Translational pauses during a spider fibroin synthesis

The large ampullate glands of Nephila clavipes synthesize a tissue-specific fibroin for the web and dragline. Its secretory product migrates as one homogeneous band of approximately 320,000 daltons in denaturing electrophoresis. Similar analyses of the secretory epithelium reveal, in addition to the final product, a step ladder array of peptides in a highly reproducible and consistent pattern. We have correlated these peptide ladders with the process of fibroin synthesis. First, the peptides are very prominent in stimulated glands and quite dim in non stimulated ones. Secondly, by offering the cultured glands different amino acids in the incubating medium, we have linked the presence of the bands with those amino acids which abound in the fibroin. The evidence correlates the peptides with active fibroin synthesis, thus they are products of translation, and possibly reflect discontinuities in the translational process such as those demonstrated during the synthesis of Bombyx fibroin.     

Features of the cell-free translation of a spider fibroin mRNA

The massive production of fibroin by the large ampullate glands of the spider, Nephila clavipes, serves as a model system in which to study the synthesis and control of a large secretory protein. Their tissue-specific product, fibroin, produced during the entire adult life of the female, is approximately 320 kilodaltons, and rich in glycine and alanine. Highly purified fibroin mRNA from the glands has been translated in a rabbit reticulocyte cell-free system with variable supplements. The translational products analyzed by SDS-PAGE display two features, tRNA modulation and discontinuous pauses during elongation. tRNA complements exert their effects both in the translational efficiency and in the size of the peptides generated. The pauses observed during translation generate subsets of smaller discrete peptides, visualized in the gels as ladders of variable relative intensities, appearing exclusively and concomitantly with the fibroin. Definitive linkage between the discrete peptides and fibroin synthesis process has been established by their selective labeling with specific radioactive amino acids.     

13C NMR of Nephila clavipes major ampullate silk gland.

The major ampullate glands of the spider Nephila clavipes contain approximately 0.2 microliter each of a highly concentrated (approximately 50%) solution of silk fibroin. Therefore, the reservoir of silk in these glands presents an ideal opportunity to observe prefolded conformations of a protein in its native state. To this end, the structure and conformation of major ampullate gland silk fibroin within the glands of the spider N. clavipes were examined by 13C NMR spectroscopy. These results were compared to those from silk protein first drawn from the spinneret and then denatured. The 13C NMR chemical shifts, along with infrared and circular dichroism data, suggest that the silk fibroin in the glands exists in dynamically averaged helical conformations. Furthermore, there is no evidence of proline residues in U-(13)C-D-glucose-labeled silk. This transient prefolded "molten fibril" state may correspond to the silk I form found in Bombyx mori silk. There is no evidence of the final beta-sheet structure in the ampullate gland silk fibroin before final silk processing. However, the conformation of silk in the glands appears to be in a highly metastable state, as plasticization with water produces the beta-sheet structure. Therefore, the ducts connecting the ampullate glands to the spinnerets play a larger role in silk processing than previously thought.     

Spider silkglands contain a tissue-specific alanine tRNA that accumulates in vitro in response to the stimulus for silk protein synthesis

The large ampullate glands of the orb-web spider, Nephila clavipes provide massive amounts of fibroin throughout the lifetime of the adult female. We have developed methods to culture the glands and manipulate their biosynthetic activity. This has allowed us to monitor a series of molecular events that precede silk production in glands excised from appropriately stimulated animals. In this paper, we demonstrate that prior to the transient dramatic production of fibroin, such glands accumulate large amounts of tRNAs cognate to the abundant amino acids in spider silk. One of these, alanine tRNA, appears to consist of two isoaccepting forms--one constitutive, and the other silkgland specific. Moreover, the silkgland-specific form appears to accumulate preferentially in response to stimulation. This phenomenon of tissue-specific tRNA production appears similar to that found in the silkglands of Bombyx mori, but the spider system has the unique property of permitting manipulation in vitro. Thus, it provides an unusual opportunity to study the mechanism of regulated tRNA synthesis.     

The cylindrical or tubiliform glands of Nephila clavipes

The cylindrical or tubiliform glands of the spider Nephila clavipes have been studied and compared to the large ampullates on which we have previously reported. The three pairs of cylindrical or tubiliform glands secrete the fibroin for the organism's egg case. Their solubilized luminar contents migrate as a homogeneous band in Sodium dodecyl sulfate polyacrylamide gel electrophoresis and turn out to be a larger protein than that produced by the large ampullates. The excised cylindrical glands remain metabolically active for several hours in a simple culture medium, where fibroin synthesis can be monitored through the incorporation of 14C alanine. The glands' response to a fibroin production stimulus does not reach the magnitude displayed by the large ampullates, but this is to be expected since their products supply different functions in this organism. This fibroin also seems to be elongated discontinuously. Translational pauses have been detected in the secretory epithelium of cylindrical and large ampullate glands of Nephila as well as in the silk glands of Bombyx mori. Since these glands produce the fibroin for the females egg case, they should prove to be an interesting model system.     

