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.     

Shotgun proteomic analysis of the Bombyx mori anterior silk gland: An insight into the biosynthetic fiber spinning process

The Bombyx mori anterior silk gland (ASG) is a natural fiber manipulator for the material provided by the middle and posterior silk glands. In view of the significant role of the ASG in the liquid–crystal spinning process, a shotgun proteomics approach was taken to study the relationship between the function of proteins in the silkworm ASG and the spinning mechanism. A total of 1132 proteins with 7647 unique peptides were identified in the ASG dataset including some involved in the cuticle, ion transportation, energy metabolism, and apoptosis. Two putative cuticle‐specific proteins were highly and specifically expressed in the ASG; therefore, the ASG dataset could provide clues for comprehensive understanding of the natural silk spinning mechanism in the silkworm. All MS data have been deposited in the ProteomeXchange with identifier PXD000090.     

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。     

Genome‐wide identification of chitin‐binding proteins and characterization of BmCBP1 in the silkworm,Bombyx mori

The insect cuticle plays important roles in numerous physiological functions to protect the body from invasion of pathogens, physical injury and dehydration. In this report, we conducted a comprehensive genome-wide search for genes encoding proteins with peritrophin A-type (ChtBD2) chitin-binding domain (CBD) in the silkworm, Bombyx mori. One of these genes, which encodes the cuticle protein BmCBP1, was additionally cloned, and its expression and location during the process of development and molting in B. mori were investigated. In total, 46 protein-coding genes were identified in the silkworm genome, including those encoding 15 cuticle proteins analogous to peritrophins with one CBD (CPAP1s), nine cuticle proteins analogous to peritrophins with three CBD (CPAP3s), 15 peritrophic membrane proteins (PMPs), four chitinases, and three chitin deacetylases, which contained at least one ChtBD2 domain. Microarray analysis indicated that CPAP-encoding genes were widely expressed in various tissues, whereas PMP genes were highly expressed in the midgut. Quantitative polymerase chain reaction and western blotting showed that the cuticle protein BmCBP1 was highly expressed in the epidermis and head, particularly during molting and metamorphosis. An immunofluorescence study revealed that chitin co-localized with BmCBP1 at the epidermal surface during molting. Additionally, BmCBP1 was notably up-regulated by 20-hydroxyecdysone treatment. These results provide a genome-level view of the chitin-binding protein in silkworm and suggest that BmCBP1 participates in the formation of the new cuticle during molting.     

Analysis of the chitin recognition mechanism of cuticle proteins from the soft cuticle of the silkworm, Bombyx mori

Insect cuticle is composed mainly of chitin, a polymer of N-acetylglucosamine, and chitin-binding cuticle proteins. Four major cuticle proteins, BMCP30, 22, 18, and 17, have been previously identified and purified from the larval cuticle of silkworm, B. mori. We analyzed the chitin-binding activity of BMCP30 by use of chitin-affinity chromatography. The pH optimum for the binding of BMCP30 to chitin is 6.4, which corresponds to hemolymph pH. Competition experiments using chitooligosaccharides suggested that BMCP30 recognizes 4-6 mer of N-acetylglucosamine in chitin fiber as a unit for binding. The comparison of the binding properties of BMCP30 with those of BMCP18 showed that their binding activities to chitin are similar in a standard buffer but that BMCP30 binds to chitin more stably than BMCP18 in the presence of urea. BMCPs possess the RR-1 form of the R&R consensus, about 70 amino acids region conserved widely among cuticle proteins mainly from the soft cuticle of many insect and arthropod species. Analysis of the binding activity using deletion mutants of BMCPs revealed that this type of conserved region also functions as the chitin-binding domain, similarly to the RR-2 region previously shown to confer chitin binding. Thus, the extended R&R consensus is the general chitin-binding domain of cuticle proteins in Arthropoda.     

Comparative proteomics analysis of silkworm hemolymph during the stages of metamorphosis via liquid chromatography and mass spectrometry

The silkworm is a lepidopteran insect that has an open circulatory system with hemolymph consisting of blood and lymph fluid. Hemolymph is not only considered as a depository of nutrients and energy, but it also plays a key role in substance transportation, immunity response, and proteolysis. In this study, we used LC‐MS/MS to analyze the hemolymph proteins of four developmental stages during metamorphosis. A total of 728 proteins were identified from the hemolymph of the second day of wandering stage, first day of pupation, ninth day of pupation, and first day as an adult moth. GO annotations and categories showed that silkworm hemolymph proteins were enriched in carbohydrate metabolism, proteolysis, protein binding, and antibacterial humoral response. The levels of nutrient, immunity‐related, and structural proteins changed significantly during development and metamorphosis. Some, such as cuticle, odorant‐binding, and chemosensory proteins, showed stage‐specific expression in the hemolymph. In addition, the expression of several antimicrobial peptides exhibited their highest level of abundance in the hemolymph of the early pupal stage. These findings provide a comprehensive proteomic insight of the silkworm hemolymph and suggest additional molecular targets for studying insect metamorphosis.     

