The advances and perspectives of recombinant protein production in the silk gland of silkworm Bombyx mori

The silk gland of silkworm Bombyx mori, is one of the most important organs that has been fully studied and utilized so far. It contributes finest silk fibers to humankind. The silk gland has excellent ability of synthesizing silk proteins and is a kind tool to produce some useful recombinant proteins, which can be widely used in the biological, biotechnical and pharmaceutical application fields. It’s a very active area to express recombinant proteins using the silk gland as a bioreactor, and great progress has been achieved recently. This review recapitulates the progress of producing recombinant proteins and silk-based biomaterials in the silk gland of silkworm in addition to the construction of expression systems. Current challenges and future trends in the production of valuable recombinant proteins using transgenic silkworms are also discussed.     

家蚕前部丝腺特异表皮蛋白Bm11721的鉴定及表达

家蚕的丝腺是其丝蛋白合成和分泌的器官,根据其形态和功能的不同分为前部、中部和后部丝腺,前部丝腺不具有合成丝蛋白的能力,是丝蛋白构象发生转变的场所。剪切力在丝蛋白构象转变中起到重要的作用,其在家蚕前部丝腺主要由前部丝腺逐渐变细的管腔结构和富含几丁质及表皮蛋白的坚硬的内壁提供。鉴定家蚕前部丝腺新的几丁质结合蛋白,并调查其在家蚕幼虫不同组织的表达特征。通过几丁质亲和层析的方法在前部丝腺筛选并鉴定到一个新的具有几丁质结合功能的表皮蛋白Bm11721,其编码基因编号为BGIBMGA011721(Gen Bank Accession No.NM-001173285.1)。利用原核表达系统成功表达了该蛋白,通过Ni-NTA亲和层析的方法获得了Bm11721的重组蛋白并制备了多克隆抗体。组织表达分析发现无论是转录水平还是蛋白水平Bm11721均只在前部丝腺特异表达,且Bm11721蛋白在5龄期的前部丝腺中恒定表达。免疫荧光定位结果显示Bm11721蛋白定位在前部丝腺的内膜中,推测其可能与前部丝腺的机械硬度有关,为丝蛋白的构象转变提供剪切力。     

Alteration of tubuliform silk gland cytoarchitecture with the reproductive cycle of the Western black widow spider,Latrodectus hesperus

The protein synthetic and secretory activity of spider tubuliform glands is known to be coordinated with the reproductive stage of the spider. For spiders that produce multiple egg cases, such as the black widow Latrodectus hesperus, this means that the cells that make up the tubuliform gland cycle from minimal to maximal silk protein synthesis and exocytosis as the spider transitions from early vitellogenesis to a gravid state and back. The impact of these transitions on the cells that form the tubuliform gland has yet to be characterized. The entire tubuliform gland undergoes an elastic deformation, doubling in size in response to the accumulation and depletion of egg case silk proteins within its lumen. Similarly, the diversity and organization of organelles within the cytoplasm of the secretory epithelial cells that make up the wall of the tubuliform gland change with the reproductive stage of the spider. Progression of a spider from early to late vitellogenesis is accompanied by decondensed nucleoli and distention of the rough endoplasmic reticulum, markers of protein synthetic activity. The presumed silk proteins that fill the lumen of the tubuliform gland of a gravid spider include a fibrous matrix with homogeneous spherical inclusions. These components are also present within the cytoplasm of the cell; however, only the fibrous material appears to be enclosed by membranous organelles. Transition of the tubuliform gland from peak silk synthesis back to a quiescent state is marked by the appearance of multivesicular bodies and organelles resembling phagophores and autophagosomes, suggestive of a role for autophagy in the process of recovery. The reproducible cellular dynamics of the tubuliform silk gland of the black widow spider makes it a potential model system for study of the regulation of silk gene expression, endomembrane transport, and exocytosis of silk proteins and autophagy.     

Silks produced by insect labial glands

Insect silks are secreted from diverse gland types; this chapter deals with the silks produced by labial glands of Holometabola (insects with pupa in their life cycle). Labial silk glands are composed of a few tens or hundreds of large polyploid cells that secrete polymerizing proteins which are stored in the gland lumen as a semi?liquid gel. Polymerization is based on weak molecular interactions between repetitive amino acid motifs present in one or more silk proteins; cross?linking by disulfide bonds may be important in the silks spun under water. The mechanism of long?term storage of the silk dope inside the glands and its conversion into the silk fiber during spinning is not fully understood. The conversion occurs within seconds at ambient temperature and pressure, under minimal drawing force and in some cases under water. The silk filament is largely built of proteins called fibroins and in Lepidoptera and Trichoptera coated by glue?type proteins known as sericins. Silks often contain small amounts of additional proteins of poorly known function. The silk components controlling dope storage and filament formation seem to be conserved at the level of orders, while the nature of polymerizing motifs in the fibroins, which determine the physical properties of silk, differ at the level of family and even genus. Most silks are based on fibroin β?sheets interrupted with other structures such as α?helices but the silk proteins of certain sawflies have predominantly a collagen?like or polyglycine II arrangement and the silks of social Hymenoptera are formed from proteins in a coiled coil arrangement.     

