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PubMedDate not supplied

Analysis of the conserved N-terminal domains in major ampullate spider silk proteins.

Major ampullate silk, also known as dragline silk, is one of the strongest biomaterials known. This silk is composed of two proteins, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2). Only partial cDNA sequences have been obtained for these proteins, and these sequences are toward the C-terminus. Thus, the N-terminal domains have never been characterized for either protein. Here we report the sequence of the N-terminal region of major ampullate silk proteins from three spider species: Argiope trifasciata, Latrodectus geometricus, and Nephila inaurata madagascariensis. The amino acid sequences are inferred from genomic DNA clones. Northern blotting experiments suggest that the predicted 5' end of the transcripts are present in fibroin mRNA. The presence of more than one Met codon in the N-terminal region indicates the possibility of translation of both a long and a short isoform. The size of the short isoform is consistent with the published, cDNA based, N-terminal sequence found in flagelliform silk. Analyses comparing the level of identity of all known spider silk N-termini show that the N-terminus is the most conserved part of silk proteins. Two DNA sequence motifs identified upstream of the putative transcription start site are potential silk fibroin promoter elements.

Amino Acid Sequence↗

PubMed2005

Use of microcoil probehead for determination of the structure of oriented silk fibers by solid-state NMR.

A microcoil probehead for solid-state NMR was developed with a two-channel radio-frequency circuit, and 13C observation with a proton-decoupling probehead was performed to obtain information on the distribution of the orientation of silk fibroin molecules in the fiber. The coil (1 mm (diameter) x 5 mm (length)) of the probehead was placed at the angles 90 degrees and 30 degrees , relative to the static magnetic field. Only 70 mug of [1-13C]Gly silk fibroin fiber was used in a magnet of 9.4 T (400 MHz for proton channel).

Alanine↗

PubMed1988

In vitro transcription of eukaryotic genes is affected differently by the degree of DNA supercoiling.

In a posterior silk gland extract, covalently closed circular (ccc) DNA is in a superhelical state that supports more transcription of fibroin gene than does linear DNA. A HeLa cell extract showed neither the supercoiling activity nor the preference for the transcription of ccc DNA over linear DNA. These activities could be added to the HeLa cell extract. Phosphocellulose fractionation of the posterior silk gland extract yielded a flow-through fraction and a 0.6 M KCl eluate fraction that were required for the supercoiling and for the efficient transcription of the ccc template in the acceptor HeLa cell extract. The 0.6 M KCl fraction had a DNA topoisomerase II activity, and the flow-through fraction contained a supercoiling factor that, with the aid of topoisomerase II, introduced negative supercoils into ccc DNA. When both fractions were added to the posterior silk gland extract, more supercoiling occurred than with the extract alone. Several genes were optimally transcribed under various extents of supercoiling. The fibroin gene and adenovirus 2 major late promoter were fully transcribed as partially supercoiled templates. The sericin gene required more supercoiling for full transcription, whereas no preference for supercoiling was seen with the transcription of hsp70. These results suggest that DNA topology plays a role in the regulation of gene expression.

Animals↗

PubMed2003

Synthesis and structural characterization of silk-like materials incorporated with an elastic motif.

Genetic engineering strategies were applied to synthesize silk-like materials, [(GVPGV)(2)GG(GAGAGS)(3)AS](n). The primary structure of these materials represents the repetitive crystalline region of Bombyx mori silk fibroins incorporated with an elastic motif selected from animal elastin. The oligonucleotides were designed to encode the desired recombinant proteins and then expressed in the Escherichi coli system. The expression and purification conditions for the production of the recombinant proteins were optimized. (13)C CP/MAS NMR was used for structural characterization in the solid state, where the isotope labeling was performed using a modified M9 medium. The secondary structures of these materials are primarily governed by the designated amino acid sequence, where the B. mori silk fibroin block, (GAGAGS)(3), tends to form the crystalline region, which is interrupted by the flexible (GVPGV)(2) block. The CD data suggested that the structure of these materials was length-dependent in the solution state, i.e., a higher molecule weight leads to a higher ordered structure.

