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.     

Protein and amino acid composition of silks from the cob weaver, Latrodectus hesperus (black widow)

The silks from the cob weaving spider, Latrodectus hesperus (black widow), have been examined with the goal of expanding our understanding of the relationship between the protein structure and mechanical performance of these unique biomaterials. The scaffolding, dragline and inner egg case silks each appear to be distinct fibers based on mole percent amino acid composition and polypeptide composition. Further, we find that the amino acid composition of dragline and egg case silk are similar to the analogous silks produced by orb weaving spiders, while scaffolding silk may represent a novel silk. The black widow silks are comprised of multiple high molecular weight polypeptides, however, the egg case and scaffolding silks also contain some smaller polypeptides.     

Ultrastructure of the major ampullate gland of the black widow spider,Latrodectus hesperus

Silk production in the spider occurs within specialized glands that are capable of the synthesis of large fibrous proteins and the post-translational processing of those proteins to form an insoluble fiber. The major ampullate gland of Latrodectus hesperus (black widow) is similar in morphology to those found in the Araneid spiders. The tail domain of this gland is highly protein synthetic, giving rise to a core, fibrous protein product. In addition to a storage function, the ampulla region also synthesizes and exports an electron dense material that appears to form a 'coat' surrounding the silk generated within the tail. The duct of the gland consists of at least two distinct cell types: one type contains 'honeycomb' vesicles of unknown function, while the other possesses elaborate apical microvilli that may be involved in the reabsorption of water and subsequent dehydration of the silk. As the silk product transits through these various stages of assembly, it can been seen to undergo a condensation or concentration, possibly reflecting the influence of both the shear forces induced by movement into the duct and the dehydration that is thought to occur there.     

Family Affects Sibling Cannibalism in the Black Widow Spider,Latrodectus hesperus

Adaptive foraging tactics are shaped by genes, the environment and gene–environment interactions. Because of relatively high levels of agonism toward conspecifics, spiders have been a popular focus for behavioral–ecological examinations of conspecific predation, or cannibalism. Surprisingly, studies examining the underlying, proximate assumption that cannibalism in spiders is a heritable trait shaped by interactions between genes and the environment are virtually non‐existent. Here, we examine the influence of family on the expression of sibling cannibalism in the post‐hatching, group‐living phase of an otherwise solitary, web‐building spider, the North American black widow (Latrodectus hesperus). Our results showed significant levels of variation in cannibalistic propensity among 26 sibships, with some families cannibalizing full sibs within 2 d and other families waiting 3 wk before resorting to cannibalism. A similar family‐level effect was evident in measures of sibling cohabitation, voracity toward cricket prey, and development speed. Negative correlations between maternal egg sac investment and offspring cannibalism suggest that this family effect may stem, at least in part, from a maternal effect, although we were not able to directly test the prediction that cannibalism is most common from spiderlings in poor condition. Thus, we present novel data suggesting family effects seem to be responsible for cannibalism in L. hesperus spiderlings; however, future work will be required to disentangle the relative importance of shared genes and shared maternal environment. We discuss several mechanisms that could explain the persistence of family‐level variation in cannibalism, a trait that seems likely to be subject to strong directional selection.     

Aciniform spidroin, a constituent of egg case sacs and wrapping silk fibers from the black widow spider Latrodectus hesperus

