[Identification and expression pattern analysis of a secretory phospholipase A2 gene in Bombyx mori].

Phospholipase A2 (PLA2) is widely distributed in animals, plants, and microorganisms, and it plays an important role in many physiological activities. In a previous study, we have identified a secretory PLA2 in Bombyx mori (BmsPLA2-1-1). In this study, we further identified four new sPLA2 genes (BmsPLA2-1-2, BmsPLA2-2, BmsPLA2-3, and BmsPLA2-4) in B. mori genome. All four genes exhibits the characteristic features of sPLA2, including the sPLA2 domain, metal binding sites, and highly conserved catalytic domain. This study completed the cloning, in vitro expression, and expression pattern analysis of the BmsPLA2-4 gene in B. mori. The full length of BmsPLA2-4 is 585 bp, and the recombinant protein obtained through prokaryotic expression has an estimated size of 25 kDa. qRT-PCR analysis revealed that the expression level of BmsPLA2-4 reached its peak on the first day of the fifth instar larval stage. Tissue expression profiling analysis showed that BmsPLA2-4 had the highest expression level in the midgut, followed by the epidermis and fat body. Western blotting analysis results were consistent with those of qRT-PCR. Furthermore, after infecting fifth instar 1-day-old larvae with Escherichia coli and Staphylococcus aureus, the expression level of the BmsPLA2-4 gene significantly increased in 24 h. The findings of this study provides a theoretical basis and valuable experimental data for future related research.

Post-ingestive stability of a mulberry Kunitz-type protease inhibitor MnKTI-1 in the digestive lumen of silkworm: dual inhibition towards α-amylase and serine protease.

BACKGROUND: Adaptation of specialist insects to their host plants and defense responses of plants to phytophagous insects have been extensively recognized while the dynamic interaction between these two events has been largely underestimated. Here, we provide evidence for characterization of an unrevealed dynamic interaction mode of digestive enzymes of specialist insect silkworm and inhibitor of its host plant mulberry tree. RESULTS: MnKTI-1, a mulberry Kunitz-type protease inhibitor, whose messenger RNA (mRNA) transcription and protein expression in mulberry leaf were severely triggered and up-regulated by tens of times in a matter of hours in response to silkworm, Bombyx mori, and other mulberry pest insects, suggesting a quick response and broad spectrum to insect herbivory. MnKTI-1 proteins were detected in gut content and frass of specialist B. mori, and exhibited significant post-ingestive stability. Recombinant refolded MnKTI-1 (rMnKTI-1) displayed binding affinity to digestive enzymes and a dual inhibitory activity to α-amylase BmAmy and serine protease BmSP2956 in digestive juice of silkworm. Moreover, data from in vitro assays proved that the inhibition of recombinant rMnKTI-1 to BmAmy can be reverted by pre-incubation with BmSP15920, an inactivated silkworm digestive protease that lack of complete catalytic triad. CONCLUSION: These findings demonstrate that mulberry MnKTI-1 has the potential to inhibit the digestive enzyme activities of its specialist insect herbivore silkworm, whereas this insect may employ inactivated proteases to block protease inhibitors to accomplish food digestion. The current work provides an insight to better understand the interacting mode between host plant Kunitz protease inhibitors and herbivorous insect digestive enzymes. © 2024 Society of Chemical Industry.

Bombyx mori-derived aldo-keto reductase AKR2E8 detoxifies aldehydes present in mulberry leaves.

Lepidopterans are agricultural pests. Since the silkworm is a model for lepidopterans, analysis of the enzymes produced by silkworms is of great interest for developing methods of pest control. The aldo-keto reductase (AKR) superfamily catalyzes the reduction of aldehydes by converting a carbonyl group to an alcohol group. Here, we characterized a new AKR present in the silkworm Bombyx mori, which has been designated as AKR2E8. Amino acid sequence and phylogenetic analyses showed that AKR2E8 is similar to human AKR1B1 and AKR1B10. Three amino acid residues in the active site were identical among AKR2E8, AKR1B1, and AKR1B10. Recombinant AKR2E8 overexpressed in Escherichia coli used nicotinamide adenine dinucleotide phosphate as a coenzyme to reduce the aldehydes present in mulberry (Morus alba) leaves. AKR2E8 was found to reduce benzaldehyde, hexanal, heptanal, nonanal, trans-2-nonenal, and citral. No nicotinamide adenine dinucleotide-dependent activity was detected. Akr2e8 mRNA was detected in the testes, ovaries, and fat body; the highest expression was found in the midgut. The substrate specificity and highest observed expression of AKR2E8 in the midgut suggests that AKR2E8 may play a major role in aldehyde detoxification in silkworms. The findings of this study may assist in the development of pest control methods for controlling the population of lepidopterans, such as silkworms, that damage crops.

