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PubMed2001

The natural silk spinning process. A nucleation-dependent aggregation mechanism?

The spinning mechanism of natural silk has been an open issue. In this study, both the conformation transition from random coil to beta sheet and the beta sheet aggregation growth of silk fibroin are identified in the B. mori regenerated silk fibroin aqueous solution by circular dichroism (CD) spectroscopy. A nucleation-dependent aggregation mechanism, similar to that found in prion protein, amyloid beta (Abeta) protein, and alpha-synuclein protein with the conformation transition from a soluble protein to a neurotoxic, insoluble beta sheet containing aggregate, is a novel suggestion for the silk spinning process. We present evidence that two steps are involved in this mechanism: (a) nucleation, a rate-limiting step involving the conversion of the soluble random coil to insoluble beta sheet and subsequently a series of thermodynamically unfavorable association of beta sheet unit, i.e. the formation of a nucleus or seed; (b) once the nucleus forms, further growth of the beta sheet unit becomes thermodynamically favorable, resulting a rapid extension of beta sheet aggregation. The aggregation growth follows a first order kinetic process with respect to the random coil fibroin concentration. The increase of temperature accelerates the beta sheet aggregation growth if the beta sheet seed is introduced into the random coil fibroin solution. This work enhances our understanding of the natural silk spinning process in vivo.

Animals↗

PubMed1976

Studies on the posterior silk gland of the silkworm Bombyx mori. VI. Distribution of microtubules in the posterior silk gland cells.

There are two microtubule systems in the posterior silk gland cells. One is a radial microtubule system in which the microtubules run radially from the basal to the apical cytoplasm and in which fibroin globules (secretory granules of fibroin) and mitochondria are arranged along these microtubules, thus composing a "canal system" which is assumed to be responsible for the intracellular transport of fibroin globules. The other is a circular microtubule system in the apical cytoplasm which is composed of bundles of microtubules and microfilaments running in a circular arrangement around the glandular lumen at an interval of approximately 4 mum at the end of the fifth instar. This system is presumably concerned with secretion and/or intraluminal transport of fibroin.

Animals↗

PubMed1981

[Gene expression in Bombyx mori silkgland cells during the last larval instar].

Poly (A)-containing RNA was isolated from the posterior silkgland of Bombyx mori just after the fourth molt, when the gland is not producing fibroin (stage V0), and at the middle of the fifth larval instar, when the fibroin is massively synthesized (stage V6). The hybridization kinetics of these mRNA with their complementary DNA (cDNA) were remarkably different. The mRNA from stage V0 consisted of two abundance classes, comprising 38 and 2 915 different species, respectively. At stage V6, fibroin mRNA (FmRNA) was separated from the rest of the poly (A)-containing RNA (F-mRNA) by centrifugation on sucrose gradients, and both preparations were analyzed separately. The kinetic complexity of FmRNA was very low as compared to its actual size. This result agrees with the existence of a short repetitive sequence accounting for 60 p. 100 of the molecule. Stage V6 F-mRNA was resolved into four classes containing 1, 20, 319 and about 2 600 species, respectively. We carried out cross-hybridization with each of the two cDNA classes from stage V0 and stage V6 F-mRNA. They demonstrated that almost all the sequences present at stage V0 were also present at stage V6, but that their abundance class distribution was modified. Our data show that the specialization of the silkgland cell for fibroin production is characterized by massive accumulation of FmRNA and another unknown species, and that stage variations of mRNA populations are related to relative quantitative variations rather than to qualitative ones.

Animals↗

PubMed2000

Small ampullate glands of Nephila clavipes.

The small ampullate glands of the orb-web spider, Nephila clavipes, have been studied and compared to other of the silk producing glands from this organism. They exhibit the same gross morphological features of the other glands. Electrophoretic analyses show that the gland's luminal contents migrate as a single band, while the contents of the secretory epithelium reveal a step-ladder array of peptides in addition to the full size product. Previous studies from our laboratory identified these peptides as products generated by translational pauses. This alternate mode of translation is typical of fibroin synthesis in all the spider glands thus far studied as well as in those of the silkworm. The correlation of the peptides to the process of fibroin synthesis is shown through experimental evidence in this paper. The gradual ultrastructural changes in Golgi vesicles elicited by the fibroin synthesis stimulus can be seen in this paper. The response to stimulation is of a higher magnitude in these glands than in any of those previously analyzed. These studies show the small ampullate glands are a promising and certainly exploitable model system for studies on the synthesis of tissue-specific protein product and its control. J. Exp. Zool. 286:114-119, 2000.