Spiders produce high performance fibers with diverse mechanical properties and biological functions. Molecular and biochemical studies of spider egg case silk have revealed that the main constituent of the large diameter fiber contains the fibroin TuSp1. Here we demonstrate by SDS-PAGE and protein silver staining the presence of a distinct approximately 300-kDa polypeptide that is found in solubilized egg case sacs. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called AcSp1-like and demonstrate that its protein product is assembled into the small diameter fibers of egg case sacs and wrapping silks from the black widow spider, Latrodectus hesperus. BLAST searches of the NCBInr protein data base using the amino acid sequence of AcSp1-like revealed similarity to AcSp1, an inferred protein proposed to be a component of wrapping silk. However, the AcSp1-like protein was found to display more nonuniformity in its internal iterated repeat modules than the putative AcSp1 fibroin. Real time quantitative PCR analysis demonstrates that the AcSp1-like gene displays an aciniform gland-restricted pattern of expression. The amino acid composition of the fibroins extracted from the luminal contents of the aciniform glands was remarkably similar to the predicted amino acid composition of the AcSp1-like protein, which supports the assertion that AcSp1-like protein represents the major constituent stored within the aciniform gland. Collectively, our findings provide the first direct molecular evidence for the involvement of the aciniform gland in the production of a common fibroin that is assembled into the small diameter threads of egg case and wrapping silk of cob weavers.     

Material properties of cobweb silk from the black widow spider Latrodectus hesperus.

We present the material analysis of scaffolding silk from the cobweb of the black widow spider Latrodectus hesperus. 30 strands were tested from the webs of nine spiders. Strands were stretched at 0.211 mm/s as force and extension were recorded. Cross-sectional area was measured under 1000 x oil-immersion light microscopy. The stress strain curve shows that cobweb silk is a distinct material from other known spider silks. The average breaking point for this cobweb silk is 1.1 +/- 0.5 GPa at 0.22 +/- 0.05 strain. All samples increased stiffness as they were stretched, but to different extents. Variation in stiffness might be due to differential crystallization or alignment of the silk proteins during stretching.     

盐离子对次壶腹腺丝蛋白重组模块的影响

为研究不同生理环境对蛛丝蛋白组装及成丝的影响,首次以MiSp序列为对象,研究其NTR1SR2CT重组模块在不同种类(浓度)盐离子条件下的聚集和成纤维特性及其在成纤维过程中二级结构的变化。基于大腹园蛛MiSp全长序列构建NTR1SR2CT模块,并在大肠杆菌Escherichia coli BL21中成功表达。借助8 mol/L尿素裂解包涵体进行变性纯化得到NTR1SR2CT重组蛋白。NTR1SR2CT重组蛋白二级结构主要为无规则卷曲(Random coil)或α螺旋(Helix),在自然成丝及冻干过程中部分random coil或helix转变为β折叠(β-sheet),甲醇能促进该转变过程。另外,钾离子和磷酸根离子有利于NTR1SR2CT重组蛋白聚集从而促进丝纤维的形成。研究结果为成丝机理研究奠定了基础,同时也为工业化生产高品质的蛛丝纤维提供了条件。     

Egg case protein-1. A new class of silk proteins with fibroin-like properties from the spider Latrodectus hesperus

Spiders produce multiple types of silk that exhibit diverse mechanical properties and biological functions. Most molecular studies of spider silk have focused on fibroins from dragline silk and capture silk, two important silk types involved in the survival of the spider. In our studies we have focused on the characterization of egg case silk, a third silk fiber produced by the black widow spider, Latrodectus hesperus. Analysis of the physical structure of egg case silk using scanning electron microscopy demonstrates the presence of small and large diameter fibers. By using the strong protein denaturant 8 M guanidine hydrochloride to solubilize the fibers, we demonstrated by SDS-PAGE and protein silver staining that an abundant component of egg case silk is a 100-kDa protein doublet. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called ecp-1, which encodes for one of the protein components of the 100-kDa species. BLAST searches of the NCBInr protein data base using the primary sequence of ECP-1 revealed similarity to fibroins from spiders and silkworms, which mapped to two distinct regions within the ECP-1. These regions contained the conserved repetitive fibroin motifs poly(Ala) and poly(Gly-Ala), but surprisingly, no larger ensemble repeats could be identified within the primary sequence of ECP-1. Consistent with silk gland-restricted patterns of expression for fibroins, ECP-1 was demonstrated to be predominantly produced in the tubuliform gland, with lower levels detected in the major and minor ampullate glands. ECP-1 monomeric units were also shown to assemble into higher aggregate structures through the formation of disulfide bonds via a unique cysteine-rich N-terminal region. Collectively, our findings provide new insight into the components of egg case silk and identify a new class of silk proteins with distinctive molecular features relative to traditional members of the spider silk gene family.     

