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PubMed1980

Fine structural aspects of silk secretion in a spider (Araneus diadematus). I. Elaboration in the pyriform glands.

Pyriform glands of Araneus diadematus which produce the silky material used for the attachment discs of the web, consist of two kinds of secretory cells. One, located in the distal half of the glands, elaborates finely fibrillar proteinic granules through an extensive rough endoplasmic reticulum; another, in the proximal half of the glands, secretes complex-structured granules in areas of the cell where Golgi and ergastoplasmic cisternae are equally developed. The opaque nascent granules of secretion appear in swollen Golgi saccules. These aggregate is superposed circular interconnected layers leaving an electron-lucent space between them; in the course of maturation the space is progressively filled with a fibrillar material. Histochemical tests suggest that the secretory product of the proximal half is mainly a protein rich in acidic groups and associated with a carbohydrate component. The two products, extruded by a merocrine process, form respectively the core and the envelope of the silk fibre. The dual composition of the pyriform gland silk, which did not appear from the results of chemical analyses, is compared to the association of fibroin and sericin in Lepidoptera silk and to certain double-layered Trichoptera silks.

Animals↗

PubMed1990

Marine adhesive proteins: natural composite thermosets.

Marine environments are severely challenging for the performance and durability of synthetic adhesives. Factors commonly associated with adhesive failure are weak boundary layers (water, oxides), adhesive erosion and swelling. For many permanently attached marine organisms such as barnacles, mussels, oysters, etc., however, underwater adhesion is 'business-as-usual'. Knowledge about the chemistry and bioprocessing of these marine adhesives will provide profound insights for the evolution of a new generation of environmentally safe, water-resistant adhesives. Despite their apparent structural diversity, marine adhesives are essentially analogous to composite thermosets, that is, the adhesive consists of fibre, filler and catalyst molecules that are dispersed in a cross-linked resin rendering it resistant to heat and solvents. The fibres and fillers in these composites are variable. e.g. collagen, fibroin, chitin present as fibres, and sand, shell, air and water present as fillers. The precured resins of seven organisms including members of the Mollusca, Annelida, and Platyhelminthes have now been isolated and partially sequenced. These are proteins with basic isoelectric points, high levels of the amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA), and an extended, flexible conformation. The DOPA functional group in particular is thought to play a key role in (a) the chemisorption of these polymers to surface underwater, and (b) covalent cross-linking or setting of the adhesive, the latter reaction catalysed by the enzyme catecholoxidase. Much more needs to be done to explore the details of the adhesive processing and delivery strategies used by these organisms.

Adhesiveness↗

PubMed1996

An alanine tRNA gene cluster from Nephila clavipes.

We report the sequence of a 2.3-kb genomic DNA fragment from the orb-web spider, Nephila clavipes (Nc). The fragment contains four regions of high homology to tRNA(Ala). The members of this irregularly spaced cluster of genes are oriented in the same direction and have the same anticodon (GCA), but their sequence differs at several positions. Initiation and termination signals, as well as consensus intragenic promoter sequences characteristic of tRNA genes, have been identified in all genes. tRNA(Ala) are involved in the regulation of the fibroin synthesis in the large ampullate Nc glands.

Animals↗

PubMed1994

Protein ubiquitination in the posterior silk glands of Bombyx mori.

Ubiquitin gene expression and the ubiquitination of proteins in the posterior silk glands (PSG) of B. mori were analyzed developmentally with respect to fibroin synthesis and degeneration. Two ubiquitin transcripts are expressed throughout larval stages, and the level of each transcript is regulated differently. The larger transcript, a polyubiquitin mRNA, was abundant during the molt stage, while levels of the smaller ubiquitin transcript increased immediately after molting. Only a single 65 kDa ubiquitinated protein was detected late in the 5th instar, and the amount increased up to spinning stage. This ubiquitinated protein may participate in the PSG degeneration.

Animals↗

PubMed2006

Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds.

