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PubMed1979

Studies on tRNA adaptation, tRNA turnover, precursor tRNA and tRNA gene distribution in Bombyx mori by using two-dimensional polyacrylamide gel electrophoresis.

Eighteen out of twenty amino acids have been used for identifying tRNAs from the silkworm Bombyx mori L. fractionated on two-dimensional polyacrylamide gel electrophoresis. 43 spots out of 53 have been identified. This mapping confirms previous results and brings new answers to some questions on the regulation of tRNA biosynthesis. 1. In addition to quantitative adaptation of tRNAs to the composition of silk proteins (fibroin from the posterior silk gland, sericin from the middle part) and of iso-tRNAs from posterior silk gland to the major codons of fibroin mRNA, we also observe adaptation of tRNA from various tissues to the average amino acid content of proteins from fat body, gut, gonads and carcass of the silkworm. 2. In the silk gland, turnover rates of several tRNA species are similar. The selective accumulation of tRNAs needed for decoding fibroin and sericin mRNAs which takes place during the Vth larval instar, cannot be explained by the occurrence of a preferential degradation of some tRNA species. 3. Under given conditions for incubating silk glands, it is possible to obtain an accumulation of precursor tRNA species, which are enriched in pre-tRNAAla and pre-tRNAGly in the posterior silk gland and pre-tRNASer in the middle part. 4. The distribution of tRNA genes is not random. tRNA genes for glycine, alanine and serine are prominent. Selective transcription of batteries of iso-tRNA genes could explain our data.

Animals↗

PubMedDate not supplied

Synthesis and characterization of chimeric silkworm silk.

A synthetic gene encoding a chimeric silklike protein was constructed that combined a polyalanine encoding region (Ala)(18), a sequence slightly longer than the (Ala)(12-13) found in the silk fibroin from the wild silkworm Samia cynthia ricini, and a sequence encoding GVGAGYGAGAGYGVGAGYGAGVGYGAGAGY, found in the silk fibroin from the silkworm Bombyx mori. A tetramer of the chimeric repeat sequence encoding a approximately 29 kDa protein was expressed as a fusion protein in Escherichia coli. In comparison to S. c. ricini silk, the chimeric protein demonstrated improved solubility because it could be dissolved in 8 M urea. The purified protein assumed an alpha-helical structure based on solid-state (13)C CP/MAS NMR and was less prone to conformational transition to a beta-sheet, unlike native silk proteins from S. c. ricini. Model peptides representing the crystalline region of S. c. ricini silk fibroin, (Ala)(12) and (Ala)(18), formed beta-sheet structures. Therefore, the solubility and structural transitions of the chimeric protein were significantly altered through the formation of this chimeric silk. This experimental strategy to the study of silk structure and function can be used to develop an improved understanding of the contributions of protein domains in repetitive silkworm and spider silk sequences to structure development and structural transitions.

Amino Acid Sequence↗

PubMedDate not supplied

Study of protein conformation and orientation in silkworm and spider silk fibers using Raman microspectroscopy.

Raman microspectroscopy has been used for the first time to determine quantitatively the orientation of the beta-sheets in silk monofilaments from Bombyx mori and Samia cynthia ricini silkworms, and from the spider Nephila edulis. It is shown that, for systems with uniaxial symmetry such as silk, it is possible to determine the order parameters P2 and P4 of the orientation distribution function from intensity ratios of polarized Raman spectra. The equations allowing the calculation of P2 and P4 using polarized Raman microspectroscopy for a vibration with a cylindrical Raman tensor were first derived and then applied to the amide I band that is mostly due to the C=O stretching vibration of the peptide groups. The shape of the Raman tensor for the amide I vibration of the beta-sheets was determined from an isotropic film of Bombyx mori silk treated with methanol. For both the Bombyx mori and Samia cynthia ricini fibroin fibers, the values of P2 and P4 obtained are equal to -0.36 +/- 0.03 and 0.19 +/- 0.02, respectively, even though the two types of silkworm fibroins strongly differ in their primary sequences. For the Nephila edulis dragline silk, values of P2 and P4 of -0.32 +/- 0.02 and 0.13 +/- 0.02 were obtained, respectively. These results clearly indicate that the carbonyl groups are highly oriented perpendicular to the fiber axis and that the beta-sheets are oriented parallel to the fiber axis, in agreement with previous X-ray and NMR results. The most probable distribution of orientation was also calculated from the values of P2 and P4 using the information entropy theory. For the three types of silk, the beta-sheets are highly oriented parallel to the fiber axis. The orientation distributions of the beta-sheets are nearly Gaussian functions with a width of 32 degrees and 40 degrees for the silkworm fibroins and the spider dragline silk, respectively. In addition to these results, the comparison of the Raman spectra recorded for the different silk samples and the polarization dependence of several bands has allowed to clarify some important band assignments.