Solid-state NMR investigation of major and minor ampullate spider silk in the native and hydrated states

Silks spun from the major (Ma) and minor (Mi) ampullate glands by the spider Nephila clavipes respond to water differently. Specifically, Ma silk supercontracts (shrinks 40-50% in length) while Mi silk does not contract at all when hydrated with water. In the present study, 1H --> 13C cross polarization magic angle spinning (CP-MAS), 13C MAS NMR collected with dipolar decoupling, and two-dimensional wide-line separation spectra are presented on Mi silk in its native and hydrated state and comparisons are made to Ma silk. This combination of NMR data demonstrates that water plasticizes Mi and Ma silk similarly, with an increase in chain dynamics observed in regions containing Gly, Glu, Ser, Tyr, Leu, and a fraction of Ala when the Mi silk is hydrated. Resonances that correspond to the poly(Ala) and poly(Gly Ala) motifs of Ma and Mi silk are predominately rigid indicating that water does not penetrate these beta-sheet domains.     

Alteration of tubuliform silk gland cytoarchitecture with the reproductive cycle of the Western black widow spider,Latrodectus hesperus

The protein synthetic and secretory activity of spider tubuliform glands is known to be coordinated with the reproductive stage of the spider. For spiders that produce multiple egg cases, such as the black widow Latrodectus hesperus, this means that the cells that make up the tubuliform gland cycle from minimal to maximal silk protein synthesis and exocytosis as the spider transitions from early vitellogenesis to a gravid state and back. The impact of these transitions on the cells that form the tubuliform gland has yet to be characterized. The entire tubuliform gland undergoes an elastic deformation, doubling in size in response to the accumulation and depletion of egg case silk proteins within its lumen. Similarly, the diversity and organization of organelles within the cytoplasm of the secretory epithelial cells that make up the wall of the tubuliform gland change with the reproductive stage of the spider. Progression of a spider from early to late vitellogenesis is accompanied by decondensed nucleoli and distention of the rough endoplasmic reticulum, markers of protein synthetic activity. The presumed silk proteins that fill the lumen of the tubuliform gland of a gravid spider include a fibrous matrix with homogeneous spherical inclusions. These components are also present within the cytoplasm of the cell; however, only the fibrous material appears to be enclosed by membranous organelles. Transition of the tubuliform gland from peak silk synthesis back to a quiescent state is marked by the appearance of multivesicular bodies and organelles resembling phagophores and autophagosomes, suggestive of a role for autophagy in the process of recovery. The reproducible cellular dynamics of the tubuliform silk gland of the black widow spider makes it a potential model system for study of the regulation of silk gene expression, endomembrane transport, and exocytosis of silk proteins and autophagy.     

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.     

Secondary structures and conformational changes in flagelliform, cylindrical, major, and minor ampullate silk proteins. Temperature and concentration effects

Orb weaver spiders use exceptionally complex spinning processes to transform soluble silk proteins into solid fibers with specific functions and mechanical properties. In this study, to understand the nature of this transformation we investigated the structural changes of the soluble silk proteins from the major ampullate gland (web radial threads and spider safety line); flagelliform gland (web sticky spiral threads); minor ampullate gland (web auxiliary spiral threads); and cylindrical gland (egg sac silk). Using circular dichroism, we elucidated (i) the different structures and folds for the various silk proteins; (ii) irreversible temperature-induced transitions of the various silk structures toward beta-sheet-rich final states; and (iii) the role of protein concentration in silk storage and transport. We discuss the implication of these results in the spinning process and a possible mechanism for temperature-induced beta-sheet formation.     

