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Subhas C. Kundu - One of the best experts on this subject based on the ideXlab platform.
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silk Fibroin film from non mulberry tropical tasar silkworms a novel substrate for in vitro fibroblast culture
Acta Biomaterialia, 2009Co-Authors: Chitrangada Acharya, Sudip Kumar Ghosh, Subhas C. KunduAbstract:The silk protein Fibroin, isolated from the cocoon of the domesticated mulberry silkworm, Bombyx mori, is used extensively in biomaterial design and in cell and tissue culture. We report here for the first time the potential application of Fibroin obtained from the cocoon of non-mulberry tropical silkworm, Antheraea mylitta, as a substrate for in vitro cell culture. The mechanical strength of A. mylitta silk fibers indicates a stronger thread composition. The contact angle of A. mylitta Fibroin films suggests that it has lower hydrophilicity and lower solubility in organic solvents compared to B. mori Fibroin films. Retention of a secondary structure of Fibroin in both A. mylitta and B. mori films is confirmed by Fourier transform infrared analysis. The adherence, growth and proliferation patterns of feline fibroblast cells on A. mylitta Fibroin films suggest that this kind of film has a greater ability to support cell growth than B. mori Fibroin films and is comparable to that of control. This study demonstrates that, as well as being non-toxic to dermal fibroblast cells, non-mulberry Fibroin might be a useful alternative substrate to the more common B. mori Fibroin for a variety of biomedical applications.
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differential expression of the Fibroin gene in developmental stages of silkworm antheraea mylitta saturniidae
Comparative Biochemistry and Physiology B, 2001Co-Authors: Abhik Datta, Ananta K. Ghosh, Subhas C. KunduAbstract:Fibroin gene expression during the larval developmental stages of the Saturniid silkworm, Antheraea mylitta, was analyzed. Northern blot analysis of larval silk gland total RNA using the Fibroin gene as a probe showed that Fibroin is expressed in the intermoult stages and repressed during the moulting stages. Abundance of Fibroin transcripts gradually increased from the third to fifth intermoult stage, reaching a peak in the fifth intermoult. Transcripts declined during the early spinning stage. Western blot analysis of Fibroin protein production with anti-Fibroin antibody confirmed the differential Fibroin expression, in accordance with Fibroin mRNA synthesis. Dot blot hybridization of genomic DNA isolated from each larval developmental stage with the labelled Fibroin gene showed that at the genomic level, the relative concentration of the Fibroin gene was constant throughout the developmental stages. Our data confirm that Fibroin gene expression in A. mylitta, like in B. mori, is transcriptionally controlled and shows differential temporal variations.
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Purification and characterization of Fibroin from the tropical Saturniid silkworm, Antheraea mylitta.
Insect biochemistry and molecular biology, 2001Co-Authors: Abhik Datta, Ananta K. Ghosh, Subhas C. KunduAbstract:The Fibroin protein isolated from the posterior silkgland of the tropical Saturniid silkworm Antheraea mylitta, was solubilized in lithium dodecyl sulfate and purified by gel filtration. The major fraction from gel filtration was analyzed by SDS-PAGE under non-reducing and reducing conditions. One major protein band of ca 395 kDa was obtained under non-reducing conditions and a doublet band of approximately 197 kDa under reducing conditions. The appearance of a single spot in two-dimensional electrophoresis confirmed the purity of the protein indicating that it may be a homodimeric protein of two similar sized polypeptides. Amino acid composition analysis showed that, like other Saturniid Fibroins, it is rich in glycine, alanine and serine amino acids. N-terminal amino acid sequence shows significant homology with other Antheraea species. The enzymatic deglycosylation analysis indicates that the Fibroin protein is glycosylated and the oligosaccharides are O-linked to the protein backbone by N-acetylgalactoseamine moiety which conforms to a Core 1 mucin-type glycosylation pattern.
Craig Vierra - One of the best experts on this subject based on the ideXlab platform.
