Dynamic light scattering of native silk fibroin solution extracted from different parts of the middle division of the silk gland of the Bombyx mori silkworm

Dynamic light scattering (DLS) measurements were performed on aqueous solutions of native silk fibroin extracted from three parts, the posterior (MP), the middle (MM), and the anterior parts (MA), of the middle division (M) of the silk gland of the Bombyx mori silkworm to study the dynamics and aggregation properties of silk fibroin. In the MP part, fibroin molecules are present as aggregates (or clusters) being composed of several large protein complexes or elementary unit (EU), which are further associated to make a large assembly connected via divalent metallic ions. In the MM part, such clusters of EU take more compact structure, and finally in the MA part, clusters disappear, but EUs are more or less aligned to keep the assembly, and the EU takes the conformation of wormlike cylinder capped with hemispheres at both ends. The overall conformational change in solution structure was interpreted as being due to the change in ionic environment in the solution. DLS study was also performed on regenerated silk fibroin solutions, which revealed that fibroin is present as a single molecule dominantly and their association behavior seems completely different from that of native samples and does not depend on types and concentration of added metallic ions.     

Studies on silk fibroin of the silkworm Bombyx mori

A procedure has been developed to obtain native fibroin in a pure state from the reservoir part of the silk gland. The purified protein has a sedimentation coefficient of 10 S as determined on sucrose density gradients and the amino acid composition is similar to that reported for fibroin from the cocoons. The effects of various solvents has been studied; lithium thiocyanate was found to be the solvent of choice. By in vivo labeling of fibroin with [³H]glycine and [¹⁴C]alanine it was demonstrated that fibroin synthesized in the posterior part of the gland and that stored in the reservoir part are identical.     

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.     

Rheology and dynamic light scattering of silk fibroin solution extracted from the middle division of Bombyx mori silkworm

Dynamic light scattering (DLS) and rheological measurements were performed on aqueous silk fibroin solutions extracted from the middle division of Bombyx mori silkworm over a wide range of polymer concentration C from 0.08 to 27.5 wt %. DLS results obtained in the dilute region of C less than 1 wt % are consistent with a model that an elementary unit is a large protein complex consisting of silk fibroin and P25 with a 6:1 molar ratio. Rheological measurements in the dilute C region reveal that those units (or clusters) with the hydrodynamic radius of about 100 nm form a network extending over the whole sample volume with small pseudoplateau modulus mainly by ionic bonding between COO(-) ions of the fibroin molecules and divalent metallic ions such as Ca(2+) or Mg(2+) ions present in the sample and also that, after a yield stress is reached, steady plastic flow is induced with viscosity much lower than the zero-shear viscosity estimated from creep and creep recovery measurements by 4-6 orders of magnitude. Angular frequency omega dependencies of the storage and the loss shear moduli, G'(omega) and G' '(omega), measured in the linear viscoelastic region, indicate that all solutions possess the pseudoplateau modulus in the low omega region and samples become highly viscoleastic for C greater, similar 4.2 wt %. Above C = 11.2 wt % another plateau appears at the high omega end accompanied by a distinct maximum of G' ' in the intermediate omega region. The relaxation motion with tau = 0.5 s corresponding to the maximum of G' ' is one of characteristic properties of the fibroin solutions in the high C region. Thermorheological behaviors of the solution with C = 27.5 wt % show that the network structure formed in the MM part of the silk gland is susceptible to temperature and a more stable homogeneous network is realized by raising the temperature up to T = 65 degrees C.     

Studies on the posterior silk gland of the silkworm Bombyx mori. V. Electron microscope localization of fibroin in the posterior silk gland at the later stage of the fifth instar

Electron microscope observations of thin sections of epoxy resin- embeded posterior silk gland cells at the later stage of the fifth instar revealed that the Golgi vacuoles and the secretory granules (fibroin globules) in the cytoplasm and the glandular lumen contain fine fibrous materials. In frozen thin sections these structures appear as electron-dense granules and electron-dense blocks, or a column, respectively. Immunoelectron microscopy has shown that ferritin particles or products of the peroxidase reaction are localized on these structures. It was concluded that the fine fibrous materials most probably represent native fibroin molecules or their aggregates.     