Carbonic anhydrase generates a pH gradient in Bombyx mori silk glands

Silk is a protein of interest to both biological and industrial sciences. The silkworm, Bombyx mori, forms this protein into strong threads starting from soluble silk proteins using a number of biochemical and physical cues to allow the transition from liquid to fibrous silk. A pH gradient has been measured along the gland, but the methodology employed was not able to precisely determine the pH at specific regions of interest in the silk gland. Furthermore, the physiological mechanisms responsible for the generation of this pH gradient are unknown.In this study, concentric ion selective microelectrodes were used to determine the luminal pH of B. mori silk glands. A gradient from pH 8.2 to 7.2 was measured in the posterior silk gland, with a pH 7 throughout the middle silk gland, and a gradient from pH 6.8 to 6.2 in the beginning of the anterior silk gland where silk processing into fibers occurs. The small diameter of the most anterior region of the anterior silk gland prevented microelectrode access in this region. Using a histochemical method, the presence of active carbonic anhydrase was identified in the funnel and anterior silk gland of fifth instar larvae. The observed pH gradient collapsed upon addition of the carbonic anhydrase inhibitor methazolamide, confirming an essential role for this enzyme in pH regulation in the B. mori silk gland. Plastic embedding of whole silk glands allowed clear visualization of the morphology, including the identification of four distinct epithelial cell types in the gland and allowed correlations between silk gland morphology and silk stages of assembly related to the pH gradient.B. mori silk glands have four different epithelial cell types, one of which produces carbonic anhydrase. Carbonic anhydrase is necessary for the mechanism that generates an intraluminal pH gradient, which likely regulates the assembly of silk proteins and then the formation of fibers from soluble silk proteins. These new insights into native silk formation may lead to a more efficient production of artificial or regenerated silkworm silk fibers.     

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.     

Comparative Proteome Analysis Reveals that Cuticular Proteins Analogous to Peritrophin‐Motif Proteins are Involved in the Regeneration of Chitin Layer in the Silk Gland of Bombyx mori at the Molting Stage

The silk gland of silkworm produces silk proteins during larval development. Many studies have long focused on the silk gland of the fifth instar larvae, but few have investigated this gland at other larval stages. In the present study, the silk gland proteomes of the fourth instar and fourth molt are analyzed using liquid chromatography–tandem mass spectrometry. In total, 2654 proteins are identified from the silk gland. A high abundance of ribosomal proteins and RR‐motif chitin‐binding proteins is identified during day 2 of the fourth instar (IV‐2) larval developmental stage, and the expression of cuticular proteins analogous to peritrophin (CPAP)‐motif chitin‐binding proteins is higher during the fourth molt (IV‐M). In all, nine enzymes are found to be involved in the chitin regeneration pathway in the silk gland. Among them, two chitinase and two chitin deacetylases are identified as CPAP‐motif proteins. Furthermore, the expression of CPAP3‐G, the most abundant CPAP‐motif cuticular protein in the silk gland during the IV‐M stage, is investigated using western blot and immunofluorescence analyses; CPAP3‐G shows a reverse changing trend with chitin in the silk gland. The findings of this study suggest that CPAP‐motif chitin‐binding proteins are involved in the degradation of the chitin layer in the silk gland. The data have been deposited to the ProteomeXchange with identifier PXD008677.     

家蚕V型ATP酶B亚基的克隆及表达特征

V型ATP酶(Vacuolar-type ATPase)是一种定位于细胞膜和细胞器膜上的氢离子转运酶。它利用ATP水解的能量将氢离子转运到液泡、囊泡或者胞外,从而维持细胞内正常的酸碱环境。V型ATP酶B亚基(V-ATPase B)作为ATP的催化位点,也有着非常重要的作用。为了探讨家蚕V-ATPase B(Bm V-ATPase B)的功能,首先从家蚕五龄幼虫的中肠c DNA中克隆了Bm V-ATPase B基因并构建原核表达载体进行原核表达,获得了重组蛋白,经质谱鉴定正确后,通过镍柱亲和层析的方法纯化了该蛋白并制备了多克隆抗体;最后分析了该蛋白在家蚕丝腺中的表达特征并利用免疫荧光对其在丝腺中的表达位置进行了定位。结果显示Bm V-ATPase B基因序列全长1 473 bp,预测蛋白分子量55 k Da,预测等电点5.3。通过Western blotting对家蚕5龄第3天和上蔟第1天幼虫丝腺的不同区段进行Bm V-ATPase B蛋白的表达特征分析,发现在两个时期该蛋白均在前部丝腺高量表达,而在中部丝腺和后部丝腺表达量相对较低。进一步对两个时期丝腺的不同区段进行免疫荧光定位,发现该蛋白在两个时期的前部丝腺、中部丝腺和后部丝腺均定位于细胞层。利用激光共聚焦显微镜对该蛋白进行进一步的定位,发现该蛋白主要在丝腺的细胞膜表达。研究结果明确了该蛋白在丝腺中的表达模式,为深入研究该蛋白在蚕丝纤维形成中的作用奠定了基础。     

