Vibrational 13C-cross-polarization/magic angle spinning NMR spectroscopic and thermal characterization of poly(alanine-glycine) as model for silk I Bombyx mori fibroin

This study focuses on the conformational characterization of poly(alanine-glycine) II (pAG II) as a model for a Bombyx mori fibroin silk I structure. Raman, IR, and 13C-cross-polarization/magic angle spinning NMR spectra of pAG II are discussed in comparison with those of the crystalline fraction of B. mori silk fibroin (chymotryptic precipitate, Cp) with a silk I (silk I-Cp) structure. The spectral data give evidence that silk I-Cp and the synthetic copolypeptide pAG II have similar conformations. Moreover, the spectral findings reveal that silk I-Cp is more crystalline than pAG II; consequently, the latter contains a larger amount of the random coil conformation. Differential scanning calorimetry measurements confirm this result. N-Deuteration experiments on pAG II allow us to attribute the Raman component at 1320 cm(-1) to the amide III mode of a beta-turn type II conformation, thus confirming the results of those who propose a repeated beta-turn type II structure for silk I. The analysis of the Raman spectra in the nuNH region confirms that the silk I structure is characterized by the presence of different types of H-bonding arrangements, in agreement with the above model.     

13C CP/MAS NMR study on structural heterogeneity in Bombyx mori silk fiber and their generation by stretching

It is important to resolve the structure of Bombyx mori silk fibroin before spinning (silk I) and after spinning (silk II), and the mechanism of the structural transition during fiber formation in developing new silk-like fiber. The silk I structure has been recently resolved by (13)C solid-state NMR as a "repeated beta-turn type II structure." Here, we used (13)C solid-state NMR to clarify the heterogeneous structure of the natural fiber from Bombyx mori silk fibroin in the silk II form. Interestingly, the (13)C CP/MAS NMR revealed a broad and asymmetric peak for the Ala Cbeta carbon. The relative proportions of the various heterogeneous components were determined from their relative peak intensities after line shape deconvolution. Namely, for 56% crystalline fraction (mainly repeated Ala-Gly-Ser-Gly-Ala-Gly sequences), 18% distorted beta-turn, 13% beta-sheet (parallel Ala residues), and 25% beta-sheet (alternating Ala residues). The remaining fraction of 44% amorphous Tyr-rich region, 22% in both distorted beta-turn and distorted beta-sheet. Such a heterogeneous structure including distorted beta-turn can be observed for the peptides (AG)(n) (n > 9 ). The structural change from silk I to silk II occurs exclusively for the sequence (Ala-Gly-Ser-Gly-Ala-Gly)(n) in B. mori silk fibroin. The generation of the heterogeneous structure can be studied by change in the Ala Cbeta peak of (13)C CP/MAS NMR spectra of the silk fibroin samples with different stretching ratios.     

Vibrational infrared conformational studies of model peptides representing the semicrystalline domains of Bombyx mori silk fibroin

The structural organization of Bombyx mori silk fibroin was investigated by infrared (IR) spectroscopy. To this aim, (AG)15 and other model peptides of varying chain length, containing tyrosine (Y), valine (V), and serine (S) in the basic (AG)n sequence were synthesized by the solid phase method and their spectroscopic properties were determined. Both the position and the relative content of Y, V, and S residues in the (AG)n model system appeared critical in determining the preferred conformation, i.e., silk I, silk II, and unordered structures. Curve fitting analysis in the amide I range showed that the model peptides with prevailing silk II structure displayed different beta-sheet content, which was dependent on the degree of interruption of the (AG)n sequence. In this regard, the bands at about 1000 and 980 cm(-1), specifically assigned to the AG sequence of the B. mori silk fibroin chain, were identified as marker of the degree of interruption of the (AG)n sequence.A stable silk I structure was observed only when the Y residue was located near the chain terminus, while a silk I --> silk II conformational transition occurred when it was positioned in the central region of the peptide.Analysis of the second-derivative spectra in the amide I range allowed us to identify a band at 1639 cm(-1) (4 --> 1 hydrogen-bonded type II beta-turns), which is characteristic of the silk I conformation.     