Animals↗

PubMed2006

Molecular mechanisms of spider silk.

Spiders spin high-performance silks through the expression and assembly of tissue-restricted fibroin proteins. Spider silks are composite protein biopolymers that have complex microstructures. Retrieval of cDNAs and genomic DNAs encoding silk fibroins has revealed an association between the protein sequences and structure-property relationships. However, before spider silks can be subject to genetic engineering for commercial applications, the complete protein sequences and their functions, as well as the details of the spinning mechanism, will require additional progress and collaborative efforts in the areas of biochemistry, molecular biology and material science. Novel approaches to reveal additional molecular constituents embedded in the spider fibers, as well as cloning strategies to manipulate the genes for expression, will continue to be important aspects of spider biology research. Here we summarize the molecular characteristics of the different spider fibroins, the mechanical properties and assembly process of spidroins and the advances in protein expression systems used for recombinant silk production. We also highlight different technical approaches being used to elucidate the molecular constituents of silk fibers.

Amino Acid Motifs↗

PubMed2005

Biosynthesis and cocoon-export of a recombinant globular protein in transgenic silkworms.

A gene construct was made by fusing the coding sequence of the red fluorescent protein (DsRed) to the exon 2 of the fibrohexamerin gene (fhx), that encodes a subunit of fibroin, the major silk protein of the silkworm Bombyx mori. The fusion gene was inserted into a piggyBac vector to establish a series of transgenic lines. The expression of the transgene was monitored during the course of larval life and was found restricted to the posterior silk gland cells as the endogenous fhx gene, in all the selected transgenic lines. The exogenous polypeptide was secreted into the lumen of the posterior silk gland together with fibroin, and further exported with the silk proteins as a foreign constituent of the cocoon fiber. The capacity of DsRed to emit fluorescence in the air-dried silk thread led to show that the recombinant protein was distributed over the whole length of the fiber. A remarkable property of the system lies in the localization of the globular protein at the periphery of the silk thread, allowing its rapid and easy recovery in aqueous solutions, without dissolving fibroin. The procedure represents a novel and promising strategy for the production of massive recombinant proteins of biomedical and pharmaceutical interest, with reduced cost.

Animals↗

PubMed1988

Temporal and spatial control of silk gene transcription analyzed by nuclear run-on assays.

In vivo transcriptional activity of the Bombyx mori fibroin and sericin genes was estimated by nuclear run-on assay. Tissue-specific and developmental stage-specific expression of the fibroin and sericin genes through larval development was shown to be primarily regulated at the level of transcription. The expression patterns of these two genes are quite different in their developmental profile, suggesting that they are probably regulated by different underlying mechanisms. A finer spatial control of transcription could be found. The sericin gene transcription is mostly restricted to the posterior region of the middle silk gland throughout the fifth larval instar. Interestingly, the fibroin gene transcription is observed only in the anterior region of the posterior silk gland on the first day of the fifth instar, and spreads toward the posterior region as the fifth instar stage proceeds.

Age Factors↗

PubMed1983

Lifespan and protein synthesis in several Bombyx mori mutants with genetic abnormalities in amino acid and protein metabolism.

Three kinds of "white eggs" and red-blooded Bombyx mutants (W1, W2, W3 and rb) which lack enzymes in tryptophan metabolism showed almost no difference in their lifespans from that of normal stock. However, other Bombyx mutants, such as Nd, Nd-s and DES-Nd, unable to synthesize fibroin, had shorter lifespans than normal. Among these fibroin-deficient mutants, only the lifespan of DES-Nd was similar to the normal. However, further analysis of the amino acid composition in the cocoon of the mutant showed that its lifespan was also dependent on its ability to synthesize fibroin-like protein. It is very likely that, different from abnormalities in amino acid metabolism, a genetic abnormality in protein metabolism affects the maintenance of adult life in silkmoths and this results in the short lifespan. Generally speaking, an abnormality in amino acid metabolism seems to have no influence on silkmoth lifespan. However, an abnormality in protein metabolism results in shortening lifespan. The greater the abnormality in protein metabolism possessed by a silkmoth, the shorter the lifespan.