Genome-wide identification of lipases in silkworm (Bombyx mori) and their spatio-temporal expression in larval midgut.

Lipases play crucial roles in food digestion by degrading dietary lipids into free fatty acids and glycerols. The domesticated silkworm (Bombyx mori) has been widely used as an important Lepidopteran model for decades. However, little is known about the lipase gene family in the silkworm, especially their hydrolytic activities as digestive enzymes. In this study, a total of 38 lipase genes were identified in the silkworm genome. Phylogenetic analysis indicated that they were divided into three major groups. Twelve lipases were confirmed to be expressed in the midgut at both transcriptional and translational levels. They were grouped into the same gene cluster, suggesting that they could have similar physiological functions. Quantitative real-time PCR (qRT-PCR) analyses indicated that lipases were mainly expressed in anterior and middle midgut regions, and their expression levels varied greatly along the length of midgut. A majority of lipases were down-regulated in the midgut when larvae stopped feeding. However, a unique lipase gene (Bmlip10583) showed low expression level during feeding stage, but it was significantly up-regulated during the larvae-pupae transition. These results demonstrated that expression of silkworm lipases was spatially and temporally regulated in the midgut during larval development. Taken together, our results provide a fundamental research of the lipase gene family in the silkworm.

Heat shock protein 70, glutamate dehydrogenase, and angiotensin-converting enzyme of Bombyx mori mediate the cell attachment of Cypovirus 1.

Dendrolimus punctatus causes great damage to pine forests worldwide. Dendrolimus punctatus cypovirus 1 (DpCPV-1) is an important pathogen of D. punctatus. However, the mechanism of DpCPV-1 cell entry has not been elucidated. In this study, we revealed that both GTase and MTase domains of VP3 (B-spike) and VP4 (A-spike) of DpCPV-1 interacted with the midgut proteins of Bombyx mori. Binding and competition assays revealed that GTase, MTase and VP4 played roles as viral attachment proteins. Far-Western blotting and LC-MS/MS analyses identified that heat shock protein 70 (BmHSP70), glutamate dehydrogenase (BmGDH), and angiotensin-converting enzyme (BmACE) in the midgut proteins as ligand candidates of the viral attachment proteins, and this was further verified by co-immunoprecipitation and fluorescence co-localization assays. Viral binding to the host midgut in vitro was inhibited by pre-treating B. mori midgut proteins with anti-BmHSP70, anti-BmGDH, anti-BmACE antibodies singly and in combination. Incubating DpCPV-1 virions with prokaryotically expressed BmHSP70, BmGDH, and BmACE also decreased viral attachment to the host midgut. In vivo bioassays revealed that viral infection in Helicoverpa armigera was partially neutralized by BmHSP70, BmGDH, and BmACE. Taking together, we concluded that HSP70, GDH, and ACE mediate DpCPV attachment and entry via binding to the viral attachment proteins, VP3 and VP4. The findings provide foundation for further understanding the entry mechanisms of cypoviruses.

Heterogeneous dissociation process of truncated RNAs by oligomerized Vasa helicase.

RNA helicases are enzymes that generally unwind double-stranded RNA using ATP hydrolysis energy, mainly involved in RNA metabolism, transcription, translation, and mRNA splicing. While the helicase core is crucial for RNA unwinding activity, N- and C-terminal extensions of specific helicases may contain an intrinsically disordered region for electrostatic interaction, resulting in the formation of droplets in the cytoplasm. However, how the disordered region of the RNA helicase contributes to RNA unwinding and dissociation remains unclear. Here, we focused on Bombyx mori Vasa, which unwinds truncated target transposon RNAs from the piRNA-induced silencing complex piRISC. In this study, we used single-molecule techniques to visualise how Vasa dynamically interacts with piRISC and investigate how Vasa oligomerization is involved in the process of piRNA amplification, named the ping-pong pathway. We found that Vasa's oligomerization is required during these processes in vitro and in vivo, and that Vasa triggers the dissociation of truncated RNA in heterogeneous pathways. Our single-molecule results suggest that oligomerized Vasa guides the timing of the process regulating overall dissociation efficiency.