In vitro production of Bombyx mori silk fibroin by organ culture of the posterior silk glands; isotope labeling and fluorination of the silk fibroin

An in vitro silk fibroin production system has been developed by culture of posterior silk glands from Bombyx mori. A large amount of the silk fibroin was produced continuously and effectively with a rotation culture procedure. Modified Grace's insect medium was used, and oxygen bubbling in the medium was performed. In addition, half of the medium was replaced with fresh medium every 6 h. The production yield of silk fibroin produced after 100 h culture was 81 mg/g wet weight of posterior silk gland. This culture system was used successfully for efficient (15)N isotope labeling of silk fibroin, which is required for (15)N solid state nuclear magnetic resonance (NMR) analysis of silk fibroin. Moreover, the introduction of fluorinated amino acids into silk fibroin was also carried out using this culture system. (c) 1993 John Wiley & Sons, Inc.     

Structural analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori

To study the minimal length required for the secretion of recombinant proteins and silkproteins in posterior silk gland,the signal peptide(SP)of the fibroin heavy chain(FibH)of silkwormBombyx mori was systematically shortened from the C-terminal.Its effect on the secretion of protein wasobserved using enhanced green fluorescent protein(EGFP)as a reporter.Secretion of EGFP fusion proteinswas examined under fluorescence microscope.FibH SPs with lengths of 20,18,16 and 12 a.a.can directthe secretion of the reporter,yet those with lengths of 11, 10, 9, 8 and 1 a.a. can not. When the FibH SP wasshortened to 12a.a., the secretion efficiency was decreased slightly and cleavage occurred within EGFP.When 16a.a.of the FibH SP were used,the secretion of fusion protein was normal and the cleavage sitewas between the Gly-Ser linker and Met,the starting amino acid of EGFP. These findings are applicable forthe expression of foreign proteins in silkworm silk gland.The cleavage site of the SP is discussed andcompared with the predictive results of the SignalP 3.0 online prediction program.     

Expression of the Japanese oak silkworm Antheraea yamamai fibroin gene in the domesticated silkworm Bombyx mori

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.     

The role of irregular unit,GAAS, on the secondary structure of Bombyx mori silk fibroin studied with 13C CP/MAS NMR and wide-angle X-ray scattering

Bombyx mori silk fibroin is a fibrous protein whose fiber is extremely strong and tough, although it is produced by the silkworm at room temperature and from an aqueous solution. The primary structure is mainly Ala-Gly alternative copolypeptide, but Gly-Ala-Ala-Ser units appear frequently and periodically. Thus, this study aims at elucidating the role of such Gly-Ala-Ala-Ser units on the secondary structure. The sequential model peptides containing Gly-Ala-Ala-Ser units selected from the primary structure of B. mori silk fibroin were synthesized, and their secondary structure was studied with (13)C CP/MAS NMR and wide-angle X-ray scattering. The (13)C isotope labeling of the peptides and the (13)C conformation-dependent chemical shifts were used for the purpose. The Ala-Ala units take antiparallel beta-sheet structure locally, and the introduction of one Ala-Ala unit in (Ala-Gly)(15) chain promotes dramatical structural changes from silk I (repeated beta-turn type II structure) to silk II (antiparallel beta-sheet structure). Thus, the presence of Ala-Ala units in B. mori silk fibroin chain will be one of the inducing factors of the structural transition for silk fiber formation. The role of Tyr residue in the peptide chain was also studied and clarified to induce "locally nonordered structure."     