Aging↗

PubMed2005

Influence of macroporous protein scaffolds on bone tissue engineering from bone marrow stem cells.

The aim of this study was to investigate the effect of three-dimensional silk fibroin scaffold preparation methods (aqueous and solvent) on osteogenic responses by human bone marrow stem cells (hMSCs). Macroporous 3D protein scaffolds with similar sized pores of 900+/-50 microm were prepared either by an organic solvent process (hexafluoro-2-propanol, HFIP) or an aqueous process. hMSCs were expanded, seeded on the scaffolds, and cultured up to 28 days under static conditions in osteogenic media. hMSCs seeded onto the water-based silk scaffolds showed a significant increase in cell numbers (p<0.01) vs. the HFIP-prepared silk scaffolds. Significantly higher (p<0.01) alkaline phosphatase (ALPase) activity and calcium deposition were apparent after 28 days of culture in the water-based silk scaffolds when compared to the HFIP-derived silk scaffolds. Transcript levels for collagen type I (Col I), ALP, and osteopontin (OP) increased (p<0.05) in the water-based silk scaffolds in comparison to the HFIP-derived materials. At early stages of culture, increased expression of OP and collagen type II (Col II) were also observed in both scaffolds. Expression of Col II, MMP 13, Col I, and OP proteins increased in the water-based silk scaffolds in comparison to the HFIP-derived scaffolds while bone sialoprotein (BSP) proteins increased in the HFIP-derived silk scaffolds in comparison to the water-based scaffolds after 28 days of culture. Histological analysis showed the development of bone-like trabeculae with cuboid cells in an extracellular matrix (ECM) in the water-based silk scaffolds with more organization than in the HFIP-derived material after 28 days of culture. Alcian blue staining demonstrated the presence of proteoglycan in the ECM formed in the water-based scaffolds but not in the HFIP-prepared silk scaffolds. The results suggest that macroporous 3D aqueous-derived silk fibroin scaffolds provide improved bone-related outcomes in comparison to the HFIP-derived systems. These data illustrate the importance of materials processing on biological outcomes, as the same protein, silk fibroin, was used in both preparations.

Bone Substitutes↗

PubMed1999

Spectroscopic investigation of tertiary fold of staphylococcal protein A to explore its engineering application.

Staphylococcal protein A is a cell wall constituent of most strains of Staphylococcus aureus, and it is characterized by its binding affinity to some immunological classes. A mutated low molecular weight type protein A (LPA; Mwt = 27 kDa) which consists of the domains, E, D, A, B and 13 residues of the C-domain was prepared in this study. Since LPA does not possess a cell wall-bound region in contrast to wild-type protein A (WPA; Mwt = 42 kDa), we have established a methodology of large scale purification of LPA without using any extracellular expression systems such as Escherichia coli. Using this relatively abundant protein, the immobilization of the LPA with silk fibroin of Bombyx mori was performed. Thermal stability of LPA immobilized with silk fibroin is higher than that of free LPA at high temperature judging from the immunoglobulin G (IgG)-binding affinity. However, the apparent value of its affinity decreased relative to that of immobilized WPA. These results indicate that structural information is essential to explore improvement of IgG-binding affinity of immobilized LPA. Therefore, secondary structure of free LPA was detected by its characteristic helical pattern in circular dichroism (CD) in aqueous solution. In addition to this, tertiary fold of four IgG-binding domains were investigated by two-dimensional 1H-NMR spectra. Four significantly high-field shifted cross-peaks attributed to methyl signals of alanine residues suggest that all four domains pack into a three helix bundle motif in solution. These structural data and properties of IgG-binding affinity suggest that spatial arrangement of four IgG-binding domains are packed into a compact globular molecular shape. This causes a certain active site of immobilized LPA to be buried in the silk fibroin fiber.