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aciniform spidroin a constituent of egg case sacs and wrapping silk fibers from the black widow spider latrodectus hesperus
Journal of Biological Chemistry, 2007Co-Authors: Keshav Vasanthavada, Anne M F Moore, Coby La Mattina, Ryan Reza, Xiaoyi Hu, Patrick R Jones, Tiffany Tuton, Arnold M Falick, Craig VierraAbstract:Abstract Spiders produce high performance fibers with diverse mechanical properties and biological functions. Molecular and biochemical studies of spider egg case silk have revealed that the main constituent of the large diameter fiber contains the Fibroin TuSp1. Here we demonstrate by SDS-PAGE and protein silver staining the presence of a distinct ∼300-kDa polypeptide that is found in solubilized egg case sacs. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called AcSp1-like and demonstrate that its protein product is assembled into the small diameter fibers of egg case sacs and wrapping silks from the black widow spider, Latrodectus hesperus. BLAST searches of the NCBInr protein data base using the amino acid sequence of AcSp1-like revealed similarity to AcSp1, an inferred protein proposed to be a component of wrapping silk. However, the AcSp1-like protein was found to display more nonuniformity in its internal iterated repeat modules than the putative AcSp1 Fibroin. Real time quantitative PCR analysis demonstrates that the AcSp1-like gene displays an aciniform gland-restricted pattern of expression. The amino acid composition of the Fibroins extracted from the luminal contents of the aciniform glands was remarkably similar to the predicted amino acid composition of the AcSp1-like protein, which supports the assertion that AcSp1-like protein represents the major constituent stored within the aciniform gland. Collectively, our findings provide the first direct molecular evidence for the involvement of the aciniform gland in the production of a common Fibroin that is assembled into the small diameter threads of egg case and wrapping silk of cob weavers.
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Molecular mechanisms of spider silk
Cellular and Molecular Life Sciences CMLS, 2006Co-Authors: X. Hu, Keshav Vasanthavada, K. Kohler, S. Mcnary, A. M. F. Moore, Craig VierraAbstract: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.
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Spider Egg Case Core Fibers: Trimeric Complexes Assembled from TuSp1, ECP-1, and ECP-2 †
Biochemistry, 2006Co-Authors: Xiaoyi Hu, Anne M F Moore, Kristin Kohler, Patrick R Jones, Arnold M Falick, Craig VierraAbstract: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 ∼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 r...
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egg case protein 1 a new class of silk proteins with Fibroin like properties from the spider latrodectus hesperus
Journal of Biological Chemistry, 2005Co-Authors: Xiaoyi Hu, Anne M F Moore, Kristin Kohler, Patrick R Jones, David O Sparkman, Arnold M Falick, Craig VierraAbstract:Abstract Spiders produce multiple types of silk that exhibit diverse mechanical properties and biological functions. Most molecular studies of spider silk have focused on Fibroins from dragline silk and capture silk, two important silk types involved in the survival of the spider. In our studies we have focused on the characterization of egg case silk, a third silk fiber produced by the black widow spider, Latrodectus hesperus. Analysis of the physical structure of egg case silk using scanning electron microscopy demonstrates the presence of small and large diameter fibers. By using the strong protein denaturant 8 m guanidine hydrochloride to solubilize the fibers, we demonstrated by SDS-PAGE and protein silver staining that an abundant component of egg case silk is a 100-kDa protein doublet. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called ecp-1, which encodes for one of the protein components of the 100-kDa species. BLAST searches of the NCBInr protein data base using the primary sequence of ECP-1 revealed similarity to Fibroins from spiders and silkworms, which mapped to two distinct regions within the ECP-1. These regions contained the conserved repetitive Fibroin motifs poly(Ala) and poly(Gly-Ala), but surprisingly, no larger ensemble repeats could be identified within the primary sequence of ECP-1. Consistent with silk gland-restricted patterns of expression for Fibroins, ECP-1 was demonstrated to be predominantly produced in the tubuliform gland, with lower levels detected in the major and minor ampullate glands. ECP-1 monomeric units were also shown to assemble into higher aggregate structures through the formation of disulfide bonds via a unique cysteine-rich N-terminal region. Collectively, our findings provide new insight into the components of egg case silk and identify a new class of silk proteins with distinctive molecular features relative to traditional members of the spider silk gene family.