Comparative proteomic analysis between the domesticated silkworm (Bombyx mori) reared on fresh mulberry leaves and on artificial diet

To gain an insight into the effects of different diets on growth and development of the domesticated silkworm at protein level, we employed comparative proteomic approach to investigate the proteomic differences of midgut, hemolymph, fat body and posterior silk gland of the silkworms reared on fresh mulberry leaves and on artificial diet. Seventy-six differentially expressed proteins were identified by MALDI TOF/TOF MS, and among them, 41 proteins were up-regulated, and 35 proteins were downregulated. Database searches, combined with GO analysis and KEGG pathway analysis revealed that some hemolymph proteins such as Nuecin, Gloverin-like proteins, PGRP, P50 and beta/-N-acetylglucosamidase were related to innate immunity of the silkworm, and some proteins identified in silkworm midgut including Myosin 1 light chain, Tropomyosin 1, Profilin, Serpin-2 and GSH-Px were involved in digestion and nutrition absorption. Moreover, two up-regulated enzymes in fat body of larvae reared on artificial diet were identified as V-ATPase subunit B and Arginine kinase which participate in energy metabolism. Furthermore, 6 down-regulated proteins identified in posterior silk gland of silkworm larvae reared on artificial diet including Ribosomal protein SA, EF-2, EF-1gamma, AspAT, ERp57 and PHB were related to silk synthesis. Our results suggested that the different diets could alter the expression of proteins related to immune system, digestion and absorption of nutrient, energy metabolism and silk synthesis poor nutrition and absorption of nutrition in silkworm. The results also confirmed that the poor nutrient absorption, weakened innate immunity, decreased energy metabolism and reduced silk synthesis are the main reasons for low cocoons yield, inferior filament quality, low survival rate of young larvae and insufficient resistance against specific pathogens in the silkworms fed on artificial diet.     

Identification and characterization of two chitin-binding proteins from the peritrophic membrane of the silkworm, Bombyx mori L

The peritrophic membrane (PM) is a semi‐permeable lining of the insect midgut, broadly analogous to the mucous lining of vertebrate gut. The PM proteins are important achievements for the function of the PM. In this study, two chitin‐binding proteins (BmPM‐P43 and BmPM‐P41) from the PM of the silkworm, Bombyx mori, were identified and cloned. These proteins showed the molecular mass of 43 and 41 kDa, respectively. The deduced amino acid sequences codes for a protein of 381 amino acid residues and 364 amino acid residues, containing 12 and 14 cysteine residues followed by similar domain, both of them have 5 cysteine residues in similar position in the C‐terminal. The confirmation of these proteins was performed by western blot analysis of recombinant BmPM‐P43 and BmPM‐P41. The chitin‐binding activity analysis showed that the BmPM‐P43 and BmPM‐P41 could bind to chitin strongly. It is concluded that BmPM‐P43 and BmPM‐P41 contains a polysaccharide deacetylase domain instead of peritrophin domain, indicated that these two proteins may belong to a new chitin‐binding protein family. © 2010 Wiley Periodicals, Inc.     

D‐3‐phosphoglycerate dehydrogenase from the silkworm Bombyx mori: Identification,functional characterization,and expression

D‐3‐phosphoglycerate dehydrogenase (PHGDH) is a key enzyme involved in the synthesis of l ‐serine. Despite the high serine content in silk proteins and the crucial role of PHGDH in serine biosynthesis, PHGDH has not been described in silkworms to date. Here, we identified PHGDH in the silkworm Bombyx mori and evaluated its biochemical properties. On the basis of the amino acid sequence and phylogenetic tree, this PHGDH has been categorized as a new type and designated as bmPHGDH. The recombinant bmPHGDH was overexpressed and purified to homogeneity. Kinetic studies revealed that PHGDH uses NADH as a coenzyme to reduce phosphohydroxypyruvate. High expression levels of bmphgdh messenger RNA (mRNA) were observed in the middle part of the silk gland and midgut in a standard strain of silkworm. Moreover, a sericin‐deficient silkworm strain displayed reduced expression of bmphgdh mRNA. These findings indicate that bmPHGDH might play a crucial role in the provision of l ‐serine in the larva of B. mori.     