Amino Acid Motifs↗

PubMed1999

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).

Animals↗

PubMed2004

Adhesion and function of rat liver cells adherent to silk fibroin/collagen blend films.

Collagen is often used in bioartificial livers as a biomimetic coating to promote liver cell adhesion and differentiation. Animal proteins are expensive and expose the host to risks of cross-species infection due to contamination with prions. Silk fibroin (SF) is a biocompatible protein produced by Bombyx mori silk worms and possibly an alternative to collagen. We prepared SF-collagen blend films with different SF content adherent to the bottom of standard tissue culture dishes, and characterized their surface morphology by SEM, their wettability and examined them for their capacity to support rat liver cell adhesion and metabolism. Cell metabolism was characterized by estimating the rate at which cells eliminated ammonia and synthesized urea for up to 48h of culture. SF-containing films were smooth, clear and more wettable than collagen. Cells readily adhered, formed junctions and small size aggregates on all films. As many cells adhered on SF as on collagen films. Cell adhesion to high collagen content blend films could not be reliably estimated because cells dwelt in the large cavities in the film. The effect of SF on cell metabolism differed with the investigated metabolic pathway. However, cells on SF-containing films eliminated ammonia and synthesized urea at rates generally comparable to, for urea synthesis at times higher than, that of cells on collagen. These results suggest that silk fibroin is a suitable substratum for liver cell attachment and culture, and a potential alternative to collagen as a biomimetic coating.

Animals↗

PubMed2000

The effects of Pva/chitosan/fibroin (PCF)-blended spongy sheets on wound healing in rats.

The effects of poly vinyl alcohol (PVA)/Chitosan/Fibroin (PCF)-blended sponge on wound healing in rats were investigated. We excised the skin of a rat, including the dermis, approximately 2 x 2 cm in size. The wound was covered with PCF-blended spongy sheets. The spongy sheets absorbed the exudate, and gained flexibility and softness. Histopathological inspection of the wound 12 d later showed an increase of vascular ingrowth and the absence of inflammatory cells. Regeneration of the skin around the wound was faster than that of the control. We also tested wound healing effects of PVA, Chitosan and Fibroin, alone or in various combinations. Wound healing was accelerated in the order of PCF-blended sponge>Chitosan/Fibroin (CF)-blended sponge>Fibroin (F) sponge > PVA/Chitosan-blended sponge (PC) > Chitosan (C) sponge.

Animals↗

PubMed1981

[tRNA adaptation and the optimization of translation].

The intracellular level of each tRNA species is adjusted to the codon frequency of the mRNA being decoded. This was first observed in such highly differentiated cells as the silk gland of Bombyx mori, which produces fibroin and sericin, and the rabbit reticulocyte. tRNA adaptation also occurs in other cell types from E. coli to mammalian cells. Regardless of the mechanism regulating tRNA biosynthesis, we believe that tRNA adaptation is the basic step optimizing chain elongation at the ribosomal level. We propose the system of trial and error as a working model for the ribosome. This model clarifies the correlations between iso-accepting tRNA levels and codon frequencies, as well as the effect of tRNA pool balance on mean elongation rate and non-uniform individual elongation rate (depending on whether codons are rare or abundant) for fibroin mRNA translated in a reticulocyte cell-free system.

Adaptation, Physiological↗

PubMed1995

Control of expression of silk protein genes.

At least three silk genes are specifically expressed in the posterior, and five other genes in middle, silk glands. The products of genes active in PSG include fibroin, L-chain fibroin and P25 protein. PSG genes as well as the Ser-1 gene, differing in structure, exhibit a striking degree of homology of their 5' flanking sequences. This suggests the presence of common regulatory mechanisms. The expression of silk protein genes is probably controlled by tissue-specific and general transcriptional factors. Hormones seem to participate in the regulation of expression of silk protein genes.