Gene expression analysis in the larval silk gland of the eri silkworm Samia ricini

Insects produce silk for a range of purposes. In the Lepidoptera, silk is utilized as a material for cocoon production and serves to protect larvae from adverse environmental conditions or predators. Species in the Saturniidae family produce an especially wide variety of cocoons, for example, large, golden colored cocoons and those with many small holes. Although gene expression in the silk gland of the domestic silkworm (Bombyx mori L.) has been extensively studied, considerably fewer investigations have focused on members of the saturniid family. Here, we established expression sequence tags from the silk gland of the eri silkworm (Samia ricini), a saturniid species, and used these to analyze gene expression. Although we identified the fibroin heavy chain gene in the established library, genes for other major silk proteins, such as fibroin light chain and fibrohexamerin, were absent. This finding is consistent with previous reports that these latter proteins are lacking in saturniid silk. Recently, a series of fibrohexamerin‐like genes were identified in the Bombyx genome. We used this information to conduct a detailed analysis of the library established here. This analysis identified putative homologues of these genes. We also found several genes encoding small silk protein molecules that are also present in the silk of other Lepidoptera. Gene expression patterns were compared between eri and domestic silkworm, and both conserved and nonconserved expression patterns were identified for the tested genes. Such differential gene expression might be one of the major causes of the differences in silk properties between these species. We believe that our study can be of value as a basic catalogue for silk gland gene expression, which will yield to the further understanding of silk evolution.     

Combined Effect of Cameo2 and CBP on the Cellular Uptake of Lutein in the Silkworm,Bombyx mori

Formation of yellow-red color cocoons in the silkworm, Bombyx mori, occurs as the result of the selective delivery of carotenoids from the midgut to the silk gland via the hemolymph. This process of pigment transport is thought to be mediated by specific cellular carotenoids carrier proteins. Previous studies indicated that two proteins, Cameo2 and CBP, are associated with the selective transport of lutein from the midgut into the silk gland in Bombyx mori. However, the exact roles of Cameo2 and CBP during the uptake and transport of carotenoids are still unknown. In this study, we investigated the respective contributions of these two proteins to lutein and β-carotene transport in Bombyx mori as well as commercial cell-line. We found that tissues, expressed both Cameo2 and CBP, accumulate lutein. Cells, co-expressed Cameo2 and CBP, absorb 2 fold more lutein (P<0.01) than any other transfected cells, and the rate of cellular uptake of lutein was concentration-dependent and reached saturation. From immunofluorescence staining, confocal microscopy observation and western blot analysis, Cameo2 was localized at the membrane and CBP was expressed in the cytosol. What’s more, bimolecular fluorescence complementation analysis showed that these two proteins directly interacted at cellular level. Therefore, Cameo2 and CBP are necessarily expressed in midguts and silk glands for lutein uptake in Bombyx mori. Cameo2 and CBP, as the membrane protein and the cytosol protein, respectively, have the combined effect to facilitate the cellular uptake of lutein.     

Dragline Silk Spinning in the Orb Web Spider Nephila clavata

Although many researches have revealed that liquid phase of silk in the ampulla is turning into the polymerized dragline silk fibers as the feedstock passes through the long duct, the exact mechanism has still been not fully understood. Spider's strongest silk fiber, dragline, is mainly produced in the large ampullate glands, the biggest silk gland in the abdomen with a distinctive yellow color. Morphologically, the duct of large ampullate gland is in its unique S‐shape with 2 loops dividing the entire duct into three limbs. In addition, the diameter of the duct showed radical decrease toward the nozzle of the duct. Therefore, it assumed that the duct is playing a significant role in the entire process of silk production allowing great strength. Here, we present some of the fine structural properties of the large ampullate gland duct in Nephila clavata using various visualizations techniques.     