Alanine↗

PubMed1975

The Bombyx mori silk proteins: characterization of large polypeptides.

Proteins taken directly from the Bombyx mori silk gland have been separated and identified as either fibroin or sericin on the basis of their location within the gland and their amino acid composition. Molecular weights of these polypeptides have been determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and by agarose-guanidine chromatography. Fibroin consists of approximately equimolar amounts of two large (350,000) polypeptide chains. These may be the products of distinct fibroin alleles present in hybrid silkworm strains. Sericin, on the other hand, is composed of at least the three largest polypeptides (130,000-220,000) present in a mixture of proteins ranging in size from about 20,000 to 220,000.

Alleles↗

PubMed2007

Some observations on the structure and function of the spinning apparatus in the silkworm Bombyx mori.

Silkworm silk has outstanding mechanical properties despite being spun at room temperature and from aqueous solution. Although it has been proposed that fiber formation is mainly induced by shearing and extensional flow in the spinneret, the detailed structure and function of the spinning apparatus of Bombyx mori silkworms are still not fully elucidated. In this paper we describe three aspects of the functional microanatomy of the spinning apparatus: changes in the diameter of the silk gland duct with distance along the duct, how the birefringence of the fibroin changes as it flows down the duct, and the detailed three-dimensional structure of the silk press and related structures. The existence of a double escaped nematic liquid crystal texture in the fibroin in a region of the duct is described. After this region the birefringence suddenly disappeared until the start of an internal draw down taper which commenced just before the silk press. In the internal draw down taper the birefringence increased dramatically to an asymptotic value as a thread was drawn from the fibroin gel. The structure of the silk press suggests that it acts as a restriction die whose diameter can be regulated.

Animals↗

PubMed2002

Transgenic silkworms produce recombinant human type III procollagen in cocoons.

We describe the generation of transgenic silkworms that produce cocoons containing recombinant human collagen. A fusion cDNA was constructed encoding a protein that incorporated a human type III procollagen mini-chain with C-propeptide deleted, a fibroin light chain (L-chain), and an enhanced green fluorescent protein (EGFP). This cDNA was ligated downstream of the fibroin L-chain promoter and inserted into a piggyBac vector. Silkworm eggs were injected with the vectors, producing worms displaying EGFP fluorescence in their silk glands. The cocoons emitted EGFP fluorescence, indicating that the promoter and fibroin L-chain cDNAs directed the synthesized products to be secreted into cocoons. The presence of fusion proteins in cocoons was demonstrated by immunoblotting, collagenase-sensitivity tests, and amino acid sequencing. The fusion proteins from cocoons were purified to a single electrophoretic band. This study demonstrates the viability of transgenic silkworms as a tool for producing useful proteins in bulk.

Animals↗

PubMed1980

Isolation of the smallest component of silk protein.

Silk proteins were solubilized from cocoons with ethylenediamine/cupric hydroxide solution. A series of polymers of the smallest component, detected by polyacrylamide-gel electrophoresis, could be converted into the smallest component by reduction and aminoethylation. Fibroin and sericin fractions were separated by precipitation of sericin at pH 5.5. On gel electrophoresis, sericin showed distinct bands but fibroin did not. The components of fibroin and sericin were fractionated by gel filtration on Sepharose 6B. The smallest component in the sericin fraction was purified by rechromatography and showed a single band on gel electrophoresis. Its mol. wt. was 24 000, and its amino acid composition was determined.

Amino Acids↗

PubMed2003

Construction of targeting vector and expression of green fluorescent protein in the silkworm, Antheraea pernyi.

The transcription initiation site of Anrheraea pernyi was identified, and a gene targeting vector was constructed. The green fluorescent protein (GFP) gene was inserted into this vector under the control of a fibroin promoter. This recombinant vector was used to target the GFP gene to the fibroin region of the silkworm genome. The DNA was injected into the testes during the pupae, and was also transferred into the egg via the sperm during fertilization. The analysis showed that the GFP gene was integrated into the fibroin gene on the genome by homologous recombination, and was expressed in the silk gland.