Tetsuo Asakura - One of the best experts on this subject based on the ideXlab platform.
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regeneration of the femoral epicondyle on calcium binding silk scaffolds developed using transgenic silk Fibroin produced by transgenic silkworm
Acta Biomaterialia, 2011Co-Authors: Aya Nagano, Hideki Sezutsu, Yumi Tanioka, Nobuko Sakurai, Noboru Kuboyama, Hideo Kiba, Yasuhiro Tanimoto, Norihiro Nishiyama, Tetsuo AsakuraAbstract:Genetically modified silk Fibroin containing a poly-glutamic acid site, [(AGSGAG)4E8AS]4, for mineralization was produced as fibers by transgenic silkworms through systematic transformation of the silkworms. The Ca binding activity and mineralization of the transgenic silk Fibroin were examined in vitro, showing that this transgenic silk Fibroin had relatively high Ca binding activity compared with native silk Fibroin. Porous silk scaffolds were prepared with the transgenic and native silk Fibroins. Healing of femoral epicondyle defects in rabbit femurs treated with the scaffolds was examined by observing changes in images of the defects using micro-computed tomography. Earlier mineralization and bone formation were observed with scaffolds of transgenic silk Fibroin compared with those of native silk Fibroin. Thus, this study shows the feasibility of using genetically modified silk Fibroin from transgenic silkworms as a mineralization-accelerating material for bone repair.
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porous silk Fibroin film as a transparent carrier for cultivated corneal epithelial sheets
Journal of Biomaterials Science-polymer Edition, 2011Co-Authors: Kazunari Higa, Tetsuo Asakura, Naomi Takeshima, Fumika Moro, Tetsuya Kawakita, Motoko Kawashima, Makoto Demura, Jun Shimazaki, Kazuo Tsubota, Shigeto ShimmuraAbstract:Biological carriers, such as the amniotic membrane and serum-derived fibrin, are currently used to deliver cultivated corneal epithelial sheets to the ocular surface. Such carriers require being transparent and allowing the diffusion of metabolites in order to maintain a healthy ocular surface. However, safety issues concerning biological agents encouraged the development of safer, biocompatible materials as cell carriers. We examined the application of porous silk Fibroin films with high molecular permeability prepared by mixing silk Fibroin and poly(ethylene glycol) (PEG), and then removal of PEG from the silk-PEG films. Molecular permeability of porous silk Fibroin film is higher than untreated silk Fibroin film. Epithelial cells were isolated from rabbit limbal epithelium, and seeded onto silk Fibroin coated wells and co-cultured with mitomycin C-treated 3T3 fibroblasts. Stratified epithelial sheets successfully engineered on porous silk Fibroin film expressed the cornea-specific cytokeratins K3 and K12, as well as the corneal epithelial marker pax6. Basement membrane components such as type-IV collagen and integrin β1 were expressed in the stratified epithelial sheets. Further more, colony-forming efficiency of dissociated cells was similar to primary corneal epithelial cells showing that progenitor cells were preserved. The biocompatibility of Fibroin films was confirmed in rabbit corneas for up to 6 months. Porous silk Fibroin film is a highly transparent, biocompatible material that may be useful as a carrier of cultivated epithelial sheets in the regeneration of corneal epithelium.