Raman study of poly(alanine-glycine)-based peptides containing tyrosine, valine, and serine as model for the semicrystalline domains of Bombyx mori silk fibroin

For a deeper insight into the structure of Bombyx mori silk fibroin, some model peptides containing tyrosine (Y), valine (V), and serine (S) in the basic (AG)n sequence were synthesized by the solid-phase method and analyzed by Raman spectroscopy in order to clarify their conformation and to evaluate the formation and/or disruption of the ordered structure typical of B. mori silk fibroin upon incorporation of Y, V, and S residues into the basic (AG)n sequence. The Raman results indicated that the silk I structure remains stable only when the Y residue is positioned near the chain terminus; otherwise, a silk I --> silk II conformational transition occurs. The peptides AGVGAGYGAGVGAGYGAGVGAGYG(AG)3 and (AG)3YG(AG)2VGYG(AG)3YG(AG)3 treated with LiBr revealed a prevalent silk II conformation; moreover, the former contained a higher amount of random coil than the latter. This result was explained in relation to the different degrees of interruption of the (AG)n sequence. The Raman analysis of the AGSGAG-containing samples confirmed that the AGSGAG hexapeptide is a good model for the silk II crystalline domain. As the number of AGSGAG repeating units decreased, the random coil content increased. The study of the Y domain (I850/I830 intensity ratio) allowed us to hypothesize that in the packing characteristic of Silk I and Silk II conformations the Y residues experience different environments and hydrogen-bonding arrangements; the packing typical of silk I structure traps the tyrosyl side chains in environments more unfavorable to phenoxyl hydrogen-bonding interactions.     

The dissolution and characterization of Bombyx mori silk fibroin in calcium nitrate-methanol solution and the regeneration of films

Bombyx mori silk fibroin from the silkworm was found to be soluble in a calcium nitrate-methanol system. Fibroin dissolves in 75% w/v Ca(NO₃)₂/MeOH solution at a temperature of 67°C. The viscometric behavior of the fibroin-salt solution was analyzed and the fibroin's secondary structures were determined via ¹³C solution nmr. Fourier transform ir, solid state ¹³C-nmr, x-ray diffraction, differential scanning calorimetry, scanning electron microscopy (SEM), and polarizing microscopy were used to characterize regenerated films and fibers. A compositional phase diagram of fibroin in the salt solution was constructed. Viscosity data indicate that there is aggregation of fibroin chains within the salt solution. The extremely high value of intrinsic viscosity of 8.7 dL/g at 298 K may be due to aggregation. Aggregation may be caused by the complexing of calcium ions with the fibroin chains at their amide linkages. The energy required for viscous flow for the fibroin solution (ΔH_vis = 9.03 kcal/mol) is greater than that of the solvent (ΔH_vis = 7.01 kcal/mol). Chain entanglements may be hindering the free motion of chains thus increasing the energy required for the viscous flow. ¹³C-nmr shows that fibroin chains exist in two independent conformational environments. While most of the molecule is in a random coil conformation, there is evidence of some order within the chains of fibroin. In as-cast regenerated films, the fibroin chains are in a random coil/α-helix conformation with some β-sheet content. Crystallinity induced by immersion of thin films in methanol is evidenced via x-ray diffraction, which shows lattice spacings at 4.042 Å. Thin films have a fibrillar morphology that is clearly shown under the SEM and the polarizing microscope. Fibers were hand pulled from the concentrated fibroin-salt solutions and coagulated with acetone and methanol. A microscopic analysis was done using the polarizer. © 1997 John Wiley & Sons, Inc. Biopoly 42: 61–74, 1997     

家蚕丝素重链启动子驱动DsRed的瞬时分泌表达

根据家蚕丝蛋白基因的启动子活性高、丝蛋白具有高效分泌的特性,克隆了家蚕丝素重链基因(Fib-H)启动子及其下游的信号肽序列(FibHS),将DsRed基因与信号肽序列融合构建了分泌型瞬时表达载体;转染细胞实验显示,该载体能在家蚕BmN细胞中瞬时表达DsRed;家蚕注射载体后,可在丝腺腔中检测到红色荧光,表明瞬时表达的DsRed分泌到丝腺腔,推测所克隆的序列具有信号肽的功能。此外,本研究为家蚕丝腺生物反应器分泌表达外源基因的研究奠定了基础。     