Natural bone consists of cortical and trabecular morphologies, the latter having variable pore sizes. This study aims at engineering different bone-like structures using scaffolds with small pores (112-224 microm) in diameter on one side and large pores (400-500 microm) on the other, while keeping scaffold porosities constant among groups. We hypothesized that tissue engineered bone-like structure resulting from silk fibroin (SF) implants is pre-determined by the scaffolds' geometry. To test this hypothesis, SF scaffolds with different pore diameters were prepared and seeded with human mesenchymal stem cells (hMSC). As compared to static seeding, dynamic cell seeding in spinner flasks resulted in equal cell viability and proliferation, and better cell distribution throughout the scaffold as visualized by histology and confocal microscopy, and was, therefore, selected for subsequent differentiation studies. Differentiation of hMSC in osteogenic cell culture medium in spinner flasks for 3 and 5 weeks resulted in increased alkaline phosphatase activity and calcium deposition when compared to control medium. Micro-computed tomography (microCT) detailed the pore structures of the newly formed tissue and suggested that the structure of tissue-engineered bone was controlled by the underlying scaffold geometry.

Biocompatible Materials↗

PubMed2006

Generation of hybrid transgenic silkworms that express Bombyx mori prolyl-hydroxylase alpha-subunits and human collagens in posterior silk glands: Production of cocoons that contained collagens with hydroxylated proline residues.

Prolyl 4-hydroxylase (P4H) is a heterotetramer enzyme consisting of alpha-subunits (P4Halpha) and beta-subunits (P4Hbeta), and is required for collagen biosynthesis. Previously, we generated transgenic silkworms that produced human type III collagen fragments (mini-collagens) in the posterior silk gland (PSG). However, prolyl 4-hydroxylation did not occur on the mini-collagens, because in spite of an abundant expression of P4Hbeta in PSGs, P4Halpha expression was quite low there, thus resulting in an insufficient activity of P4H. In this study we aimed at generating hybrid transgenic silkworms whose PSGs are capable of producing mini-collagens and enough P4H for their prolyl 4-hydroxylation. Isolated PSGs were bombarded with fibroin L-chain gene promoter-driven vectors containing Bombyx mori P4Halpha (BmP4Halpha) cDNAs and were transplanted into the hemolymphatic cavity. The P4H activity in the PSG cells significantly increased, indicating that the expressed BmP4Halpha formed active tetramers with endogenous BmP4Hbeta. Using germ-line transgenesis technology, silkworms were generated that synthesized BmP4Halpha in PSG cells. The P4H activity in the transgenic silkworms was 130-fold higher than that of wild-type counterparts. Finally, we generated hybrid transgenic silkworms that expressed cDNAs of both BmP4Halpha and mini-collagen in PSG cells. They spun cocoons that contained mini-collagens whose appropriate proline residues had been adequately hydroxylated.

Animals↗

PubMed2004

The Acrolepiopsis assectella silk cocoon: kairomonal function and chemical characterisation.

Two soluble sericin-like polypeptides, B1 and B2, from leek moth (Acrolepiopsis assectella) cocoons trigger host-acceptance behaviour in the parasitoid, Diadromus pulchellus (Proc. Roy. Soc. London B 269 (2002) 1879). We found that these polypeptides were particularly cysteine-rich and lost their ability to trigger host-acceptance behaviour after being denatured and purified. This suggests that inter-disulphide bonds and the secondary structure of B1 and B2 are important for their biological activity. We also isolated six insoluble polypeptides (or polypeptides of low solubility) from A. assectella cocoons. At least four of these polypeptides triggered host-acceptance behaviour. The strongest responses were observed with P22, a light-chain fibroin or a seroin-peptide, and P100, a sericin-like polypeptide that is probably more strongly associated with the silk core than are B1 and B2. In conclusion, several polypeptides from different parts of the A. assectella silk-cocoon (the insoluble core and coating of the silk thread) are able to elicit host-acceptance behaviour in D. pulchellus females. These polypeptides belong to different silk protein families and are used as kairomones by this specialist parasitoid.

Animals↗

PubMed2005

Gene expression changes in Ephestia kuehniella caterpillars after parasitization by the endoparasitic wasp Venturia canescens analyzed through cDNA-AFLPs.

To understand the transcriptional response of Ephestia kuehniella caterpillars to parasitization by the endoparasitic wasp Venturia canescens, we examined patterns of gene expression in parasitized and unparasitized host caterpillars using the cDNA-AFLP (cDNA-amplified fragment length polymorphism) technique. Among the approximately 1860 cDNA-fragments detected by using 88 different AFLP primer combinations, we identified 13 transcripts in parasitized caterpillars that showed a suppressed expression pattern with cDNA-AFLP results being confirmed by virtual Northern blot or RT-PCR analysis. Apparently, two different wasp genotypes (RP, RM) seem to differ in their ability to regulate host physiology with regard to suppression of certain genes. Sequence analysis of the cDNA fragments revealed that some of the respective genes are likely to be involved in energy metabolism, protein synthesis, or in signal transduction. In addition, one sequence corresponds to a gene encoding the lepidopteran silk protein fibroin. The putative function of these genes in the interactions between endoparasitoids and their host insects is discussed.