家蚕精巢蛋白质的双向电泳及质谱分析

精巢是雄性家蚕Bombyx mori的生殖腺,它的主要功能是产生精子,全面检测和鉴定精巢器官的蛋白分布将为分析家蚕雄性个体的发育和繁殖奠定基础。本研究利用双向聚丙烯酰胺凝胶电泳和蛋白硝酸银染色技术对家蚕5龄第5天幼虫的精巢组织进行了蛋白检测,利用基质辅助激光解析质量飞行时间质谱（MALDI-TOF-MS）对表达量较高的蛋白点进行了肽质量指纹图谱鉴定。结果表明：家蚕精巢蛋白质可以检测出1 000个以上的蛋白点,这些蛋白点主要集中在分子量为15～90 kD区域,等电点3.5～9之间,其中60个蛋白点得到了成功鉴定,按照已知或推测的蛋白功能,将其分为8类,包括：细胞骨架和细胞结构蛋白,膜蛋白或信号相关蛋白,大量应激反应蛋白（伴侣蛋白）,线粒体和能量产生相关蛋白,转录调控和翻译及DNA/RNA结合相关蛋白,酶和少量血液组成蛋白。其中很多蛋白可能在鞭毛形成、能量代谢及减数分裂过程中有重要作用。这些结果为进一步认识家蚕精子形成过程提供了重要的生物学信息。     

A structural model of the chitin-binding domain of cuticle proteins

The nature of the interaction of insect cuticular proteins and chitin is unknown even though about half of the cuticular proteins sequenced thus far share a consensus region that has been predicted to be the site of chitin binding. We previously predicted the preponderance of beta-pleated sheet in the consensus region and proposed its responsibility for the formation of helicoidal cuticle (Iconomidou et al., Insect Biochem. Mol. Biol. 29 (1999) 285). Consequently, we have also verified experimentally the abundance of antiparallel beta-pleated sheet in the structure of cuticle proteins (Iconomidou et al., Insect Biochem. Mol. Biol. 31 (2001) 877). In this work, based on sequence and secondary structure similarity of cuticle proteins, and especially that of the consensus motif, to that of bovine plasma retinol binding protein (RBP), we propose by homology modelling an antiparallel beta-sheet half-barrel structure as the basic folding motif of cuticle proteins. This folding motif may provide the template for elucidating cuticle protein-chitin interactions in detail and reveal the precise geometrical formation of cuticle's helicoidal architecture. This predicted motif is another example where nature utilizes an almost flat protein surface covered by aromatic side chains to interact with the polysaccharide chains of chitin.     

Fibroin silk proteins from the nonmulberry silkworm Philosamia ricini are biochemically and immunochemically distinct from those of the mulberry silkworm Bombyx mori

Silk proteins were isolated from the cocoons of the nonmulberry silkworm, Philosamia ricini. Three polypeptides of 97, 66, and 45 kDa were identified. The 66-kDa molecule represented sericin, whereas the 97-kDa and the 45-kDa polypeptides linked together through a disulfide bond constituted the fibroin protein. Antibodies raised against the 97-kDa P. ricini fibroin heavy chain reacted specifically with this molecule and did not recognize fibroin heavy chain from another nonmulberry silkworm, Antheraea assama or from the mulberry silkworm, Bombyx mori, suggesting the presence of P. ricini species-specific determinants in this heavy chain. Antibodies generated against fibroin light chain of P. ricini also showed similar reactivity pattern. Immunoblot analysis with proteins isolated from the silk glands of P. ricini at different stages of larval development showed that the expression of fibroin heavy chain was developmentally and spatially regulated. The protein was most abundant in the 5th instar larva, and could be detected in the middle and the posterior but not the anterior silk glands. The amino acid composition of the 97-kDa fibroin protein showed abundance of glutamic acid and did not contain (Gly-Ala)(n) motifs, a characteristic feature of B. mori fibroin heavy chain. Our study reveals significant differences between the nonmulberry silkworm P. ricini and the mulberry silkworm B. mori in the biochemical composition and immunochemical characteristics of fibroin heavy chain. These differences might be responsible for the differences seen in the quality of silk produced by these two silkworms.     

Analysis of expression and chitin‐binding activity of the wing disc cuticle protein BmWCP4 in the silkworm,Bombyx mori

The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin‐binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic “R&R” chitin‐binding domain (CBD). Western blotting showed that BmWCP4 was predominately expressed in the wing disc‐containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full‐length BmWCP4 protein, “R&R” CBD peptide (CBD), non‐CBD peptide (BmWCP4‐CBD^?), four single site‐directed mutated peptides (M₁, M₂, M₃ and M₄) and four‐sites‐mutated peptide (M_F) were generated and purified, respectively, for in vitro chitin‐binding assay. The results indicated that both the full‐length protein and the “R&R” CBD peptide could bind with chitin, whereas the BmWCP4‐CBD^? could not bind with chitin. The single residue mutants M₁, M₂, M₃ and M₄ reduced but did not completely abolish the chitin‐binding activity, while four‐sites‐mutated protein M_F completely lost the chitin‐binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva‐to‐pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein.     

Chitin in the Silk Gland Ducts of the Spider Nephila edulis and the Silkworm Bombyx mori

Here we report the detection and localisation of chitin in the cuticle of the spinning ducts of both the spider Nephila edulis and the silkworm Bombyx mori. Our observations demonstrate that the duct walls of both animals contain chitin notwithstanding totally independent evolutionary pathways of the systems. We conclude that chitin may well be an essential component for the construction of spinning ducts; we further conclude that in both species chitin may indicate the evolutionary origin of the spinning ducts.