Animals↗

PubMed2005

Effect of metallic ions on silk formation in the Mulberry silkworm, Bombyx mori.

A protein conformation transition from random coil and/or helical conformation to beta-sheet is known to be central to the process used by silk-spinning spiders and insects to convert concentrated protein solutions to tough insoluble threads. Several factors including pH, metallic ions, shear force, and/or elongational flow can initiate this transition in both spiders and silkworms. Here, we report the use of proton induced X-ray emission (PIXE), inductively coupled plasma mass spectroscopy (ICP-MS) and atomic adsorption spectroscopy (AAS) to investigate the concentrations of six metal elements (Na, K, Mg, Ca, Cu, and Zn) at different stages in the silk secretory pathway in the Bombyx mori silkworm. We also report the use of Raman spectra to monitor the effects of these six metallic ions on the conformation transition of natural silk fibroin dope and concentrated regenerated silk fibroin solution at concentrations similar to the natural dope. The results showed that the metal element contents increased from the posterior part to the anterior part of silk gland with the exception of Ca which decreased significantly in the anterior part. We show that these changes in composition can be correlated with (i) the ability of Mg2+, Cu2+, and Zn2+ to induce the conformation transition of silk fibroin to beta-sheet, (ii) the effect of Ca2+ in forming a stable protein network (gel), and (iii) the ability of Na+ and K+ to break down the protein network.

Animals↗

PubMed1981

Differential usage of iso-accepting tRNASer species in silk glands of Bombyx mori.

The rapid development of the silk glands of Bombyx mori during the last larval instar shows two phases. During the first 4 days, in both the middle and posterior parts of the silk glands, the ribosomal machinery is assembled and the synthesis of housekeeping proteins starts. During the second phase (the last 4 days), the middle part of the gland synthesis approximately 45 mg of the silk protein sericin (31% serine) and the posterior part of the gland synthesizes approximately 130 mg of the silk protein fibroin (46% glycine, 29% alanine and 12% serine). Silk fibroin and sericin are detectable by the second day and represent 80 and 50% respectively of the total proteins produced at day 8 (refs 1--4). It is known that the tRNA population of the posterior part of the gland is quantitatively adapted to fibroin codon frequency during this period but little is known about the situation in the middle part except for the observation that it contains more tRNASer than does the posterior part. We show here that the two parts contain, and presumably use, different iso-accepting species of tRNASer, the middle part using tRNASer1, which recognizes AGU and AGC codons, and the posterior part using tRNASer2 which recognizes UCA. We also suggest that this differential adaptation of the tRNASer species is under transcriptional control as the two species are accumulated at different rates, but degraded at the same rate.

Animals↗

PubMed1997

Tough tendons. Mussel byssus has collagen with silk-like domains.

The primary structure of the alpha-chain of preCol-D (molecular mass = 80 kDa), a tanned collagenous protein predominating in the distal portion of the byssal threads of the mussel Mytilus edulis, was deduced from cDNA to encode an unprecedented natural block copolymer with three major domain types: a central collagen domain flanked by fibroin-like domains and followed by histidine-rich termini. The fibroin-like domains have sequence motifs that strongly resemble the crystalline polyalanine-rich and amorphous glycine-rich regions of spider dragline silk fibroins. The terminal regions resemble the histidine-rich domains of a variety of metal-binding proteins. The silk domains may toughen the collagen by increasing its strength and extensibility. PreCol-D expression is limited to the mussel foot, which contains a longitudinal gradient of preCol-D mRNA. This gradient increases linearly in the proximal to distal direction and reaches a maximum just before the distal depression of the foot.

Amino Acid Sequence↗

PubMed1991

DNA superhelicity affects the formation of transcription preinitiation complex on eukaryotic genes differently.