Animals↗

PubMed2001

Modeling AFM-induced PEVK extension and the reversible unfolding of Ig/FNIII domains in single and multiple titin molecules.

Molecular elasticity is associated with a select number of polypeptides and proteins, such as titin, Lustrin A, silk fibroin, and spider silk dragline protein. In the case of titin, the globular (Ig) and non-globular (PEVK) regions act as extensible springs under stretch; however, their unfolding behavior and force extension characteristics are different. Using our time-dependent macroscopic method for simulating AFM-induced titin Ig domain unfolding and refolding, we simulate the extension and relaxation of hypothetical titin chains containing Ig domains and a PEVK region. Two different models are explored: 1) a series-linked WLC expression that treats the PEVK region as a distinct entropic spring, and 2) a summation of N single WLC expressions that simulates the extension and release of a discrete number of parallel titin chains containing constant or variable amounts of PEVK. In addition to these simulations, we also modeled the extension of a hypothetical PEVK domain using a linear Hooke's spring model to account for "enthalpic" contributions to PEVK elasticity. We find that the modified WLC simulations feature chain length compensation, Ig domain unfolding/refolding, and force-extension behavior that more closely approximate AFM, laser tweezer, and immunolocalization experimental data. In addition, our simulations reveal the following: 1) PEVK extension overlaps with the onset of Ig domain unfolding, and 2) variations in PEVK content within a titin chain ensemble lead to elastic diversity within that ensemble.

Amino Acid Sequence↗

PubMed1996

Presence of phosphorus in Nephila clavipes dragline silk.

Solid-state 31P-NMR of Nephila clavipes dragline silk indicates the presence of phosphorus in at least two chemically distinct environments. Amino acid analyses of acid-hydrolyzed silk confirm the presence of phosphotyrosine as one of the phosphorus-containing components. The unusual chemical shift (18.9 ppm downfield from 85% H3PO4), proton chemical shift, and acid lability of a second component suggest that it is part of a strained five-membered cyclic phosphate that might be found on a beta-D-ribose. The five-membered cyclic phosphate is not removed from the silk fibers by exhaustive aqueous extraction. It is absent in nascent silk fibroin from the glands, suggesting that its formation is part of the fiber processing that occurs in the ducts leading to the spinnerets. High-resolution NMR spectra of silk dissolved in propionic acid/12 N HCl (50:50 v/v) show five phosphorus sites assigned to phosphorylated tyrosine residues, phosphorylated serine residues, inorganic phosphate, and two hydrolysis products of the cyclic phosphate compound. The observed posttranslational phosphorylation may be important in the processing and modulation of the physical properties of dragline silk.

Animals↗

PubMed2002

Functional genomics of gam56: characterisation of the role of a 56 kilodalton sexual stage antigen in oocyst wall formation in Eimeria maxima.

Gam56 (M(r) 56,000) is an antigen found in the sexual (macrogametocyte) stage of the intestinal parasite Eimeria maxima that is implicated in protective immunity. The gene (gam56) encoding this protein was cloned and sequenced. It is a single-copy, intronless gene, that localises to a 1,754 bp transcript, and is first detected at 120 h p.i. The gene predicts two distinct protein domains; a tyrosine-serine rich region, composed of amino acids implicated in oocyst wall formation in Eimeria spp., and a proline-methionine rich region often detected in extensins, protein components of plant cell walls. The tyrosine-serine rich region predicts a secondary structure commonly seen in the structural protein fibroin, a component of the cocoon of the caterpillar Bombyx mori. The inference that gam56 is a structural component of the oocyst wall was confirmed when a specific antibody to gam56 recognised the wall forming bodies in macrogametocytes, and the walls of oocysts and sporocysts. Together, these data identify a developmentally regulated, sexual stage gene in E. maxima that shares primary and secondary structure features in common with intrinsic structural proteins in other parasites such as Schistosoma mansoni and Fasciola hepatica, and other organisms across different phyla, including the caterpillar Bombyx mori. In addition, these findings provide evidence for the molecular mechanisms underlying oocyst wall formation in Eimeria and the role of gametocyte antigens in this process.