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comparative study of silk Fibroin porous scaffolds derived from salt water and sucrose hexafluoroisopropanol in cartilage formation
Journal of Bioscience and Bioengineering, 2009Co-Authors: Kumiko Makaya, Shinichi Terada, Kosuke Ohgo, Tetsuo AsakuraAbstract:Abstract The purpose of this study is to create a new silk Fibroin scaffold with sufficient three-dimensional morphology and porous structure for cartilage formation. We have applied sucrose particles sized around 300 to 500 μm as porogens compared to equal-sized salt particles. After the porogen was leached out with water, scaffolds were prepared with Fibroin derived from sucrose/hexafluoroisopropanol (Su/H) or salt/water (Sa/W) based composites. A compression test indicated that the Sa/W Fibroin was much harder than the Su/H Fibroin, but a protease enzyme digested the Sa/W Fibroin more quickly than Su/H Fibroin. Rabbit ear chondrocytes were seeded onto the scaffolds for 4–8 week in vitro culture and histological analyses were performed. The distribution of cartilage formation in Safranin O staining was more homogenous in Su/H Fibroin than that of Sa/W Fibroin. The overall amount of cartilage was significantly better in the Su/H Fibroin than that in the Sa/W Fibroin. However, the inner structure of pore wall in the Sa/W Fibroin was rough and microporous with cartilage matrix deposition, while that in the Su/H Fibroin was thin and homogenous. Since mature cartilage gradually regenerates to fill the porous space, slowly degradable Su/H Fibroin should be a better candidate for cartilage formation.
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Production and characterization of a silk-like hybrid protein, based on the polyalanine region of Samia cynthia ricini silk Fibroin and a cell adhesive region derived from fibronectin
Biomaterials, 2004Co-Authors: Tetsuo Asakura, Chikako Tanaka, Mingying Yang, Juming Yao, Masato KurokawaAbstract:Abstract There are a variety of silkworms and silk Fibroins produced by them. Silks have many inherent suitable properties for biomaterials. In this paper, a novel silk-like hybrid protein, [DGG(A) 12 GGAASTGRGDSPAAS] 5 , which consists of polyalanine region of silk Fibroin from a wild silkworm, Samia cynthia ricini , and cell adhesive region including Arg-Gly-Asp (RGD) sequence, derived from fibronectin, was designed and produced. The genes encoding the hybrid protein were constructed and expressed in Escherichia coli . The main conformation of the polyalanine region, that is, either α -helix or β -sheet, could be easily controlled by treatment with different acidic solvents, trifluoroacetic acid or formic acid, respectively. This structural change was monitored with 13 C CP/MAS NMR. Higher cell adhesive and growth activities of the hybrid protein compared with those of collagen were obtained.
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Structural analysis of silk with 13C NMR chemical shift contour plots.
International Journal of Biological Macromolecules, 1999Co-Authors: Tetsuo Asakura, Makoto Demura, M Iwadate, M P WilliamsonAbstract:Abstract The polymorphic structures of silk Fibroins in the solid state were examined on the basis of a quantitative relationship between the 13 C chemical shift and local structure in proteins. To determine this relationship, 13 C chemical shift contour plots for Cα and Cβ carbons of Ala and Ser residues, and the Cα chemical shift plot for Gly residues were prepared using atomic co-ordinates from the Protein Data Bank and 13 C NMR chemical shift data in aqueous solution reported for 40 proteins. The 13 C CP/MAS NMR chemical shifts of Ala, Ser and Gly residues of Bombyx mori silk Fibroin in silk I and silk II forms were used along with 13 C CP/MAS NMR chemical shifts of Ala residues of Samia cynthia ricini silk Fibroin in β-sheet and α-helix forms for the structure analyses of silk Fibroins. The allowed regions in the 13 C chemical shift contour plots for Cα and Cβ carbons of Ala and Ser residues for the structures in silk Fibroins, i.e. Silk II, Silk I and α-helix, were determined using their 13 C isotropic NMR chemical shifts in the solid state. There are two area of the φ,Ψ map which satisfy the observed Silk I chemical shift data for both the Cα and Cβ carbons of Ala and Ser residues in the 13 C chemical shift contour plots.
Toshiki Tamura - One of the best experts on this subject based on the ideXlab platform.