The embryonic origin of the ampullate silk glands of the spider Cupiennius salei

Silk production in spiders is considered a key innovation, and to have been vital for the diversification of the clade. The evolutionary origin of the organs involved in spider silk production, however, and in particular of the silk glands, is poorly understood. Homologies have been proposed between these and other glands found in arachnids, but lacking knowledge of the embryonic development of spider silk glands hampers an evaluation of hypotheses. This study focuses on the embryonic origin of the largest silk glands of the spider Cupiennius salei, the major and minor ampullate glands. We show how the ampullate glands originate from ectodermal invaginations on the embryonic spinneret limb buds, in relation to morphogenesis of these buds. Moreover, we visualize the subsequent growth of the ampullate glands in sections of the early postembryonic stages. The invaginations are shown to correlate with expression of the proneural gene CsASH2, which is remarkable since it has been proposed that spider silk glands and their nozzles originate from sensory bristles. Hence, by confirming the ectodermal origin of spider silk glands, and by describing the (post-)embryonic morphogenesis of the ampullate glands, this work provides a starting point for further investigating into the genetic program that underlies their development.     

Correlation between mRNA structure of the coding region and translational pauses.

Discontinuous translational elongation of polypeptides is observed during spider dragline silk fibroin synthesis (1,2). The repeating segment of one of the two subunit proteins constituting spider major ampullate (dragline) silk of Nephila clavipes, Spidroin 2, consists of alternate alanine-rich and proline-rich regions (3). It was found that the calculated free energy of the secondary structure of Spidroin 2 mRNA per nucleotide for the alanine-rich region is about the same as that for the successive proline-rich region. The small stability changes of local mRNA secondary structures along the mRNA chain suggest that the translational pauses observed for dragline silk fibroin synthesis may not be correlated with Spidroin 2 mRNA structure, in contrast to Spidroin 1 mRNA structure which may explain the translational pauses (4).     

Stimulation of fibroin synthesis elicits ultrastructural modifications in spider silk secretory cells

Electron microscopic studies of unstimulated and stimulated spider fibroin glands show that the fibroin synthesis stimulus evokes visible changes in both the endoplasmic reticulum and Golgi apparatus of their secretory epithelium. Gradual increase in distension of the reticulum accompanies the increase of evoked fibroin synthetic activity. The flattened translucent Golgi vesicles, seen in inactive cells, display a gradual increase in size and number, also with time. The stimulation also elicits a gradual transition in the gland's luminal membrane, during which the microvilli on the lining gradually disappear acquiring an electron dense appearance. Correlations of the observed transitions to the gland's increase in rate of elicited synthetic activity are discussed. The parallelisms between the ultrastructural modifications observed in the spider secretory cells with those described in the silkworm glands during their progression through the fifth instar have been stressed.     

Spider minor ampullate silk proteins are constituents of prey wrapping silk in the cob weaver Latrodectus hesperus

Spiders spin high performance fibers with diverse biological functions and mechanical properties. Molecular and biochemical studies of spider prey wrapping silks have revealed the presence of the aciniform silk fibroin AcSp1-like. In our studies we demonstrate the presence of a second distinct polypeptide present within prey wrapping silk. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called MiSp1-like and demonstrate that its protein product is a constituent of prey wrap silks from the black widow spider, Latrodectus hesperus. BLAST searches of the NCBInr protein database using the amino acid sequence of MiSp1-like revealed similarity to the conserved C-terminal domain of silk family members. In particular, MiSp1-like showed the highest degree of sequence similarity to the nonrepetitive C-termini of published orb-weaver minor ampullate fibroin molecules. Analysis of the internal amino acid sequence of the black widow MiSp1-like revealed polyalanine stretches interrupted by glycine residues and glycine-alanine couplets within MiSp1-like as well as repeats of the heptameric sequence AGGYGQG. Real-time quantitative PCR analysis demonstrates that the MiSp1-like gene displays a minor ampullate gland-restricted pattern of expression. Furthermore, amino acid composition analysis, coupled with scanning electron microscopy of raw wrapping silk, supports the assertion that minor ampullate silks are important constituents of black widow spider prey wrap silk. Collectively, our findings provide direct molecular evidence for the involvement of minor ampullate fibroins in swathing silks and suggest composite materials play an important role in the wrap attack process for cob-weavers.     

Upgraded expression of 5S rRNA preludes the production of fibroin by spider glands

We have developed the large ampullate glands of the orb-web spider Nephila clavipes as a model system in which to study the production of a tissue-specific secretory protein. Through simple manipulations, the glands' fibroin production can be practically abolished and subsequently elicited into high levels of synthesis through a mechanical stimulus applied to the organism. The tissue specific responses evoked by the stimulus can be monitored through time-course studies. The latter have revealed an orchestrated series of tissue and time specific macromolecular syntheses, which optimize the glandular tissues with components of the protein synthesis machinery. This work shows the upgraded accumulation of 5S rRNA in the glands as response to the stimulus within the earliest of the prelude events. Further enquiries on this accumulation must be conducted at the level of differential gene expressions, a chore we have initiated. A DNA fragment containing a single copy 5S rRNA gene has been isolated, cloned, sequenced, and transcribed in a cell-free system. We enclose a discussion on the similarity between the genomic organization of this gene to that of a 5S rRNA gene of Bombyx mori. Our studies have revealed a considerable number of similarities in the silk production strategies of Nephila clavipes and the silkworm Bombyx mori, some of them rather unusual.     