Spider silk protein refolding is controlled by changing pH

Spidroins, the major silk proteins making up the spider's dragline silk, originate in two distinct tissue layers (A and B) in the spider's major ampullate gland. Formation of the complex thread from spidroins occurs in the lumen of the duct connected to the gland. Using pH-sensitive microelectrode probes, we showed that the spidroins traveling through the gland and duct experience a monotonic decrease in pH from 7.2 to 6.3. In addition, circular dichroism spectroscopy of material extracted from the gland showed a structural refolding concomitant with position in the gland and post-extraction changes in pH. We demonstrate that lowering the pH in vitro causes a dramatic conformational change in the protein from the A zone, converting it irreversibly from a coil to a predominantly beta-sheet structure. Furthermore, amino acid analyses have indicated that there are at least two distinct, though similar, proteins secreted in the A and B zones suggesting a potential factor in the progressive acidification as well as a pH sensitivity of the folding of spidroins in the gland. Thus, we provide, for the first time, a quantitative map of the pH value and position correlated with molecular structural folding in the silk gland characterizing the crucial role that pH plays in spider silk formation.     

Liquid crystals and flow elongation in a spider's silk production line

Our observations on whole mounted major ampullate silk glands suggested that the thread is drawn from a hyperbolic die using a pre-orientated lyotropic liquid crystalline feedstock. Polarizing microscopy of the gland''s duct revealed two liquid crystalline optical textures: a curved pattern in the feedstock within the ampulla of the gland and, later in the secretory pathway, the cellular texture previously identified in synthetic nematic liquid crystals. The behaviour of droplet inclusions within the silk feedstock indicated that elongational flow at a low shear rate occurs in the gland''s duct and may be important in producing an axial molecular orientation before the final thread is drawn. Our observations suggested that the structure of the spider''s silk production pathway and the liquid crystalline feedstock are both involved in defining the exceptional mechanical properties of spider dragline silk.     

DNA replication events during larval silk gland development in the silkworm, Bombyx mori

The silk gland is an important organ in silkworm as it synthesizes silk proteins and is critical to spinning. The genomic DNA content of silk gland cells dramatically increases 200-400 thousand times for the larval life span through the process of endomitosis. Using in vitro culture, DNA synthesis was measured using BrdU labeling during the larval molt and intermolt periods. We found that the cell cycle of endomitosis was activated during the intermolt and was inhibited during the molt phase. The anterior silk gland, middle silk gland, and posterior silk gland cells asynchronously exit the endomitotic cycle after day 6 in 5th instar larvae, which correlated with the reduced expression of the cell cycle-related cdt1, pcna, cyclin E, cdk2 and cdk1 mRNAs in the wandering phase. Additional starvation had no effect on the initiation of silk gland DNA synthesis of the freshly ecdysed larvae.     

Secondary structures and conformational changes in flagelliform, cylindrical, major, and minor ampullate silk proteins. Temperature and concentration effects

Orb weaver spiders use exceptionally complex spinning processes to transform soluble silk proteins into solid fibers with specific functions and mechanical properties. In this study, to understand the nature of this transformation we investigated the structural changes of the soluble silk proteins from the major ampullate gland (web radial threads and spider safety line); flagelliform gland (web sticky spiral threads); minor ampullate gland (web auxiliary spiral threads); and cylindrical gland (egg sac silk). Using circular dichroism, we elucidated (i) the different structures and folds for the various silk proteins; (ii) irreversible temperature-induced transitions of the various silk structures toward beta-sheet-rich final states; and (iii) the role of protein concentration in silk storage and transport. We discuss the implication of these results in the spinning process and a possible mechanism for temperature-induced beta-sheet formation.     

Novel structures in secreting the androgenic gland hormone

The secretory granules in the androgenic gland of the terrestrial isopod Armadillidium vulgare, which have been indistinct for long time because of vulnerable structures, were revealed by using the rapid-freezing and freeze-substitution method. The fine structure of the androgenic gland is conspicuous by the distribution of numerous particular organelles in the cytoplasm consisting of the endoplasmic reticulum and the Golgi complex, and by having a number of highly organized structures developed between the androgenic gland cells. The structures connect to the intercellular space, which is seen as intercellular canaliculi for exporting the androgenic gland hormone. The plasma membranes near the particular structure of the intercellular canaliculi in the androgenic gland are often specialized to form cellular junctions. The secretory granules including the electron-dense materials, which are supposed to be peptides of androgenic gland hormone, are distributed beside the particular structure of the intercellular canaliculi. Some of the granules are seen to fuse with the plasma membranes. This observation suggests that, in the Armadillidium vulgare, the secretory granules containing androgenic gland hormone are transferred to the extracellular space through the intercellular canaliculi particularly developed for exporting the peptide hormone. This is the first evidence to show the secretory mechanism of the androgenic gland hormone in the Isopoda.