly homogeneous repeats that are 184 amino acids in length. The long stretches of polyalanine and glycine-alanine subrepeats, which account for the crystalline regions of minor ampullate and major ampullate fibers, are very poorly represented in TuSp1. However, polyserine blocks and short polyalanine stretches were highly iterated within the primary sequence, and (13)C NMR spectroscopy demonstrated that the majority of alanine was found in a beta-sheet structure in post-spun egg case silk. The TuSp1 repeat unit does not display substantial sequence similarity to any previously described fibroin genes or proteins, suggesting that TuSp1 is a highly divergent member of the spider silk gene family.