In vitro transcription was reconstituted with HeLa cell transcription factors and RNA polymerase II, which were essentially free from DNA topoisomerase activities. DNA templates with defined negative superhelical densities were tested for transcription activity. Transcription of the Bombyx mori fibroin gene increases and plateaus from templates of increasing superhelicity, and transcription from the adenovirus 2 major late promoter rises and then falls, while transcription of the Drosophila hsp70 gene remains unchanged. Dissection of transcription into pre and post-initiation steps by the use of Sarkosyl reveals that formation of a preinitiation complex on the fibroin gene or the adenovirus 2 major late promoter is slow on relaxed DNA and accelerated by DNA superhelicity. On the contrary, the preinitiation complex assembles rapidly on the hsp70 gene irrespective of DNA topology. As is the case with the fibroin gene promoter, DNA superhelicity appears to facilitate the interaction of transcription factor IID to the adenovirus 2 major late promoter.

Animals↗

PubMed2006

Orientation-insensitive spectra for Raman microspectroscopy.

Separating effects due to molecular conformation from those due to orientation in the spectra of oriented samples obtained by Raman microspectroscopy is a complex issue. To solve this problem, we propose a procedure to calculate an orientation-insensitive spectrum (so-called isotropic spectrum) from polarized spectra obtained by Raman microspectroscopy that is valid for systems that exhibit a uniaxial symmetry. The method has first been tested on highly oriented samples of high-density polyethylene (HDPE). Polarized and isotropic spectra of a highly oriented HDPE cylindrical rod and an isotropic HDPE sample have been compared. The differences in the relative intensities, which occur in the polarized spectra and are due to orientation of the polyethylene chains, are nearly cancelled in the isotropic spectra, showing that the orientation-insensitive spectrum adequately represents the molecular conformation without contributions of orientation. Second, spectra of silk fibroins have been compared in the amide I region for Bombyx mori cocoon silk fibers and methanol-treated regenerated fibroin films. The similarity of the shape of the amide I band of the isotropic spectra indicates that the secondary structure of the fibroins is very close in both samples. These experimental results support the conclusion that the molecular conformation can be efficiently characterized from the intensity and the shape of Raman bands in the orientation-insensitive spectrum.

Animals↗

PubMed1999

Fluorescent Transgenic Silkworm.

The replacement of fibroin heavy chain gene in silkworm by site directed homologous recombination was studied The DNA fragment consisting of an IE promoter driving green fluorescent protein (GFP) gene as reporter flanked by pieces of the 5' and 3' sequences of the fibroin heavy chain gene of silkworm at two sides was transferred into silkworm eggs by electroporation Green fluorescent flecks were seen on three silkworms fifth instar among five thousand silkworms under UV light PCR analysis proved that the GFP gene was integrated into the genome of silkworm Southern hybridization of genomic DNA of one transgenic silkworm showed that the fibroin heavy chain gene was successfully knocked out and replaced by the reporter gene The transgenic silkworms could grow up to the fifth instar as normal but they could not spin silk while control ones do.

Journal Article↗

PubMed1976

[Glycine and alanine synthesis enzymes in the tissues of the silkworm during its development].

The activity of enzymes of glycine and alanine synthesis (glutamate-pyruvate aminotransferase, aspartate-beta-decarboxylase, threonine aldolase, serine hydroxymethyltransferase, alanine-glyoxylate aminotransferase, aspartate aminotransferase) is studied in haemolymph, fat body, fibroin and sericine divisions of silk gland of silkworm Bombyx mori at terminal period of larva development. Alanine-glyoxylate aminotransferase activity in fibroin division of silk gland (34,6 mu mole of glycine/mg of protein/min-10(-3)), alanine aminotransferase--in sericine division (36,0 mu mole of alanine/mg of protein/min-10(-3)) aspartate aminotransferase 27,3 mu mole of glutamic acid/mg of protein/min-10(-3)) and alanine aminotransferase (35,8 mu mole of alanine/mg of protein/min-10(-3)) on fat body. The ratio of alanine-glyoxylate aminotransferase/glutamate-pyruvate aminotransferase activities in posterior division of silk gland is near to glycine/alanine ratio in silk fibroin. The character of the enzymes activity in silkworm tissues correlates with the silk formation rate.

Alanine Transaminase↗

PubMed2006

Covalently immobilized enzyme gradients within three-dimensional porous scaffolds.