Amino Acid Sequence↗

PubMedDate not supplied

A comparison of the composition of silk proteins produced by spiders and insects.

Proteins that are highly expressed and composed of amino acids that are costly to synthesize are likely to place a greater drain on an organism's energy resources than proteins that are composed of ingested amino acids or ones that are metabolically simple to produce. Silks are highly expressed proteins produced by all spiders and many insects. We compared the metabolic costs of silks spun by arthropods by calculating the amount of ATP required to produce their component amino acids. Although a definitive conclusion requires detailed information on the dietary pools of amino acids available to arthropods, on the basis of the central metabolic pathways, silks spun by herbivorous, Lepidoptera larvae require significantly less ATP to synthesize than the dragline silks spun by predatory spiders. While not enough data are available to draw a statistically based conclusion, comparison of homologous silks across ancestral and derived taxa of the Araneoidea seems to suggest an evolutionary trend towards reduced silk costs. However, comparison of the synthetic costs of dragline silks across all araneomorph spiders suggests a complicated evolutionary pattern that cannot be attributed to phylogenetic position alone. We propose that the diverse silk-producing systems of the araneoid spiders (including three types of protein glues and three types of silk fibroin), evolved through intra-organ competition and that taxon-specific differences in the composition of silks drawn from homologous glands may reflect limited or fluctuating amino acid availability. The different functional properties of spider silks may be a secondary result of selection acting on different polypeptide templates.

Amino Acids↗

PubMed1979

Effect of starvation on tRNA synthesis, amino acid pool, tRNA charging levels and aminoacyl-tRNA synthetase activities in the posterior silk gland of Bombyx mori L.

Changes in the translational machinery components of the Bombyx mori posterior silk gland were analysed during starvation and refeeding and compared to the regularly fed larvae. During starvation, tRNA and ribosomal RNA synthesis are stopped. The amounts of different RNA classes and of the different tRNA species slow down at the same rate. Thus various tRNA show similar half-lifes and the preexisting tRNA adaptation to fibroin mRNA translation persists during starvation. Similarly, the tRNA/rRNA ratio is constant during starvation and refeeding (12 tRNA molecules for one ribosome) as in silk glands of control animals. Aminoacyl-tRNA synthetases and tRNA charging levels are decreased during starvation. The maximal tRNA charging level obtained during maximal protein synthesis in control animals is regained after 24 h refeeding of starved larvae. Changes observed in the free amino acid pool are not similar from one amino acid to another and levels reached after starvation do not differ strongly from the controls. Our results suggest that the production of translation apparatus components is coordinated and adjusted to the protein synthesis activity. Whether this coordination occurs in the silk gland is discussed on the basis of the "metabolic regulation", primarily described in prokaryotes and Yeast. Transfer RNA charging levels seem to play a key role in the process of regulation and could be implicated in the mechanism of tRNA adaptation if this phenomenon results as expected from a transcriptional control.

Amino Acids↗

PubMed1981

Does quantitative tRNA adaptation to codon content in mRNA optimize the ribosomal translation efficiency? Proposal for a translation system model.

Neither a dynamic nor an energetic approach of the translation process has taken into account that intracellular levels of iso-tRNA species are adapted or adjusted to the codon frequency of mRNA being decoded (Bombyx mori silk gland, rabbit reticulocyte). A critical study of available experimental data suggests that the average elongation rate of a protein is maximized in the presence of an adapted tRNA population, usually an homologous tRNA. In addition, the amount of synthesized protein parallels that of corresponding mRNA. Other evidences--including in vitro and in vivo elongation assays with fibroin mRNA--show that individual elongation rates are not uniform. Pauses occur at certain sites of the mRNA chain. The relative lifetime of these pauses depends on the tRNA pool used. Finally, it appears that translation accuracy also depends on the balanced tRNA population. We propose to explain these different effects by using a codon-anticodon recognition model, called "trial and error system" based on a stochastic processing of the ribosome. Accordingly, various acylated tRNA species which surround a ribosome randomly encounter the receptor A site. Every trapped tRNA species is tested for a proper pairing with the codon to be recognized at the level of a comparator or discriminator function. If the pairing is correct, transpeptidation becomes irreversible. If not, the aminoacyl-tRNA is rejected and another randomly trapped tRNA is processed in turn. Mathematical analysis of this model shows that the mean number of trials used for translating the whole sequence of a mRNA is minimized when the proportion of different iso-tRNA species is correlated with the square root of codon frequency. Quantitations of reticulocyte tRNA support such a parabolic relation. Our translation system model brings some light into the role of tRNA adaptation for optimizing translation efficiency, i.e. maximizing both speed and accuracy. Some consequences of the model are discussed.