Animals↗

PubMed2006

Tyrosinase-catalyzed grafting of sericin peptides onto chitosan and production of protein-polysaccharide bioconjugates.

The capability of Agaricus bisporus tyrosinase to catalyze the oxidation of tyrosine residues of silk sericin was studied under homogeneous reaction conditions, by using sericin peptides purified from industrial wastewater as the substrate. Tyrosinase was able to oxidize about 57% of sericin-bound tyrosine residues. The reaction rate was higher than with silk fibroin, but lower than with other silk-derived model peptides, i.e. tryptic and chymotryptic soluble peptide fractions of silk fibroin, suggesting that the size and the molecular conformation of the substrate influenced the kinetics of the reaction. The concentration of tyrosine in oxidized sericin samples decreased gradually with increasing the enzyme-to-substrate ratio. The average molecular weight of sericin peptides significantly increased by oxidation, indicating that cross-linking occurred via self-condensation of o-quinones and/or coupling with the free amine groups of lysine and, probably, with sulfhydryl groups of cysteine. The high temperature shift of the main thermal transitions observed in the differential scanning calorimetry curves confirmed the formation of peptide species with higher molecular weight and higher thermal stability. Fourier transform-infrared spectra of oxidized sericin samples showed slight changes related to the loss of tyrosine and formation of oxidation products. Oxidized sericin peptides were able to undergo non-enzymatic coupling with chitosan. Infrared spectra provided clear evidence of the formation of sericin-chitosan bioconjugates under homogeneous reaction conditions. Spectral changes in the NH stretching region seem to support the formation of bioconjugates via the Michael addition mechanism.

Agaricus↗

PubMed1998

Action of estradiol-17beta on the synthetic activity of the silk gland in Bombyx mori L.

The effects of estradiol-17beta (E(2)) were studied on several metabolic parameters in the silk gland of Bombyx mori L. race Nistari. Topical application of different doses (0.05-4.0&mgr;g/g body weight) of E(2) on the first and second day of the fifth instar larvae showed a dose dependent effect when studied on the fifth day. A significant increase in silk gland weight and fibroin content was observed between the doses 0.05 and 0.1, and 0.1 and 1.0&mgr;g/g of E(2). A similar pattern of dose-dependent rise in DNA and RNA content of posterior silk gland (PSG) was observed with the doses of E(2) when the contents were expressed per pair of PSG. Higher doses of E(2) (2.0 or 4.0&mgr;g/g) demonstrated relatively less increase, unchanged level or a decrease in the above parameters in comparison to the control values. The glutamate-pyruvate transaminase of PSG showed a significant increase from 0.1 to 2.0&mgr;g/g of E(2) doses in comparison to the control value. Simultaneous injection of ICI-182780 (1.0&mgr;g/g), a very pure and specific antiestrogenic compound, with E(2) (1.0&mgr;g/g) caused a significant counteraction of E(2)-induced increase in silk gland activity, which was reflected in DNA and RNA content of PSG, wet weight and fibroin content of silk gland, and on glutamate-pyruvate transaminase activity. Cycloheximide (0.5&mgr;g/g), a protein synthesis blocker, caused a significant inhibition of the E(2) (1.0&mgr;g/g)-induced silk gland activity when treated along with estradiol. From this study it appears that estradiol has a specific effect on silk gland function and that it may act in a nuclear mediated way.

Journal Article↗

PubMedDate not supplied

Silk I structure in Bombyx mori silk foams.