Horseradish peroxide (HRP) was covalently coupled to three-dimensional (3D) silk fibroin scaffolds using water-soluble carbodiimide. Stable, bilaterally symmetrical immobilized HRP gradient patterns were generated within 3D silk fibroin scaffolds using the principles of diffusion. Gradients of immobilized HRP activity were controlled using variables of volume and concentration of HRP solution activated by the carbodiimide. The method developed can be extended to immobilize a variety of proteins and small molecules on several types of porous, interconnected materials. This technique of patterning enzymes and proteins in a gradient manner offers new options in the field of chemotaxis, tissue engineering, and biosensors.

Animals↗

PubMed2003

Production of interferon-beta by fibroblast cells on membranes prepared with RGD-containing peptides.

The production of interferon-beta by NB1-RGB fibroblast cells cultured on protein and peptide membranes prepared from silk fibroin, motif peptides of silk fibroin [(AG)(n)] containing arginine-glycine-aspartic acid (RGD) peptide, and Pronectin was investigated. The cell density on various protein and peptide membranes was approximately the same, although the production of interferon-beta depended significantly on the membranes where the cells were cultured. The highest production of interferon-beta was observed when the cells were cultured on (AG)(6)RGD(AG)(7) membranes prepared with hexafluoroacetone (HFA) as the casting solvent. On RGD-containing peptide membranes more centrally located in the peptides, the cells produced more interferon-beta when the peptide membranes were prepared with HFA as the casting solvent. However, there was no enhanced production of interferon-beta by cells on (AG)(6)RGD(AG)(7) membranes prepared with 9 mol/L LiBr or 4.5 mol/L LiClO(4) solution as the casting solvent. Therefore, both the chemical composition and the secondary and higher order structure of the peptide membranes are important for enhanced production of interferon-beta. The blocking of integrin beta(1) on the cells by anti-integrin beta(1) antibody prevented the enhanced production of interferon-beta on (AG)(6)RGD(AG)(7) membranes prepared with HFA. We suggest that the cells must bind to the RGD sequence having the appropriate conformation through their integrin beta(1) for enhanced production of interferon-beta.

Amino Acid Sequence↗

PubMed1990

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.

Animals↗

PubMed2006

Osteogenesis by human mesenchymal stem cells cultured on silk biomaterials: comparison of adenovirus mediated gene transfer and protein delivery of BMP-2.

Bone tissue engineering, gene therapy based on human mesenchymal stem cells (MSCs) and silk fibroin biomaterials were combined to study the impact of viral transfection on MSC osteogenic performance in vitro. MSCs were transduced with adenovirus containing a human BMP-2 (Ad-BMP-2) gene at clinically reasonable viral concentrations and cultured for 4 weeks. Controls with nontransfected MSCs, but exposed to exogenous BMP-2 concentrations on an analogous time profile as that secreted by the Ad-BMP-2 group, were compared. Both the Ad-BMP-2 MSC group and the exogenous protein BMP-2 group strongly expressed osteopontin and bone sialoprotein. Cells secreted a matrix that underwent mineralization on the silk fibroin scaffolds, forming clusters of osseous material, as determined by micro-computed tomography. The expression of osteogenic marker proteins and alkaline phosphatase was significantly higher in the Ad-BMP-2 MSC group than in the exogenous protein BMP-2 group, and no significant differences in mineralization were observed in two of the three MSC sources tested. The results demonstrate that transfection resulted in higher levels of expression of osteogenic marker genes, no change in proliferation rate and did not impact the capacity of the cells to calcify tissues on these protein scaffolds. These findings suggest additional options to control differentiation where exogenous additions of growth factors or morphogens can be replaced with transfected MSCs.

Adenoviridae↗

PubMed2004

Frictional properties of regenerated cartilage in vitro.

Although tribological function is the most important mechanical property of articular cartilage, few studies have examined this function in tissue-engineered cartilage. We investigated changes in the frictional properties of cartilage regenerated from the inoculation of rabbit chondrocytes into fibroin sponge. A reciprocating friction-testing apparatus was used to measure the friction coefficient of the regenerated cartilage under a small load. The specimen was slid against a stainless steel plate in a water vessel filled with physiological saline. The applied load was 0.03 N, the stroke length was 20 mm, and the mean sliding velocity was 0.8 mm/s. The friction coefficient of the regenerated cartilage decreased with increasing cultivation time, because a hydrophilic layer of synthesized extracellular matrix was formed on the fibroin sponge surface. The friction coefficient of the regenerated cartilage was as low as that of natural cartilage in the early stages of the sliding tests, but it increased with increasing duration of sliding owing to exudation of interstitial water from the surface layer.