Structural basis for juvenile hormone biosynthesis by the juvenile hormone acid methyltransferase.

Juvenile hormone (JH) acid methyltransferase (JHAMT) is a rate-limiting enzyme that converts JH acids or inactive precursors of JHs to active JHs at the final step of JH biosynthesis in insects and thus presents an excellent target for the development of insect growth regulators or insecticides. However, the three-dimensional properties and catalytic mechanism of this enzyme are not known. Herein, we report the crystal structure of the JHAMT apoenzyme, the three-dimensional holoprotein in binary complex with its cofactor S-adenosyl-l-homocysteine, and the ternary complex with S-adenosyl-l-homocysteine and its substrate methyl farnesoate. These structures reveal the ultrafine definition of the binding patterns for JHAMT with its substrate/cofactor. Comparative structural analyses led to novel findings concerning the structural specificity of the progressive conformational changes required for binding interactions that are induced in the presence of cofactor and substrate. Importantly, structural and biochemical analyses enabled identification of one strictly conserved catalytic Gln/His pair within JHAMTs required for catalysis and further provide a molecular basis for substrate recognition and the catalytic mechanism of JHAMTs. These findings lay the foundation for the mechanistic understanding of JH biosynthesis by JHAMTs and provide a rational framework for the discovery and development of specific JHAMT inhibitors as insect growth regulators or insecticides.

Cloning, expression, and characteristic analysis of the novel ß-galactosidase from silkworm, Bombyx mori.

ß-Galactosidase is a critical exoglycosidase involved in the hydrolysis of lactose, the modification and degradation of glycoprotein in vivo. In this study, the ß-galactosidase gene of silkworm (BmGal), whose cDNA comprises 11 exons and contains an intact ORF of 1,821 bp, was cloned. The protein sequence of BmGal showed high similarity with other known insect ß-galactosidases. No activity of the BmGal expressed in Escherichia coli or Pichia pastoris was detected while it was successfully expressed with high enzyme activity in baculovirus expression system in silkworm, and the electrophoresis result revealed that the BmGal showed activity in oligomer mode. Enzyme activity assay showed that its optimum pH was 8.4 and its optimum temperature was 40 °C. What is more, we found that iron ions can stimulate the activity of the enzyme while cobalt, nickel, or lead ions can inhibit its activity significantly. Besides, the temporal-spatial transcription pattern of the BmGal mRNA level was analyzed, which showed that BmGal was transcribed at the highest level in the fifth larval instar but relatively low level in the pupal and adult stage, and the highest transcriptional level of BmGal was found in testis among all the tissues concerned.

A Matrix Metalloproteinase Mediates Tracheal Development in Bombyx mori.

The trachea of insects is a tubular epithelia tissue that transports oxygen and other gases. It serves as a useful model for the studying of the cellular and molecular events involved in epithelial tube formation. Almost all of the extracellular matrix can be degraded by Matrix metalloproteinases (MMPs), which is closely related to the processes of development and regeneration. The regulation of trachea by MMPs is roughly known in previous studies, but the detailed regulation mechanism and involved gene function are not fully explored. In this article, we found MMP1 expressed highly during tracheal remodeling, and knocked out it makes the tracheal branch number reduced in Bombyx mori. In trachea of transgenic BmMMP1-KO silkworm, the space expanding of taenidium and epidermal cells and the structure of apical membrane were abnormal. To explore the underlying mechanism, we detected that DE-cadherin and Integrin ß1 were accumulated in trachea of transgenic BmMMP1-KO silkworm by immunohistochemistry. Moreover, 5-Bromo-2'-Deoxyuridine (BrdU) labeling showed that knockout of BmMMP1 in silkworm inhibited tracheal cell proliferation, and BmMMP1 also regulated the proliferation and migration of BmNS cells. All of the results demonstrated that BmMMP1 regulates the development of the tracheal tissue by expanding the space of tracheal cuticles and increases the number of tracheal branches by degrading DE-cadherin and Integrin ß1.