Codon↗

PubMed1997

Characterization of a Schistosoma mansoni gene encoding a homologue of the Y-box binding protein.

We have cloned and characterized a Schistosoma mansoni cDNA encoding a basic protein homologous to the human Y-box binding protein 1 (YB-1). The 1.3-kb S. mansoni YB-1 transcript, which was shown to be expressed in various stages of the parasite life cycle, codes for a protein of 217 amino acids containing, towards its N-terminus, a nucleic acid binding motif, known as the cold-shock domain (CSD). This domain is 64% identical to the cold-shock domain of other members of the Y-box binding protein family and 43% identical to the cold-shock protein CspA of Escherichia coli. In S. mansoni YB-1, the cold-shock domain possess some structural characteristics that permit dimer formation as occurs in the Bacillus subtilis cold-shock protein CspB. The C-terminal region of S. mansoni YB-1 differs from the other Y-box binding proteins because of the presence of tandem repeats of Arg and Gly, suggesting the formation of a fibroin-like beta-sandwich structure. This novel folding pattern for the C-terminus of S. mansoni YB-1 might suggest a distinct specific function for this protein in the parasite.

Amino Acid Sequence↗

PubMed1999

A spider tRNA(Ala) requires a far upstream sequence element for expression.

Within the series of timed differential accumulations of small RNAs we have shown to prelude the synthesis of fibroin in the large ampullate glands of Nephila clavipes (Nc), we are currently directing our attention to the alanine tRNAs. This work reports the subcloning of the members of a tRNAAla gene cluster and the optimization of their transcription in a heterologous cell-free system derived from Bombyx mori (Bm) silkglands. Our data show that the heterologous cell-free system supports the faithful and differential transcription of the individual spider alanine tRNA genes. We are thus making use of the extract to characterize the individual genes with respect to flank-contained regulatory elements through cell-free transcription of gene derivatives. The work has been initiated with pNTA3 because of its high transcriptional activity. Interestingly, the transcription of this gene requires a far upstream sequence, an uncommon modality in tRNA genes.

Animals↗

PubMed1997

Application of NMR microscopy to the morphological study of the silkworm, Bombyx mori, during its metamorphosis.

Zero (ZQ) and double (DQ) quantum 2D chemical shift selective and spin-echo 3D NMR imaging at microscopy resolution, has been applied to the morphological study of silkworm, Bombyx mori, during its metamorphosis. Attention had been focused on the evolution of the internal structure of the insect during its postembryonal life occurring through the larval, pupal and adult development. A major objective of this work was the characterization of the silk glands, responsible for the synthesis and secretion of fibroin and sericin, through the changes of distribution and mobility of water, by imaging the water protons during postembryonal life stages. Moreover, alanine deriving from silk gland proteins was imaged during the last life stage of Bombyx mori.

Animals↗

PubMed2006

Novel molecular and mechanical properties of egg case silk from wasp spider, Argiope bruennichi.

Araneoid spiders use specialized abdominal glands to produce up to seven different protein-based silks/glues that have various mechanical properties. To date, the fibroin sequences encoding egg case fibers have not been fully determined. To gain further understanding of a recently reported spider silk protein gene family, several novel strategies were utilized in this study to isolate two full-length cDNAs of egg case silk proteins, cylindrical silk protein 1 (CySp1, 9.1 kb) and cylindrical silk protein 2 (CySp2, 9.8 kb), from the wasp spider, Argiope bruennichi. Northern blotting analysis demonstrated that CySp1 and CySp2 are selectively expressed in the cylindrical glands. The amino acid composition of raw egg case silk was closely consistent with the deduced amino acid composition based on the sequences of CySp1 and CySp2, which supports the assertion that CySp1 and CySp2 represent two major components of egg case silk. CySp1 and CySp2 are primarily composed of remarkable homogeneous assemble repeats that are 180 residues in length and consist of several complex subrepeats, and they contain highly homologous C-termini and markedly different N-termini. Our results suggest a possible link between CySp1 and CySp2. In addition, comparisons of stress/strain curves for dragline and egg case silk from Argiope bruennichi showed obvious differences in ultimate strength and extensibility, and similarities in toughness.

Amino Acid Sequence↗