Proteome analysis of silk gland proteins from the silkworm, Bombyx mori

The silk gland of Bombyx mori is an organ specialized for the synthesis and secretion of silk proteins. We report here the resolution of silk gland proteins by 2-DE and the identification of many of those proteins. This was accomplished by dissecting the glands into several sections, with each exhibiting more than 400 protein spots by 2-DE, of which 100 spots were excised and characterized by in-gel digestion followed by PMF. Ninety-three proteins were tentatively identified. These were then categorized into groups involved in silk protein secretion, transport, lipid metabolism, defense, etc. Western blotting of a 2-DE gel using an antibody of the carotenoid binding protein confirmed the presence of this protein in the silk gland. Proteins including fibroin L-chain and P25 were found as multiple isoforms, some of which contained differential amounts of phosphate residues as analyzed by on-probe dephosphorylation. The current analysis contributes to our understanding of proteins expressed by the silk gland not only of the model lepidopteran B. mori, but also to proteins from other silk-producing insects such as Philosamia cynthia ricini.     

Predominant synthesis of fibroin heavy and light chains on the membrane-bound polysomes prepared from the posterior silk gland of the silkworm, Bombyx mori

Membrane-bound polysomes were prepared from the posterior silk gland of the silkworm, Bombyx mori, on the fourth to fifth day in the fifth larval instar. The polysomes, when supplemented with a soluble fraction from the posterior silk gland, exhibited the elongation reaction of the growing polypeptide-chains, but the initiation reaction of polypeptide synthesis was not demonstrated in this system. The predominant products synthesized on the membrane-bound polysomes were fibroin heavy chain (H-chain) and light chain (L-chain), while polypeptides of heterogeneous size classes were synthesized on the 105,000 X g-sedimentable polysomes. A substantial fraction of the fibroin L-chain synthesized was bound to the H-chain by disulfide bond. Most of the newly synthesized fibroin H- and L-chains on the membrane-bound polysomes were proved to be present within microsomal membrane vesicles because of their insensitivity to digestion with proteases in the absence of Triton X-100.     

pH induced changes in the rheology of silk fibroin solution from the middle division of Bombyx mori silkworm

The rheological properties of fibroin silk solutions extracted from the middle division of Bombyx mori silkworms were examined. Acidification of the solutions with acetic acid vapor gelled the material, a process which at short time scales could be reversed by exposure to ammonia vapor. The solution could also be converted to sol from the gel state by the addition of EDTA. The possible mechanisms for gel formation in fibroin solutions is discussed as are the implications for the process of spinning silk fibers.     

Studies on middle and posterior silk glands of silkworm (Bombyx mori) using two-dimensional electrophoresis and mass spectrometry

Silk glands, present in the larval stage of the silkworm, produce threads of silky material to form the cocoon and are mainly composed of three parts: the anterior, the middle, and the posterior silk glands, each playing different roles in silk secretion. High-resolution two-dimensional polyacrylamide gel electrophoresis and computer-assisted analysis were used to investigate quantitative and qualitative differences between the middle and posterior silk glands. Silver staining revealed over 600 spots for each sample, mostly distributed from 15 to 100 kDa with pH 4-7. Computer-assisted image analysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and post-source decay technology suggested that there were significant differences in spot distribution and expression between the middle and posterior silk glands. In addition, 98 spots from the posterior silk gland were excised and further investigated following trypsin digestion. The results suggested that more than 20% of the 88 proteins identified were related to heat-shock proteins and chaperones. Redox system and DNA replication proteins involved in silk protein synthesis were also detected in the posterior silk gland. Interestingly, two novel serpin proteins were identified in the middle silk gland, and to a lesser extent in the posterior gland, which were presumed to be involved in regulation of proteolytic activity and protection of silk proteins from degradation.     