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DOI 10.1007/s00239-009-9234-5 Conservation of Silk Genes in Trichoptera and Lepidoptera
2013Co-Authors: Toshiki Tamura Æ Frantisˇek Sehnal, Naoyuki Yonemura, Toshiki Tamura, Kazuei Mita, Frantisek SehnalAbstract:Ó The Author(s) 2009. This article is published with open access at Springerlink.com Abstract Larvae of the sister orders Trichoptera and Lepidoptera are characterized by silk secretion from a pair of labial glands. In both orders the silk filament consists of heavy (H)- and light (L)-chain Fibroins and in Lepidoptera it also includes a P25 glycoprotein. The L-Fibroin and H-Fibroin genes of Rhyacophila obliterata and Hydropsyche angustipennis caddisflies have exon/intron structuring (seven exons in L-Fibroin and two in H-Fibroin) similar to that in their counterparts in Lepidoptera. Fibroin cDNAs are also known in Limnephilus decipiens, representing the third caddisfly suborder. Amino acid sequences of deduced L-Fibroin proteins and of the terminal H-Fibroin regions are about 50 % identical among the three caddisfly species but their similarity to lepidopteran Fibroins is \25%. Positions of some residues are conserved, including cysteines that were shown to link the L-Fibroin and H-Fibroin by a disulfide bridge in Lepidoptera. The long internal part of H-Fibroins is composed of short motifs arranged in speciesspecific repeats. They are extremely uniform in R. obliterata. Motifs (SX)n, GGX, and GPGXX occur in both Trichoptera and Lepidoptera. The trichopteran H-Fibroins further contain charged amphiphilic motifs but lack the strings of alanines or alanine-glycine dipeptides that are typical lepidopteran motifs. On the other hand, sequences composed of a motif similar to ERIVAPTVITR surrounded by the (SX)4-6 strings and modifications of th
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expression of the japanese oak silkworm antheraea yamamai Fibroin gene in the domesticated silkworm bombyx mori
Insect Science, 2009Co-Authors: Isao Kobayashi, Katsura Kojima, Hideki Sezutsu, Keiro Uchino, Toshiki TamuraAbstract:Abstract To understand the evolutionary conservation of the gene expression mechanism and secretion machinery between Antheraea and Bombyx Fibroins, we introduced the genomic A. yamamai Fibroin gene into the domesticated silkworm, B. mori. The spliced A. yamamai Fibroin mRNA appeared only in the posterior region of the silk gland of the transgenic silkworm, suggesting that the functions of the Fibroin promoter region and the splicing machinery are conserved between these two species. The A. yamamai Fibroin protein was detected in the lumen of the silk gland of the transgenic silkworm, albeit at lower levels compared with the B. mori-type Fibroin. We found a strong degeneration of the posterior region of the silk gland of the transgenic silkworm. As a result, the cocoon shell weight was much lower in the transgenic silkworm than in the non-transgenic line. These results indicate that the promoter function and splicing machinery are well conserved between A. yamamai and B. mori but that the secretion mechanism of Fibroin is diversified between the two.
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Conservation of Silk Genes in Trichoptera and Lepidoptera
Journal of Molecular Evolution, 2009Co-Authors: Naoyuki Yonemura, Toshiki Tamura, Kazuei Mita, Frantisek SehnalAbstract:Larvae of the sister orders Trichoptera and Lepidoptera are characterized by silk secretion from a pair of labial glands. In both orders the silk filament consists of heavy (H)- and light (L)-chain Fibroins and in Lepidoptera it also includes a P25 glycoprotein. The L-Fibroin and H-Fibroin genes of Rhyacophila obliterata and Hydropsyche angustipennis caddisflies have exon/intron structuring (seven exons in L-Fibroin and two in H-Fibroin ) similar to that in their counterparts in Lepidoptera. Fibroin cDNAs are also known in Limnephilus decipiens , representing the third caddisfly suborder. Amino acid sequences of deduced L-Fibroin proteins and of the terminal H-Fibroin regions are about 50% identical among the three caddisfly species but their similarity to lepidopteran Fibroins is
Frantisek Sehnal - One of the best experts on this subject based on the ideXlab platform.
