Functional Analysis Of Metal Ions And Ion-transporting Proteins On Silk Fiber Formation

As early as 5000 years ago, the ancient Chinese began planting mulberry trees for feeding silkworms. The silks produced by the silkworms were reeled and used for making silk fabrics. In ancient China, silks had been widely used in textile industry, art, and printing, which greatly enriched the human social life. Until now, the silks are still in the service of development of human society. In addition to the traditional textile industry, the silks are also expected to be used in medicine and health, beauty, optical, military, nanotechnology, environmental protection and other emerging industries.With respect to the applications of silk, the studies of how the silk fiber forms in silkworm（Bombyx mori） are much weaker. Currently, the specific mechanism for the silk fiber formation is still not fully elucidated. According to the present evidences, silk fiber formation is a process that the liquid silk protein changes its conformations and forms solid silk fiber. Based on the studies conducted in vitro, researchers had proposed a hypothesis for silk fiber formation. Secreted by silk gland, the silk protein is stored in liquid state. When silk protein flows into the anterior silk gland（ASG）, the narrow duct can provide the shearing forces on the protein. The shearing forces induce the rearrangement of silk protein molecules and make the molecules packed closely. The pH within the ASG duct is acidic. Under this acidic environment, the silk protein is dehydrated and concentrated. Meanwhile, the N-terminal dimerization occurs between silk fibroin molecules. The ASG duct also has a moderate ionic strength. Metal ions can electrostatically interact with the amino acid residues within the silk fibroin. The N-terminal dimerization and electrostatic interaction further make the molecules arranged closely, and the liquid silk protein changes to liquid crystalline state. When the silk protein flows into the spinneret, the powerful shearing forces produced by the muscle contraction act on the silk proteins. The silk protein is further dehydrated. Then, the silk protein is pulled out of the spigot by the swing of silkworm head. In this process, the silk protein molecules closely pack along the long axis of the fiber filaments. Finally, the solid silk fiber forms.Based on the above hypothesis, it can be easily found that the ASG and spinneret are the places for silk fiber formation. The studies for these two tissues are instructive for elucidating the mechanism of silk fiber formation. Thus, our group firstly introduced the proteomic techniques to study the protein components of ASG in 2013, which laid the foundation for our present study. Silkworm has several organs associated with the synthesis and secretion of silks such as silk gland, spinneret, and Filippi’s gland（FG）. However, due to its small size, the studies of the spinneret have so far only been inferred from structural analysis. A tiny gland called FG in the spinneret communicates with the ASG, and its function is unclear. The gene expression profiles and physiological roles of the spinneret and FG in the silk fiber formation are still unknown. Due to the complexity to simulate the biochemical and physiological environments for silk fiber formation, all the previous studies for silk fiber formation were operated in vitro. The mechanism of how silk protein changes its conformations and which factors are involved in the process in vivo are not clear. Whether metal ions are capable of binding with silk protein via electrostatic interactions and changing the conformations of silk proteins in vivo is still unknown. The mechanical properties of silk fiber are determined by the secondary structures of silk protein. The silk fiber formation is a process that silk protein changes its secondary structures, and also a process that silk fiber achieves its mechanical properties. As a result, the mechanical properties of silk fiber can be easily modified by artificially changing the secondary structures of silk protein. This is the main idea of the current study.Using a variety of techniques of molecular biology, chemistry, and mechanics of materials, our study is focus on the roles of metal ions in the silk fiber formation and the mechanical properties of silk fiber. Using the transgenic methods, the ionic environment of silk fiber formation was genetically interrupted. Furthermore, we obtained the mechanical properties improved silk fibers and established a novel method to modify the mechanical properties of silk fibers. The major achievements and conclusions are as following: 1. Comparative transcriptome analysis of spinneret and FGFirstly, we profiled more than 11000 transcripts from the spinneret and FG of silkworm larvae on day 3 of the fifth instar and wandering stage. Using COG and GO annotation, we found that most of these genes were involved in ion binding and transporting, chitin binding and metabolism, and cuticle construction. Thus we assumed that these genes might take parts in the silk fiber formation. A total of 59 ion-transporting protein genes were identified and 7 of them were calcium transporter. The most abundant ion-transporting protein gene in the spinneret encoded sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase（BmSERCA）. A large numbers of H⁺-transporting protein genes, including vacuolar-type proton ATPase（V-ATPase） genes, were found in spinneret. We also identified genes for transporting Na⁺, K⁺, Zn²⁺, Cu²⁺, and PO43-. These results indicated that the spinneret had an active ion transporting process and provided a suitable physiological and biochemical environment for silk fiber formation.Furthermore, differential expression analysis and GO enrichment of the differential expressed genes in the FG suggested a possible role of FG in transporting small molecules（such as water and ions） to the silk gland. Using the transcriptomic data, microarray database and RT-PCR analysis, we found that BGIBMGA013432 was one of the FG-specific genes. This gene was annotated as a putative transporter.2. Proteomic analysis of silkworm spinneretOn the basis of the transcriptomic data, our study further investigated the protein components of the spinneret from the two developmental stages mentioned above by LC-MS/MS. We totally identified 1355 and 1315 proteins in these two stages. The