Localization of the genes for silk fibroin in silk gland cells of Bombyx mori.

Radioactive iodinated silk fibroin messenger RNA and ribosomal RNA have been used as probes to localize their genes in tissue sections of Bombyx mori by in situ hybridization. From filter hybridization experiments it is inferred that the majority of the grains produced by in situ hybridization with fibroin mRNA represents specific hybridization to fibroin genes. Sections of the posterior silk gland where silk is synthesized have been compared with those of the middle gland which does not synthesize fibroin. Glands have been analyzed from the second through the fifth (last) larval instar during feeding and moulting periods. During later stages when the gland cells increase their DNA content by polyploidization, serial sections were required to follow the distribution of grains through entire nuclei. At all stages, both ribosomal DNA and fibroin genes are distributed randomly throughout the nuclei without a preferred relationship to any nuclear structure.     

Ultrastructure of fibroin in the silk gland of larval Bombyx mori

The fibroin molecules stored in Golgi vacuoles in the posterior silk gland cells of 72-h-old, fifth instar larvae of Bombyx mori L. were observed electron-microscopically. The fibers which float in the Golgi vacuoles often have their ends attached to the limiting membrane. The fibers are helical bundles about 130 Å in diameter composed of 5–7 threads, each 20–30 Å thick.     

Death commitment in the anterior silk gland of the silkworm, Bombyx mori

The insect steroid hormone, 20-hydroxyecdysone (20E) triggers the programmed cell death (PCD) of the anterior silk glands (ASGs) of the silkworm, Bombyx mori. We tried to determine the time of commitment to die (death commitment) by examining ASG responses to 20E and juvenile hormone analogue (JHA) in vivo as well as in vitro. The ASGs obtained late on day 6 of the fifth instar completed PCD when cultured with 20E, while the ASGs obtained on day 4 and cultured with 20E did not undergo PCD. The ASGs became competent to respond to 20E at mid-day 5. The ASGs with responsiveness to 20E were not sensitive to JHA, indicating that the ASGs were committed to die before becoming capable of responding to 20E. Topical application of JHA on day 4 suppressed 20E-induced PCD, but that on day 5 failed to do so, indicating that the death commitment might occur between day 4 and 5. We also determined the time of death commitment after allatectomy of the fourth instar larvae, a procedure that induced the precocious PCD. Timed application of JHA and culture of ASGs with 20E in the presence of JHA showed that the ASGs had lost their sensitivity to JHA between 72 and 96 h after allatectomy, i.e. 24-48 h before precocious gut purge in the allatectomized larvae. This result is similar to that obtained in the fifth instar. We conclude that the cellular commitment to die takes place one day before the ASGs become competent to respond to 20E.     

Recognition of signal peptide by protein translocation machinery in middle silk gland of silkworm Bombyx mori

To investigate the functions of signal peptide in protein secretion in the middle silk gland of silkworm Bombyx mori, a series of recombinant Autographa californica multiple nucleopolyhedroviruses containing enhanced green fluorescent protein （egfp） gene, led by sericin-1 promoter and mutated signal peptide coding sequences, were constructed by region-deletions or single amino acid residue deletions. The recombinant Autographa californica multiple nucleopolyhedroviruses were injected into the hemocoele of newly ecdysed fifth-instar silkworm larvae. The expression and secretion of EGFP in the middle silk gland were examined by fluorescence microscopy and Western blot analysis. Results showed that even with a large part （up to 14 amino acid residues） of the ser-1 signal peptide deleted, the expressed EGFP could still be secreted into the cavity of the silk gland. Western blot analysis showed that shortening of the signal peptide from the C-terminal suppressed the maturation of pro-EGFP to EGFP. When 8 amino acid residues were deleted from the C-terminal of the signal peptide （mutant 13 aa）, the secretion of EGFP was incomplete, implicating the importance of proper coupling of the h-region and c-region. The deletion of amino acid residue（s） in the h-region did not affect the secretion of EGFP, indicating that the recognition of signal peptide by translocation machinery was mainly by a structural domain, but not by special amino acid residue（s）. Furthermore, the deletion ofArg^2 or replacement with Asp in the n-region of the signal peptide did not influence secretion of EGFP, suggesting that a positive charge is not crucial.     