Conformational and orientational transformation of silk proteins in the major ampullate gland of Nephila clavipes spiders

The orientational and conformational transformation of the native liquid silk into a solid fiber in the major ampullate gland of the spider Nephila clavipes has been studied by Raman spectromicroscopy. The spectra show that the conformation of silk proteins in the glandular sac contains several secondary structure elements, which is consistent with intrinsically unfolded proteins. A few alpha-helices are also present and involve some alanine residues located in the polyalanine segments of the spidroin sequence. The conversion of the silk solution in the major ampullate gland appears to be a two-state process without intermediate states. In the first and second limbs of the duct, silk is isotropic and spidroins are generally native-like. beta-Sheets start to develop between the second and the third limb of the duct, suggesting that early beta-sheets are generated by shear forces. However, most of the beta-sheets are formed between the draw down taper and the valve. The early beta-sheets formed upward of the draw down taper might play the role of nucleation sites for the subsequent beta-sheet aggregation. The alignment of the polypeptides chains occurs near the valve, revealing that orientational and conformational changes do not occur simultaneously. Extensional flow seems to be the driving force to produce the orientational order, which in turn is associated with the formation of the major part of the beta-sheets. The slow evolution of the spidroin conformation up to the draw down taper followed by the rapid transformation between the drawn down taper and the valve may be important to achieve the optimal structure of the final fiber.     

In situ conformation of spider silk proteins in the intact major ampullate gland and in solution

To understand the spinning process of dragline silk by spiders, the protein conformation before spinning has to be determined. Raman confocal spectromicroscopy has been used to study the conformation of the proteins in situ in the intact abdominal major ampullate gland of Nephila clavipes and Araneus diadematus spiders. The spectra obtained are typical of natively unfolded proteins and are very similar to that of a mixture of recombinant silk proteins. Vibrational circular dichroism reveals that the conformation is composed of random and polyproline II (PPII) segments with some alpha-helices. The alpha-helices seem to be located in the C-terminal part whereas the repetitive sequence is unfolded. The PPII structure can significantly contribute to the efficiency of the spinning process in nature.     

Conserved C-termini of Spidroins are secreted by the major ampullate glands and retained in the silk thread

The C-termini of Spidroins produced in the major and minor ampullate glands of spiders are highly conserved. Despite this conservation, no corresponding peptides have been identified in the spinning dopes or the silk filaments so far. To prove their presence or absence, polyclonal antibodies derived against fusion proteins containing the conserved C-terminal regions of both Spidroin 1 and 2 from the spider Nephila clavipes were generated. The antibodies reacted with high molecular weight polypeptides of the corresponding gland extracts and solubilized major ampullate filament and in addition to filament cross-sections. This demonstrates the existence of C-terminal specific peptides in the spinning dope and the mature Spidroins. Both the fusion proteins as well as the proteins contained within the gland lumen showed a reduction in their size under reducing conditions indicating the presence of disulfide bonds. Their high conservation and the biochemical data suggest crucial roles the C-termini play in the formation and/or structure of the corresponding silk filaments.     

Short and long range order of the morphology of silk from Latrodectus hesperus (Black Widow) as characterized by atomic force microscopy.

The surfaces of both stretched and unstretched silk threads from the cobweb weaver, Latrodectus hesperus (Black Widow) have been examined by atomic force microscopy (AFM). AFM images of cobweb scaffolding threads show both unordered and highly ordered regions. Two types of fibers within the threads were observed: thicker (approximately 300 nm in diameter) fibers oriented parallel to the thread axis and thinner (10-100 nm) fibrils oriented across the thread axis. While regions which lacked parallel fibers or fibrils were observed on threads at all strain values, the probability of observing fibers and/or fibrils increased with strain. High-resolution AFM images show that with increasing strain, both mean fiber and fibril diameters decrease and that fibrils align themselves more closely with the thread axis. The observation of fibers and fibrils within the cobweb threads has implications for current models of the secondary and tertiary structure and organization of spider silk.