Trypsin-type serine protease p37k hydrolyzes CPAP3-type cuticle proteins in the molting fluid of the silkworm Bombyx mori.

Cuticular proteins analogous to peritrophin 3 (CPAP3)-type cuticle proteins constitute a family of proteins with three chitin-binding domains (CBDs) that play an important role in cuticle formation by associating with chitin. In our previous study, we identified CPAP3-type cuticle proteins in the silkworm genome, of which we characterized CPAP3-A2 (BmCBP1), a protein highly expressed in the epidermis. In this study, to elucidate the digestion mechanism of CPAP3-type cuticle proteins, we incubated CPAP3-A2 with molting fluid in vitro and found that its hydrolysis, which was inhibited by serine and cysteine protease inhibitors, produced two major bands with a molecular weight of approximately 22 kD and 11 kD. A trypsin-type serine protease, p37k, was presumed to be responsible for hydrolyzing CPAP3-A2 based on liquid chromatography-tandem mass spectrometry analysis of naturally purified molting fluid. To verify this, p37k was subsequently expressed in Sf9 cells using the Bac-to-Bac baculovirus expression system. In its active form, the recombinant protease could successfully hydrolyze CPAP3-A2. Finally, we analyzed the CPAP3-A2 molting fluid digestion site. When arginine 169 of CPAP3-A2 was mutated to alanine, a weaker hydrolysis of mutant CPAP3-A2 was observed compared to that of normal CPAP3-A2. Collectively, we identified a trypsin-type serine protease that is involved in the degradation of CPAP3-type cuticle proteins, including CPAP3-A2, suggesting that this protease plays an important role during molting in Bombyx mori. These findings provide the basis for further elucidation of the mechanisms underlying insect molting and metamorphosis.

Milligram scale expression, refolding, and purification of Bombyx mori cocoonase using a recombinant E. coli system.

Silk is one of the most versatile biomaterials with signature properties of outstanding mechanical strength and flexibility. A potential avenue for developing more environmentally friendly silk production is to make use of the silk moth (Bombyx mori) cocoonase, this will at the same time increase the possibility for using the byproduct, sericin, as a raw material for other applications. Cocoonase is a serine protease utilized by the silk moth to soften the cocoon to enable its escape after completed metamorphosis. Cocoonase selectively degrades the glue protein of the cocoon, sericin, without affecting the silk-fiber made of the protein fibroin. Cocoonase can be recombinantly produced in E. coli, however, it is exclusively found as insoluble inclusion bodies. To solve this problem and to be able to utilize the benefits associated with an E. coli based expression system, we have developed a protocol that enables the production of soluble and functional protease in the milligram/liter scale. The core of the protocol is refolding of the protein in a buffer with a redox potential that is optimized for formation of native and intramolecular di-sulfide bridges. The redox potential was balanced with defined concentrations of reduced and oxidized glutathione. This E.coli based production protocol will, in addition to structure determination, also enable modification of cocoonase both in terms of catalytic function and stability. These factors will be valuable components in the development of alternate silk production methodology.

Identification of the in vitro antiviral effect of BmNedd2-like caspase in response to Bombyx mori nucleopolyhedrovirus infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious pathogens in sericulture, and the underlying antiviral mechanism in silkworm is still unclear. Bombyx mori Nedd2-like caspase (BmNc) has been identified as a candidate antiviral gene from previous transcriptome data, since it is differentially expressed in the midgut of differentially resistant silkworm strains following BmNPV infection. However, the molecular mechanism by which BmNc responds to BmNPV is unknown. In this study, the relationship between BmNc and BmNPV was confirmed by its significantly different expression in different tissues of differentially resistant strains after BmNPV infection. Moreover, the antiviral role of BmNc was confirmed by the significantly higher fluorescence signals of BV-eGFP after knockdown of BmNc in BmN cells, and a reduced signal after overexpression. This was further verified by the capsid gene vp39 expression, DNA copy number, and GP64 protein level in the RNAi and overexpression groups. Furthermore, the antiviral phenomenon of BmNc was found to be associated with apoptosis. In brief, BmNc showed a relatively high expression level in the metamorphosis stages, and the effect of BmNc on BmNPV infection following RNAi and overexpression was eliminated after treatment with the inducer, Silvestrol, and the inhibitor, Z-DEVD-FMK, respectively. Therefore, it is reasonable to conclude that BmNc is involved in anti-BmNPV infection via the mitochondrial apoptosis pathway. The results provide valuable information for elucidating the molecular mechanism of silkworm resistance to BmNPV infection.