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DOI 10.1007/s00239-009-9234-5 Conservation of Silk Genes in Trichoptera and Lepidoptera
2013Co-Authors: Toshiki Tamura Æ Frantisˇek Sehnal, Naoyuki Yonemura, Toshiki Tamura, Kazuei Mita, Frantisek SehnalAbstract:Ó The Author(s) 2009. This article is published with open access at Springerlink.com Abstract Larvae of the sister orders Trichoptera and Lepidoptera are characterized by silk secretion from a pair of labial glands. In both orders the silk filament consists of heavy (H)- and light (L)-chain Fibroins and in Lepidoptera it also includes a P25 glycoprotein. The L-Fibroin and H-Fibroin genes of Rhyacophila obliterata and Hydropsyche angustipennis caddisflies have exon/intron structuring (seven exons in L-Fibroin and two in H-Fibroin) similar to that in their counterparts in Lepidoptera. Fibroin cDNAs are also known in Limnephilus decipiens, representing the third caddisfly suborder. Amino acid sequences of deduced L-Fibroin proteins and of the terminal H-Fibroin regions are about 50 % identical among the three caddisfly species but their similarity to lepidopteran Fibroins is \25%. Positions of some residues are conserved, including cysteines that were shown to link the L-Fibroin and H-Fibroin by a disulfide bridge in Lepidoptera. The long internal part of H-Fibroins is composed of short motifs arranged in speciesspecific repeats. They are extremely uniform in R. obliterata. Motifs (SX)n, GGX, and GPGXX occur in both Trichoptera and Lepidoptera. The trichopteran H-Fibroins further contain charged amphiphilic motifs but lack the strings of alanines or alanine-glycine dipeptides that are typical lepidopteran motifs. On the other hand, sequences composed of a motif similar to ERIVAPTVITR surrounded by the (SX)4-6 strings and modifications of th
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Molecular Evolution of Lepidopteran Silk Proteins: Insights from the Ghost Moth, Hepialus californicus
Journal of Molecular Evolution, 2010Co-Authors: Matthew A. Collin, Frantisek Sehnal, Kazuei Mita, Cheryl Y HayashiAbstract:Silk production has independently evolved in numerous arthropod lineages, such as Lepidoptera, the moths and butterflies. Lepidopteran larvae (caterpillars) synthesize silk proteins in modified salivary glands and spin silk fibers into protective tunnels, escape lines, and pupation cocoons. Molecular sequence data for these proteins are necessary to determine critical features of their function and evolution. To this end, we constructed an expression library from the silk glands of the ghost moth, Hepialus californicus , and characterized light chain Fibroin and heavy chain Fibroin gene transcripts. The predicted H. californicus silk Fibroins share many elements with other lepidopteran and trichopteran Fibroins, such as conserved placements of cysteine, aromatic, and polar amino acid residues. Further comparative analyses were performed to determine site-specific signatures of selection and to assess whether Fibroin genes are informative as phylogenetic markers. We found that purifying selection has constrained mutation within the Fibroins and that light chain Fibroin is a promising molecular marker. Thus, by characterizing the H. californicus Fibroins, we identified key functional amino acids and gained insight into the evolutionary processes that have shaped these adaptive molecules.