Single crystals of Bombyx mori silk fibroin in the metastable silk I polymorph have been produced using a new foaming technique. Foams of silk protein are generated by bubbling pure nitrogen gas through an aqueous solution of regenerated silk fibroin. The foamed material is collected, dried, and then sonicated to yield individual crystals which were examined using transmission electron microscopy and electron diffraction. It is found that slightly acidic conditions in the solution from which the foam was generated favor the formation of silk II while neutral to slightly basic solutions favor silk I formation. More dilute solutions favor the formation of silk II while more concentrated solutions (about 7 wt.% or greater) favor the formation of silk I. X-ray powder diffraction patterns from the dried silk I foams displayed features highly indicative of silk I. We also report the first single crystal electron diffraction patterns of silk I. These patterns indicate a large unit cell, possibly 22.66 x 5.70 x 20.82 A. with six chains of six residues, Gly-Ala-Gly-Ala-Gly-Ser. Although we have not fully characterized this complex structure it appears that the chain is nearly fully extended and thus our data is consistent with models possessing general features similar to those proposed by Fossey SA, Nemethy G, Gibson KD, Scheraga HA. (Biopolymers 1991;31:1529-1541).

Animals↗

PubMedDate not supplied

Molecular orientation behavior of silk sericin film as revealed by ATR infrared spectroscopy.

This paper reports the structure-dependent molecular orientation behavior of sericin, an adhesive silk protein secreted by silkworm, Bombyx mori. Although application of sericin as a biomaterial is anticipated because of its unique characteristics, sericin's physicochemical properties remain unclear, mainly because of its vulnerability to heat or alkaline treatment during separation from fibroin threads. This study employed intact sericin obtained from fibroin-deficient mutant silkworm to investigate the relationship between molecular orientation and the secondary structure of sericin. Sericin films were artificially stretched after moistening with aqueous ethanol of various concentrations. The resulting molecular orientation was analyzed using polarized infrared spectroscopy. These analyses indicated that formation of aggregated strands among extended sericin chains induced by ethanol treatment is the key to generating molecular orientation. Strong intermolecular hydrogen bonds are inferred to allow aggregated strands' stretching-force transmission, thereby causing molecular orientation.

Animals↗

PubMed2003

Mechanism of silk processing in insects and spiders.

Silk spinning by insects and spiders leads to the formation of fibres that exhibit high strength and toughness. The lack of understanding of the protein processing in silk glands has prevented the recapitulation of these properties in vitro from reconstituted or genetically engineered silks. Here we report the identification of emulsion formation and micellar structures from aqueous solutions of reconstituted silkworm silk fibroin as a first step in the process to control water and protein-protein interactions. The sizes (100-200 nm diameter) of these structures could be predicted from hydrophobicity plots of silk protein primary sequence. These micelles subsequently aggregated into larger 'globules' and gel-like states as the concentration of silk fibroin increased, while maintaining solubility owing to the hydrophilic regions of the protein interspersed among the larger hydrophobic regions. Upon physical shearing or stretching structural transitions, increased birefringence and morphological alignment were demonstrated, indicating that this process mimics the behaviour of similar native silk proteins in vivo. Final morphological features of these silk materials are similar to those observed in native silkworm fibres.

Animals↗

PubMed1998

Identification of a novel type of silk protein and regulation of its expression.

The silk of lepidopteran insects has been studied extensively as proteins of two categories: the fibroins, which are produced in the posterior section of silk glands, and the sericins, which are secreted in the middle section. We now describe a third category that is named seroins to accentuate the fact that both the sericin- and the fibroin-producing cells participate in seroin secretion. Using a probe derived from the N-terminal sequences of a 23-kDa components of Galleria mellonella silk, we isolated silk gland-specific cDNA encoding 167 amino acids, of which 17 constitute the signal peptide. The following 14 residues match the N-terminal sequences of the 23- and 22.5-kDa silk proteines. The reaction of these proteins with concanavalin A and the presence of two glycosylation sites in the seroin peptide sequence indicate that seroin is secreted in two forms that both contain a mannose-rich sugar moiety. Seroin is distinguished from other silk proteins by high proline content (34 residues or 20.26% by weight), lack of cysteines, and the presence of two kinds of short amino acid repeats. The seroin gene is expressed in both the posterior and middle silk gland sections. The expression fluctuates during development in correlation with the feeding regime and the changes in hormone titers: seroin mRNA is high in the silk glands of feeding larvae, declines at ecdysis, reaches a maximum during cocoon spinning, and thereafter rapidly drops to an undetectable level. In vivo and in vitro experiments showed that the drop is caused by ecdysteroid hormones and is prevented by juvenile hormones. N-terminal sequencing of several silk proteins of Bombyx mori revealed that the 8- and 13-kDa proteins share 5 or 6 out of 10 identified amino acids with the N terminus of Galleria seroin and obviously represent seroin homologues. The result suggests that seroin-type proteins are a general component of lepidopteran silk.