The crystal structure of Bombyx mori silk fibroin at the air–water interface

A new crystalline polymorph of Bombyx mori silk, which forms at the air–water interface, has been characterized. A previous study found this structure to be trigonal, and to be distinctly different than the two previously observed silk crystal structures, silk I and silk II. This new structure was named silk III. Identification of this new silk polymorph was based on evidence from transmission electron microscopy and electron diffraction, coupled with molecular modeling. In the current paper, additional data enables us to refine our model of the silk III structure. Some single crystal electron diffraction patterns indicate a deviation in symmetry away from a perfect trigonal unit cell to monoclinic unit cell. The detailed shape of the powder diffraction peaks also supports a monoclinic cell. The monoclinic crystal structure has an nonprimitive unit cell incorporating a slightly distorted hexagonal packing of silk molecular helices. The chains each assume a threefold helical conformation, resulting in a crystal structure similar to that observed for polyglycine II, but with some additional sheet-like packing features common to the threefold helical crystalline forms of many glycine-rich polypeptides. © 1997 John Wiley & Sons, Inc. Biopoly 42: 705–717, 1997     

Effect of polyamines on mechanical and structural properties of Bombyx mori silk

Silkworm, Bombyx mori (B. mori) belongs to the Lepidoptera family. The silk produced from this insect, mulberry silk, gained lot of importance as a fabric. Silk is being exploited as a biomaterial due to its surprising strength and biocompatibility. Polyamines (PA) are important cell growth regulators. In the present work the effect of treatment of polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm) on the quantity and quality of silk produced was assessed. Results showed that exogenous feeding of Spd at a concentration of 50 µM increased fiber length significantly. Analysis by Fourier transform infrared (FTIR) on the properties of silk obtained from Spd treated silkworms revealed an increase in percentage of absorption with no difference in peak positions of amide I and amide III groups. Scanning electron microscopy (SEM) revealed an increase in diameter of silk. Further, analysis at molecular level showed an increase in fibroin expression in Spd treated silk glands. However, the Spd treatment showed no significant difference with respect to fibroin to sericin ratio per unit weight of cocoon, silk tenacity, and percent elongation. Thus, the present results show that polyamine treatment would influence silk quality at structural, mechanical, and molecular level in the Bombyx mori, which can be exploited in silk biomaterial production.     

Copper in the silk formation process of Bombyx mori silkworm

Evidence is presented here that cupric ions play a part in the natural spinning of Bombyx mori silk. Proton induced X-ray emission studies revealed that the copper content increased from the posterior part to the anterior part of silk gland, and then further increased in the silk fiber. Spectrophotometric analysis demonstrated that cupric ions formed coordination complexes with silk fibroin chains while Raman spectroscopy indicated that they induced a conformation transition from random coil/helix to beta-sheet. Taken together these findings indicate that copper could play a role in the natural spinning process in silkworms.     

Determination of intermolecular distance for a model peptide of Bombyx mori silk fibroin, GAGAG, with rotational echo double resonance

Rotational echo double resonance NMR spectroscopy is applied for the determination of the distance of intermolecular chains of pentapeptide, GAGAG (G: Gly, A: Ala), a model typical of the crystalline domain in Bombyx mori silk fibroin. 1:4 mixture of G[1-(13)C]AGAG and GAG[(15)N]AG with antiparallel beta-sheet structure was used to determine the distance of intermolecular hydrogen bonding between adjacent molecules within pleated sheet and the (13)C-(15)N interatomic distance was determined to be 4.3 A. On the other hand, 1:4 mixture of GAG[1-(13)C]AG and GAG[(15)N]AG gave information on the interpleated sheet arrangement. When we assumed the same distances between two interpleated sheets, the distance was calculated to be 5.3 A and the angle (15)N-(13)C-(15)N was 180 degrees.     