silk fiber formation related proteins, such as cuticle proteins, ion-transporting proteins, muscular movement associated proteins, and energy metabolic proteins, were abundant in the spinneret. By analyzing the signal pathways, we discovered that the energy metabolism and protein metabolism and degradation processes were active. Using label-free quantification, we compared the protein differences among these two stages and identified 232 differential expressed proteins. Among them, 184 were up-regulated and 48 were down-regulated. These differential expressed proteins were involved in energy metabolism, chitin binding and cuticle construction. The active energy metabolism might provide abundant energy for the muscle contraction, ion and water transporting. The chitin binding and cuticle construction process might provide sufficient shear force and self-protection for the silk fiber formation. Our results further confirmed that the spinneret provided a suitable physiological and biochemical environment for silk fiber formation. 3. Functional analysis of BmSERCABmSERCA was the most abundant ion-transporting protein gene in the spinneret. The bioinformatics analysis revealed that BmSERCA was highly conserved with SERCAs from other species. Then, we cloned this gene. A fragment encoding an intracellular part of BmSERCA was used for prokaryotic expression. Recombinant protein was expressed, purified, and used for generating a polyclonal anti-BmSERCA antibody. We also obtained the full-length recombinant BmSERCA by the baculovirus expression vector system. The recombinant proteins were about to express in cells 48 h post infection and highly expressed 96 h in cells post infection. Ca²⁺-ATPase activities assays were used to evaluate the enzyme activities of recombinant BmSERCA and found that the enzyme activities increased significantly after infection. We used qRT-PCR, western blotting and Ca²⁺-ATPase activities assays to analyze the expression pattern of BmSERCA and found that this gene was highly expressed in the ASG. To study the function of BmSERCA in silk fiber formation, we injected a specific inhibitor of SERCA（Thapsigargin, TG） into the silkworm. After injection of 2 nmol TG, silkworms showed a silk-spinning deficiency and their cocoons had higher calcium content compared to that of controls. The results provide evidence that BmSERCA has a key function in calcium transportation between silk and silk gland. Moreover, FTIR analysis revealed that the levels of α-helix and random coil structures increased in silk fibers from TG-injected silkworms compared to those of controls, while the levels of intermediate and β-turn structures decreased. These results suggested that BmSERCA was involved in the conformational transitions of silk protein. 4. Metal ions regulate the conformational transition and mechanical properties of silkWe investigated the metal ion content and the expression patterns of ion-transporting protein genes in different parts of silk gland. Our results showed that the content of Na, K, Cu, Mg, and Zn increased from the middle silk gland（MSG） to ASG, but calcium content decreased. Using qRT-PCR, we found most of the H⁺ transporters, Na⁺/K⁺ transporters, Ca²⁺ transporters, and Cu²⁺ transporters were highly expressed in the spinneret and ASG.Then, we explored the effects of metal ions on silk conformations and mechanical properties of silk fibers by adding them into the silk fibroin solutions or injecting them into the silkworms. CD analysis was used to monitor the conformational transitions of silk fibroin with metal ions. Our results showed that K⁺ dramatically promoted the formation of β-sheet structure, whereas Na⁺ had no obvious effects on the content of β-sheet structure. The effects of Ca²⁺ and Cu²⁺ were similar. They induced the β-sheet formation only in a particular concentration. To test whether metal ions were involved in the conformational transition of silk fibroin in vivo, we injected these metal ions into silkworms. Using FTIR, the structural changes were quantitatively evaluated. We found that after injection of K⁺ or Cu²⁺, the degree of β-sheet structure increased significantly. The silk fibers were stronger and stiffer. FTIR analysis and mechanical testing revealed that injection of Ca²⁺ significantly increased the levels of helical and random coil structures of silk proteins. In addition, the extension of silk fibers increased. 5. Modifying the mechanical properties of silk fiber by metal ionsUsing gene manipulation, we modified the mechanical properties of silk fiber by genetically disrupting the ionic environment for silk formation. We overexpressed BmSERCA and silkworm Na⁺/K⁺-ATPase（BmNKA） in the ASG by the spinning duct specific promoter BmCP231, respectively. After isolation of the transgenic silkworms, we found that the metal ion content of transgenic silk fibers changed accordingly. This illustrated that the ion-transporting process was disrupted after overexpression of these genes. The morphological differences of cocoons and fibers between transgenic silkworms and wild type were examined, and no remarkable differences were found. Overexpression of SERCA in ASG decreased the calcium content of silks. As a consequence, silk fibers had more helical and β-sheet conformations. Their tenacity increased 21%, extension increased 54%, and the toughness increased 73%. However, overexpression of BmNKA decreased the content of β-sheet structure, making the silk fibers weaker than the wild type fibers. These findings showed that changing the ionic environment for silk fiber formation is a good and convenient way to generate the silk fibers with improved mechanical properties, thus providing a novel method for modifying silk fiber properties.In conclusion, using novel perspective, systemic strategies, and multidisciplinary techniques, this dissertation revealed the expression of genes and proteins in the spinneret and FG of silkworm, and presented the solid evidences that the metal ions were responsible for silk fiber formation in vivo. The ionic environment for silk fiber formation was genetically disrupted by overexpression of ion-transporting proteins. Then we improved the mechanical properties of silk fiber without changing the gene sequence of the silk. Our innovations here will be a novel hope for elucidating the mechanism of silk fiber formation and the developments of new silk fibers.