Animals↗

PubMed1991

Comparison of several promoters and polyadenylation signals for use in heterologous gene expression in cultured Drosophila cells.

We have directly compared the ability of four promoters and three polyadenylation (poly(A)) signals to direct heterologous gene expression in stably transfected Drosophila melanogaster S2 cells. We compared two constitutive Drosophila promoters, the actin 5C distal promoter and the alpha 1-tubulin promoter, with the tightly regulated Drosophila metallothionein (Mtn) promoter and the Bombyx mori fibroin promoter. We find that the actin 5C and induced Mtn promoters generate comparable high levels of RNA and protein in this system. The alpha 1-tubulin promoter generates about four-fold lower levels, and the fibroin promoter shows no detectable activity in S2 cells. Interestingly, genes expressed from the constitutive actin 5C and alpha 1-tubulin promoters are consistently present at three- to four-fold lower copy numbers than genes expressed from the inducible Mtn promoter or the inactive fibroin promoter. Poly(A) signals of both mammalian (SV40) and Drosophila (Mtn) origin efficiently directed stable RNA synthesis in S2 cells, and, as in mammalian cells, the SV40 late poly(A) signal was more efficient than the SV40 early poly(A) signal. Thus the process of polyadenylation appears to be conserved between mammalian and Drosophila cells.

Actins↗

PubMed1983

Developmental variations of a nonfibroin mRNA of Bombyx mori silkgland, encoding for a low-molecular-weight silk protein.

The characterization of a new silk protein mRNA (P25 mRNA) in posterior silkgland cells (PSG) and the developmental variations of its cell molecular concentration versus that of fibroin mRNA are described. A 80% pure P25 cDNA was obtained by class separation of total nonfibroin cDNA from PSG and used to identify the mRNA in blotted PSG mRNA as a single 1100 nucleotide long species. When purified from agarose gel and translated in a reticulocyte cell-free system, P25 mRNA yielded a 25-kD polypeptide (P25), identical to a 25-kD protein of the cocoon in terms of pI value and partial peptide mapping pattern. Moreover, this protein comigrated with an abundant polypeptide of the posterior silkgland (PSG) and of the middle silkgland (MSG). When tritiated leucine was injected in vivo, labeled P25 showed up in the PSG after a 2-hr pulse but appeared in the MSG only after 24 hr of labeling. Since MSG cells were found to be devoid of P25 mRNA, we concluded that P25 is exclusively synthesized in the PSG, that it accumulates in the MSG lumen and that it is spun out in the same way as fibroin. Specific probes were used to measure the concentrations of P25 mRNA and also fibroin mRNA in PSG total RNA by hybridization with an excess of cDNA. Both species are highly degraded in the few hours following the physiological arrest of feeding which precedes the fourth molting period. Their subsequent accumulation during the fifth intermolt is triggered by food uptake and proceeds in such a way that a constant 1:1 molar ratio is maintained during the period of silk secretion.

Animals↗

PubMedDate not supplied

Orientation of silk III at the air-water interface.

A threefold helical crystal structure of Bombyx mori silk fibroin has been observed in films prepared from aqueous silk fibroin solutions using the Langmuir Blodgett (LB) technique. The films were studied using a combination of transmission electron microscopy and electron diffraction techniques. Films prepared at a surface pressure of 16.7 mN/m have a uniaxially oriented crystalline texture, with the helical axis oriented perpendicular to the plane of the LB film. Films obtained from the air-water interface without compression have a different orientation, with the helical axes lying roughly in the plane of the film. In both cases the d-spacings observed in electron diffraction are the same and match a threefold helical model crystal structure, silk III, described in previous publications. Differences in the relative intensities of the observed reflections in both types of oriented samples, as compared to unoriented samples, allows estimations of orientation distributions and the calculations of orientation parameters. The orientation of the fibroin chain axis in the plane of the interfacial film for uncompressed samples is consistent with the amphiphilic behavior previously postulated to drive the formation of the threefold helical silk III conformation.

Air↗