Amino Acid Sequence↗

PubMed2001

Cryoprotective effect of the serine-rich repetitive sequence in silk protein sericin.

The silk proteins, fibroin and sericin, are produced in the silk gland of Bombyx mori, and hydrophilic sericin envelops fibroin with successive sticky layers in the formation of a cocoon. To study the biological functions of sericin, we focused on the serine-rich sericin peptide consisting of 38 amino acids, which is a highly conserved and internally repetitive sequence of a sericin protein. The corresponding gene was chemically synthesized, and the PCR-amplified gene was ligated to oligomerize sericin peptide and fused at the amino terminus to a His-tagged and proteolytic cleavage sequence in an inducible expression vector. When the dimers of sericin peptides were overexpressed in Escherichia coli, the transformants showed a prominent increase in cell viability after freezing in medium. Further, the purified dimeric sericin peptide from E. coli was found to be effective in protecting lactate dehydrogenase from denaturation caused by freeze-thaw. Both of these protective effects against freezing stress in cells and proteins were also observed with sericin hydrolysate. These results indicate that this unique sericin peptide, like sericin, has a high cryoprotective activity and will be valuable as a new biomaterial for industrial use.

Amino Acid Sequence↗

PubMed2003

Evaluation of dynamic visco-elastic properties during cartilage regenerating process in vitro.

The dynamic visco-elastic properties of regenerated cartilage tissue were measured to evaluate its mechanical function during cultivation. Harvested chondrocytes from 4-week-old Japanese white rabbits were inoculated into fibroin sponge at a cell concentration of about 5 x 10(7) cells/ml. Dynamic visco-elasticity measurements were performed under compressive loading to evaluate the load bearing function of the articular cartilage. The dynamic modulus and the dynamic loss of the regenerated cartilage increased and the peak value of tandelta, as well as the frequency at the peak, decreased with increasing cultivation time. The pores of the fibroin sponge became filled with newly formed tissue as cultivation time increased. These changes in the visco-elastic properties of the regenerated cartilage were compared with those of a model system, ethylene propylene diene monomer sponge with interstitial fluid, and appear to be a result of increased fluid flow resistance and internal loss. We conclude that the changes in the dynamic visco-elastic properties of the regenerated cartilage were caused because of narrowing of the fluid path by synthesized extracellular matrix.

Animals↗

PubMed2005

[Immobilization of von Willebrand factor antibody on solid host membranes].

This study aimed at immobilizing the antibodies on the surfaces of the solid host membranes in order to improve the property of the biomaterial. The von Willebrand factor (vWf) antibodies were immobilized on the surface of Bombyx mori silk fibroin and PLA (Poly Lactic Acid) membrane by NH3 plasma treatment followed by covalent cross-linking reaction. The immobilization efficiency was evaluated by two methods including the antibody surplus and enzyme-linked immunosorbent assay (ELISA). The in vitro antithrombogenicity representing the activity of immobilized vWf antibody was determined by the method of Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT) and Thrombin Time (TT) test. The results demonstrate that the vWf antibodies are immobilized on silk fibroin and PLA membranes in an efficient way with the efficiency of antibody surplus up to 23.88% and ELISA reaction is positive. APTT and TT exceeded the upper limits distinctly, but the value of PT did not change noticeably. The in vitro antithrombogenicity represented the is activity-retaining form of antibodies. These results extend the application of antibody immobilization technique and provide a new idea about the design of biomaterials relating to the coagulation factors.

Antibodies↗