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Conservation of Silk Genes in Trichoptera and Lepidoptera
Journal of Molecular Evolution, 2009Co-Authors: Naoyuki Yonemura, Toshiki Tamura, Kazuei Mita, Frantisek SehnalAbstract:Larvae of the sister orders Trichoptera and Lepidoptera are characterized by silk secretion from a pair of labial glands. In both orders the silk filament consists of heavy (H)- and light (L)-chain Fibroins and in Lepidoptera it also includes a P25 glycoprotein. The L-Fibroin and H-Fibroin genes of Rhyacophila obliterata and Hydropsyche angustipennis caddisflies have exon/intron structuring (seven exons in L-Fibroin and two in H-Fibroin ) similar to that in their counterparts in Lepidoptera. Fibroin cDNAs are also known in Limnephilus decipiens , representing the third caddisfly suborder. Amino acid sequences of deduced L-Fibroin proteins and of the terminal H-Fibroin regions are about 50% identical among the three caddisfly species but their similarity to lepidopteran Fibroins is
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The Design of Silk Fiber Composition in Moths Has Been Conserved for More Than 150 Million Years
Journal of Molecular Evolution, 2006Co-Authors: Naoyuki Yonemura, Frantisek SehnalAbstract:The silk of caterpillars is secreted in the labial glands, stored as a gel in their lumen, and converted into a solid filament during spinning. Heavy chain Fibroin (H-Fibroin), light chain Fibroin (L-Fibroin), and P25 protein constitute the filament core in a few species that have been analyzed. Identification of these proteins in Yponomeuta evonymella , a moth from a family which diverged from the rest of Lepidoptera about 150 million years ago, reveals that the mode of filament construction is highly conserved. It is proposed that association of the three proteins is suited for long storage of hydrated silk dope and its rapid conversion to filament. Interactions underlying these processes depend on conserved spacing of critical amino acid residues that are dispersed through the L-Fibroin and P25 and assembled in the short ends of the H-Fibroin molecule. Strength, elasticity, and other physical properties of the filament are determined by simple amino acid motifs arranged in repetitive modules that build up most of the H-Fibroin. H-Fibroin synergy with L-Fibroin and P25 does not interfere with motif diversification by which the filament acquires new properties. Several types of motifs in complex repeats occur in the silks used for larval cobwebs and pupal cocoons. Restriction of silk use to cocoon construction in some lepidopteran families has been accompanied by simplification of H-Fibroin repeats. An extreme deviation of the silk structure occurs in the Saturniidae silkmoths, which possess modified H-Fibroin and lack L-Fibroin and P25.
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unique molecular architecture of silk Fibroin in the waxmoth galleria mellonella
Journal of Biological Chemistry, 2002Co-Authors: Michal Zurovec, Frantisek SehnalAbstract:Proteins of silk fibers are characterized by reiterations of amino acid repeats. Physical properties of the fiber are determined by the amino acid composition, the complexity of repetitive units, and arrangement of these units into higher order arrays. Except for very short motifs of 6–10 residues, the length of repetitive units and the number of these units concatenated in higher order assemblies vary in all spider and lepidopteran silks analyzed so far. This paper describes an exceptional silk protein represented by the 500-kDa heavy chain Fibroin (H-Fibroin) of the waxmoth,Galleria mellonella. Its non-repetitive N-terminal (175 residues) and C-terminal (60 residues) parts, the overall gene organization, and the nucleotide sequence around the TATA box show that it is homologous to the H-Fibroins of other Lepidoptera. However, over 95% of the protein consists of highly ordered repetitive structures that are unmatched in other species. The repetitive region includes 11 assemblies AB1AB1AB1AB2(AB2)AB2of remarkably conserved polypeptide repeats A (63 amino acid residues), B1 (43 residues), and B2 (18 residues). The repeats contain a high proportion of Gly (31.6%), Ala (23.8%), Ser (18.1%), and of residues with long hydrophobic side chains (16% for Leu, Ile, and Val combined). The presence of the GLGGLG and SSAASAA(AA) motifs suggests formation of pleated β-sheets and their stacking into crystallites. Conspicuous conservation of the apolar sequence VIVI followed by DD or ED is interpreted as indicating the importance of hydrophobicity and electrostatic charge in H-Fibroin cross-linking. The environment of G. mellonella larvae within bee cultures requires continuous production of silk that must be both strong and elastic. The spectacular arrangement of the repetitive H-Fibroin region apparently evolved to meet these requirements.