Quantitative measurements of fibroin messenger RNA synthesis in the posterior silk gland of normal and mutant Bombyx mori

The amount of newly synthesized and accumulated fibroin messenger RNA has been measured quantitatively at various stages of posterior silk gland development in Bombyx mori. The two-step method involves fractionation on a Bio-Gel column which excludes the large mRNA, followed by RNAase T₁ digestion, and fractionation of the oligonucleotides on DEAE-Sephadex. Larvae in the feeding stages of the third and fourth instar synthesize and accumulate fibroin mRNA to about 2% of cellular RNA; this corresponds to 0.2 and 2 μg per pair of posterior glands in the third and fourth instars, respectively. More than 70% of this mRNA is degraded in vivo during the third and fourth moulting stages. Fibroin mRNA synthesis resumes again within the first 24 hours of the fifth instar; the mRNA accumulates and predominates over other DNA-like RNAs as the stage proceeds until finally it comprises about 3.5% of cellular RNA in a mature larva (170 μg per pair of posterior glands). These results indicate that more than 99% of the fibroin mRNA detected in the fifth instar is synthesized during this stage.Four spontaneous mutants of B. mori which synthesize very low levels of fibroin have been analyzed for their RNA content in the middle fifth instar. The total cellular RNA of the posterior gland is reduced to 4 to 7% of normal. Fibroin mRNA is more severely reduced to 1% of normal. In three heterozygotes, which have mutant phenotypes with respect to fibroin production, only slight increases of total cellular RNA and fibroin mRNA were observed. Thus, the primary biochemical lesion in these mutants is still unknown.The presumed ancestor to B. mori, the wild silkworm B. mandarina, was also analyzed for its fibroin mRNA. The mRNA isolated from fifth instar larvae of B. mandarina is indistinguishable from that of B. mori with respect to its nucleotide sequence, molecular weight and fraction of total cellular RNA.     

U6 snRNA variants isolated from the posterior silk gland of the silk moth Bombyx mori

Five U6 small nuclear RNA (snRNA) isoforms were detected and characterized from the posterior silk gland (PSG) of the silk moth Bombyx mori (Nistari strain). Using the currently accepted U6 secondary structure model as a basis for comparison, the variants were analyzed for nucleotide differences across the sequence with a focus on known functional domains. Differences were observed primarily in single-stranded areas of which sixty percent were found in the highly conserved U4-U6 binding sites. In the Nistari strain, the U6A variant was found to be approximately four times more abundant as part of high molecular weight spliceosomal complexes when compared with U6A in the total unfractionated PSG cell lysate. Additionally, the European 703 B. mori strain total cell lysate U6 snRNA was analyzed and only the dominant U6A isoform initially identified in Nistari was found. Due to U6's essential role in pre-mRNA processing, variants may modulate assemblage of the catalytic core and in doing so potentially affect the rate of splicing. Phylogenetic analysis of the U6 snRNA sequences indicate an ancient divergence of U6 from the self-splicing group II intron module and a high degree of evolutionary conservation across species possibly due to functional constraints on the gene. Using in silico analysis, 35 full-length U6 variants were observed in the recently released Whole Genome Shotgun (WGS) database of the p50T strain. The consensus sequence of these U6 genes from p50T is identical to U6A identified in the Nistari strain. Furthermore p50T variant 1, which is represented in 14 genes, is equivalent to Nistari U6A.