Bmcas-1 plays an important role in response against BmNPV infection in vitro.

Apoptosis, as one kind of innate immune system, is involved in host response against pathogens innovation. Caspases play a vital role in the execution stage of host cell apoptosis. It has been reported that Bmcaspase-1 (Bmcas-1) has a close relationship with Bombyx mori nucleopolyhedrovirus (BmNPV) infection for its differentially expressed patterns after viral infection. However, its underlying response mechanism is still unclear. The significant differential expression of Bmcas-1 in different tissues of differentially resistant strains revealed its vital role in BmNPV infection. To further validate its role in BmNPV infection, budded virus (BV)-eGFP was analyzed after knockdown and overexpression of Bmcas-1 by small interfering RNA and the pIZT-mCherry vector, respectively. The reproduction of BV-eGFP obviously increased at 72 h after knockdown of Bmcas-1, and decreased after overexpression in BmN cells. Moreover, the conserved functional domain of Cas-1 among different species and the closed evolutionary relationship of Cas-1 in Lepidoptera hinted that Bmcas-1 might be associated with apoptosis, and this was also validated by the apoptosis inducer, Silvestrol, and the inhibitor, Z-DEVD-FMK. Therefore, Bmcas-1 plays an essential antiviral role by activating apoptosis, and this result lays a fundament for clarifying the molecular mechanism of silkworm in response against BmNPV infection and breeding of resistant strains.

Bombyx mori ß1,4-N-acetylgalactosaminyltransferase possesses relaxed donor substrate specificity in N-glycan synthesis.

N-Glycosylation is one of the most important post-translational protein modifications in eukaryotic cells. Although more than 200 N-glycogenes contributing to N-glycan biosynthesis have been identified and characterized, the information on insect N-glycosylation is still limited. Here, focusing on insect N-glycosylation, we characterized Bombyx mori N-acetylgalactosaminyltransferase (BmGalNAcT) participating in complex N-glycan biosynthesis in mammals. BmGalNAcT localized at the Golgi and was ubiquitously expressed in every organ and in the developmental stage of the middle silk gland of fifth instar larvae. Analysis of recombinant BmGalNAcT expressed in Sf9 cells showed that BmGalNAcT transferred GalNAc to non-reducing terminals of GlcNAcß1,2-R with ß1,4-linkage. In addition, BmGalNAcT mediated transfer of galactose and N-acetylglucosamine residues but not transfer of either glucose or glucuronic acid from the UDP-sugar donor substrate to the N-glycan. Despite this tri-functional sugar transfer activity, however, most of the endogenous glycoproteins of insect cells were present without GalNAc, Gal, or GlcNAc residues at the non-reducing terminal of ß1,2-GlcNAc residue(s). Moreover, overexpression of BmGalNAcT in insect cells had no effect on N-acetylgalactosaminylation, galactosylation, or N-acetylglucosaminylation of the major N-glycan during biosynthesis. These results suggested that B. mori has a novel multifunctional glycosyltransferase, but the N-glycosylation is highly and strictly regulated by the endogenous N-glycosylation machineries.

Gene mapping reveals the association between tyrosine protein kinase Abl1 and the silk yield of Bombyx mori.

The Z chromosome of the silkworm contains a major gene that influences silk yield. This major locus on chromosome Z accounts for 35.10% of the phenotypic variance. The location and identification of the gene have been a focus of silkworm genetics research. Unfortunately, identification of this gene has been difficult. We used extreme phenotype subpopulations and selected from a backcross population, BC1 M, which was obtained using the high-yield strain 872B and the low-yield strain IS-Dazao as parents, for mapping the gene on the chromosome Z. The candidate region was narrowed down to 134 kb at the tip of the chromosome. BmAbl1 in this region correlated with silk gland development by spatiotemporal expression analysis. This gene was differentially expressed in the posterior silk glands of the high- and low-yield strains. In BmAbl1, an insertion-deletion (indel) within the 10th exonic region and an SNP within the 6th intronic region were detected and shown to be associated with cocoon shell weight in 84 Bombyx mori strains with different yields. Nucleotide diversity analysis of BmAbl1 and its 50 kb flanking regions indicated that BmAbl1 has experienced strong artificial selection during silkworm domestication. This study is the first to identify the genes controlling silk yield in the major QTL of the Z chromosome using forward genetics.