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.     

Microstructure of the silk apparatus of the comb-footed spider, Achaearanea tepidariorum (Araneae: Theridiidae)

The microstructural organization of the silk‐spinning apparatus of the comb‐footed spider, Achaearanea tepidariorum, was observed by using a field emission scanning electron microscope. The silk glands of the spider were classified into six groups: ampullate, tubuliform, flagelliform, aggregate, aciniform and pyriform glands. Among these, three types of silk glands, the ampullate, pyriform and aciniform glands, occur only in female spiders. One (adult) or two (subadult) pairs of major ampullate glands send secretory ductules to the anterior spinnerets, and another pair of minor ampullate glands supply the median spinnerets. Three pairs of tubuliform glands in female spiders send secretory ductules to the median (one pair) and posterior (two pairs) spinnerets. Furthermore, one pair of flagelliform glands and two pairs of aggregate glands together supply the posterior spinnerets, and form a characteristic spinning structure known as a “triad” spigot. In male spiders, this combined apparatus of the flagelliform and the aggregate spigots for capture thread production is not apparent, instead only a non‐functional remnant of this triad spigot is present. In addition, the aciniform glands send ductules to the median (two pairs) and the posterior spinnerets (12–16 pairs), and the pyriform glands feed silk into the anterior spinnerets (90–100 pairs in females and 45–50 pairs in males).     

Selected aspects of spinning apparatus development in Araneus cavaticus (Araneae,Araneidae)

In the first half of this century, several workers observed small, seemingly glandular structures attached to the ampullate glands of spiders. Hence, they were termed accessory ampullate glands. In juvenile Araneus cavaticus, two pairs of these structures are present (starting at least with third instars), one pair attached to the major ampullate (MaA) glands and the other pair attached to the minor ampullate (MiA) glands. In adults, two pairs of accessory MaA glands and two pairs of accessory MiA glands are present. The two latter-formed pairs of accessory ampullate glands are clearly the remnants of those ampullate glands which atrophy shortly after adulthood is reached. Morphological similarities between these accessory ampullate glands and those present in juveniles provide an indication that the latter also have their origin in functional ampullate glands. A reduction in the number of ampullate glands following the last molt occurs in many spiders. The reason(s) for these reductions is unknown. In penultimate spiders close to ecdysis, we have observed that while the larger pairs of MaA and MiA glands (those that are retained in the adult) are undergoing molt-related changes which apparently render them nonfunctional, their smaller counterparts are seemingly unaffected and functional. This raises the possibility that the principal role of the smaller ampullate glands may be to assume functions during the pre-ecdysial period which are normally in the domain of the larger ampullate glands. If true, then their degeneration after the last molt would make economic sense. The presence of cylindrical spigots in juvenile females starting with fourth instars is documented.     

Spider silk fibroin enhances insulin secretion and reduces blood glucose levels in diabetic mice

Fibroin silk proteins make up the cocoons of silkworms and spider webs and are rich in glycine and alanine residues. Recent studies have shown that silk fibroin hydrolysate from silkworms improves blood glucose and lipid metabolism. In the present study, we investigated the anti-diabetic effects of spider silk fibroin protein in type 2 diabetic mice. Recombinant AvMaSp-R, which consists of the 240 amino acid repetitive domain of major ampullate silk protein (AvMaSp) from the spider Araneus ventricosus, was produced in baculovirus-infected insect cells. We tested the effects of oral AvMaSp-R administration on serum insulin and blood glucose levels in diabetic mice and found that AvMaSp-R increases serum insulin levels and reduces blood glucose levels in diabetic mice. Consequently, our results are the first to provide evidence that silk fibroin protein from spiders enhances insulin secretion, which leads to reduced blood glucose levels in type 2 diabetic mice.     

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.     

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.     

Microstructure of the silk spigots of the green crab spider Oxytate striatipes (Araneae: Thomisidae)

The genus Oxytate L. Koch, 1878 comprises a homogeneous group of nocturnal crab spiders that have silk apparatuses even though they do not spin webs to trap prey. We examined the microstructure of the silk spinning apparatus of the green crab spider Oxytate striatipes, using field emission scanning electron microscopy. The silk glands of the spider were classified into three types: ampullate, pyriform and aciniform. The spigots of these three types of silk gland occur in both sexes. Two pairs of major ampullate glands send secretory ductules to the anterior spinnerets, and another two pairs of minor ampullate glands supply the median spinnerets. In addition, the pyriform glands send ductules to the anterior spinnerets (45 pairs in females and 40 pairs in males), and the aciniform glands feed silk into the median (9–12 pairs in females and 7–10 pairs in males) and the posterior (30 pairs in both sexes) spinnerets. The spigot system of O. striatipes is simpler and more primitive than other wandering spiders: even the female spiders possess neither tubuliform glands for cocoon production nor triad spigots for web‐building.