Structural evolution of regenerated silk fibroin under shear: combined wide- and small-angle x-ray scattering experiments using synchrotron radiation

The structural evolution of regenerated Bombyx mori silk fibroin during shearing with a Couette cell has been studied in situ by synchrotron radiation small- and wide-angle x-ray scattering techniques. An elongation of fibroin molecules was observed with increasing shear rate, followed by an aggregation phase. The aggregates were found to be amorphous with beta-conformation according to infrared spectroscopy. Scanning x-ray microdiffraction with a 5 microm beam on aggregated material, which had solidified in air, showed silk II reflections and a material with equatorial reflections close to the silk I structure reflections, but with strong differences in reflection intensities. This silk I type material shows up to two low-angle peaks suggesting the presence of water molecules that might be intercalated between hydrogen-bonded sheets.     

Titanium nanoparticles influence the Akt/Tor signal pathway in the silkworm,Bombyx mori,silk gland

Various nanoparticles, such as silver nanoparticles (AgNPs) and titanium nanoparticles (TiO₂NPs) are increasingly used in industrial processes. Because they are released into the environment, research into their influence on the biosphere is necessary. Among its other effects, dietary TiO₂NPs promotes silk protein synthesis in silkworms, which prompted our hypothesis that TiO₂NPs influence protein kinase B (Akt)/Target of rapamycin (Tor) signaling pathway (Akt/Tor) signaling in their silk glands. The Akt/Tor signaling pathway is a principle connector integrating cellular reactions to growth factors, metabolites, nutrients, protein synthesis, and stress. We tested our hypothesis by determining the influence of dietary TiO₂NPs (for 72 h) and, separately, of two Akt/Tor pathway inhibitors (LY294002 and rapamycin) on expression of Akt/Tor signaling pathway genes and proteins in the silk glands. TiO₂NPs treatments led to increased accumulation of mRNAs for Akt, Tor1 and Tor2 by 1.6‐, 12.1‐, and 4.8‐fold. Dietary inhibitors led to 2.6‐ to 4‐fold increases in mRNAs encoding Akt and substantial decreases in mRNAs encoding Tor1 and Tor2. Western blot analysis showed that dietary TiO₂NPs increased the phosphorylation of Akt and its downstream proteins. LY294002 treatments led to inhibition of Akt phosphorylation and its downstream proteins and rapamycin treatments similarly inhibited the phosphorylation of Tor‐linked downstream proteins. These findings support our hypothesis that TiO₂NPs influence Akt/Tor signaling in silk glands. The significance of this work is identification of specific sites of TiO₂NPs actions.     

Effects of starvation and hormones on DNA synthesis in silk gland cells of the silkworm,Bombyx mori

Silk gland cells of silkworm larvae undergo multiple cycles of endomitosis for the synthesis of silk proteins during the spinning phase. In this paper, we analyzed the endomitotic DNA synthesis of silk gland cells during larval development, and found that it was a periodic fluctuation, increasing during the vigorous feeding phase and being gradually inhibited in the next molting phase. That means it might be activated by a self‐regulating process after molting. The expression levels of cyclin E, cdt1 and pcna were consistent with these developmental changes. Moreover, we further examined whether these changes in endomitotic DNA synthesis resulted from feeding or hormonal stimulation. The results showed that DNA synthesis could be inhibited by starvation and re‐activated by re‐feeding, and therefore appears to be dependent on nutrition. DNA synthesis was suppressed by in vivo treatment with 20‐hydroxyecdysone (20E). However, there was no effect on DNA synthesis by in vitro 20E treatment or by either in vivo or in vitro juvenile hormone treatment. The levels of Akt and 4E‐BP phosphorylation in the silk glands were also reduced by starvation and in vivo treatment with 20E. These results indicate that the activation of endomitotic DNA synthesis during the intermolt stages is related to feeding and DNA synthesis is inhibited indirectly by 20E.     

家蚕BmlncR2036调控P25基因启动子活性及参与中肠发育调控

[目的]长链非编码RNA (long non-coding RNA,lncRNA)对家蚕Bombyx mori发育具有重要调控作用.我们在前期研究中发现一个位于家蚕丝素蛋白基因P25附近的lncRNA BmlncR2036.本研究旨在进一步探索BmlncR2036调控家蚕P25基因表达的分子机制.[方法]qPCR检测B...