Animals↗

PubMed2006

Silk microspheres for encapsulation and controlled release.

A method was developed to prepare silk fibroin microspheres using lipid vesicles as templates to efficiently load protein drugs in active form for controlled release. The lipid was subsequently removed by methanol or sodium chloride treatments, resulting in silk microspheres consisting of beta-sheet structure and about 2 mum in diameter. NaCl treated microspheres had smoother surfaces compared to the methanol treatments based on SEM analysis, and both types of microspheres had a mixture of multilamellar and unilamellar structures. A model protein drug, horseradish peroxidase, was encapsulated in the microspheres. Freeze-thaw cycles during preparation led to higher loading of the peroxidase due to improved mixing between the silk and drug, while without this process the drug and silk remained in separate layers or domains in microspheres. This partitioning was determined with fluorescein-labeled silk and rhodamine-labeled dextran. Small molecules such as the enzyme substrate 3,3',5,5'-tetramethylbenzidine, Mw=240 Da, and its oxidized product freely diffused through the MeOH- and NaCl-processed silk microspheres so that enzyme loading and activity could be determined. Enzyme activity was retained during processing and in the final microspheres. The enzyme release profile depended on the NaCl-process used in microsphere preparation. The physically cross-linked beta-sheet structure of silk fibroin and the residual lipids in the microspheres played important roles in controlling enzyme release profiles. The silk microspheres have the potential for diverse applications where controlled protein release from biocompatible, mechanically tough, and slowly biodegradable carriers is desirable.

Delayed-Action Preparations↗

PubMed2003

Modelling self assembly of natural silk solutions.

We present a tentative interpretation of the origin of nematic liquid crystalline order exhibited by various natural silk fibroin solutions, notably those of orb-weaving spiders and the domestic silkworm Bombyx mori. It is thought that liquid crystalline rheology is exploited during the spinning process. We discuss in this approach the response of the liquid crystalline phase diagram to equilibrium physiological conditions and to parameters characterising the amino acid sequence of the fibroin molecules. The phase diagram is sensitive in this latter respect to sequence mutations, such that it may constitute a source of evolutionary selection pressure.

Animals↗

PubMed2006

Spider egg case core fibers: trimeric complexes assembled from TuSp1, ECP-1, and ECP-2.

Spider silk proteins are well-known for their extraordinary mechanical properties, displaying remarkable strength and toughness. In this study, matrix-assisted laser desorption ionization (MALDI) tandem time-of-flight (TOF) mass spectrometry (MS/MS) and reverse genetics were used to isolate a new cDNA sequence that encodes for a protein assembled into egg case silk from the black widow spider, Latrodectus hesperus. Analysis of the primary sequence of this protein reveals approximately 52% identity to the egg case protein 1 (ECP-1) fibroin-like family member. On the basis of the similarity in the primary sequence and expression pattern, we have named this factor egg case protein 2 (ECP-2). Alignments of ECP-1 and ECP-2 demonstrate highly conserved N termini, with 16 Cys residues found within the first 153 amino acids. Traditional ensemble repeats found within reported fibroins were poorly represented in the primary sequence of ECP-2, but scattered blocks of polyalanine were present, along with a C terminus rich in GA repeats. Reverse transcription quantitative PCR analysis showed that ECP-2 is predominantly expressed in the tubuliform gland. Relative to ECP-1, ECP-2 mRNA levels were determined to be >2-fold higher. MALDI MS/MS analysis of peptide fragments generated from the large-diameter core fiber after enzymatic digestion and acid hydrolysis demonstrated the presence of a fiber that is trimeric in nature, containing tubuliform spidroin 1 (TuSp1), ECP-1, and ECP-2. We also report an additional primary sequence for TuSp1, demonstrating that TuSp1 contains two Cys residues within a nonrepetitive N-terminal region. In combination with the distinctive protein architectures of ECP-1 and ECP-2, along with their co-localization with TuSp1 in the core fiber, our findings suggest that ECP-1 and ECP-2 play important structural roles in the egg case silk fiber.

Amino Acid Motifs↗