The mutation of SPI51, a protease inhibitor of silkworm, resulted in the change of antifungal activity during domestication.

Domestication of silkworm has led to alterations in various gene expression patterns. For instance, many protease inhibitors were significantly downregulated in the domestic silkworm cocoon compared to its wild progenitor. Considering that SPI51 is the most abundant protease inhibitor in silkworm cocoons, herein, we compared the gene structures and sequences of SPI51 from B. mori (BmoSPI51) and B. mandarina (BmaSPI51). Comparing to the "RGGFR" active site in BmaSPI51, that of BmoPI51 is "KGSFP" and the C-terminal "YNTCECSCP" tail sequence is lost in the latter. To investigate the effect elicited by the active site and tail sequences on the function of SPI51, we expressed two mutated forms of BmoSPI51, namely, BmoSPI51 + tail and BmoSPI51M. BmoSPI51, BmoSPI51 + tail and BmoSPI51M were compared and found to have similar levels of inhibitory activity against trypsin. However, the BmoSPI51 + tail and BmoSPI51M proteins exhibited significantly stronger capacities to inhibit fungi growth, compared to BmoSPI51. We concluded that the specific amino acid sequence of the active site, as well as its the disulfide bond formed by C-terminal sequence in the BmaSPI51, represent the key factors responsible for its higher antifungal activity. This study provided new insights into the antifungal mechanisms elicited by protease inhibitors in the cocoons of silkworms.

Expression of protein tyrosine phosphatases and Bombyx embryonic development.

Protein phosphorylation is an integral component of signal transduction pathways within eukaryotic cells, and it is regulated by coordinated interactions between protein kinases and protein phosphatases. Our previous study demonstrated differential expressions of serine/threonine protein phosphatases (PP2A and calcineurin) between diapause and developing eggs in Bombyx mori. In the present study, we further investigated expression of protein tyrosine phosphatases (PTPs) in relation to the Bombyx embryonic development. An immunoblot analysis showed that eggs contained the proteins of the 51-kDa PTP 1B (PTP1B), the 55-kDa phosphatase and tensin homologue (PTEN), and the 70-kDa Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2), which undergo differential changes between diapause and developing eggs. Protein level of PTP1B and PTEN in eggs whose diapause initiation was prevented by HCl gradually increased toward embryonic development. The protein level of SHP2 also showed a dramatic increase on days 7 and 8 after HCl treatment. However, protein levels of PTP1B, PTEN, and SHP2 in diapause eggs remained at low levels during the first 9 days after oviposition. These differential changing patterns in protein levels were further confirmed using both non-diapause eggs and eggs in which diapause had been terminated by chilling of diapausing eggs at 5 °C for 70 days and then were transferred to 25 °C. Direct determination of PTP enzymatic activities showed higher activities in developing eggs (HCl-treated eggs, non-diapause eggs, and chilled eggs) compared to those in diapause eggs. Examination of temporal changes in mRNA expression levels of PTP1B, PTEN, and SHP2 did not show significant differences between diapause eggs and HCl-treated eggs except high expression in SHP2 variant B during the later embryonic development in HCl-treated eggs. These results demonstrate that higher protein levels of PTP1B, PTEN, and SHP2 and increased tyrosine phosphatase enzymatic activities in developing eggs are likely related to embryonic development of B. mori.

Investigation of the Substrate-Binding Site of a Prostaglandin E Synthase in Bombyx mori.

Prostaglandin E synthase (PGES) catalyzes the conversion of prostaglandin H2 to prostaglandin E2 in the presence of glutathione (GSH) in mammals. Amid the limited knowledge on prostaglandin and its related enzymes in insects, we recently identified PGES from the silkworm Bombyx mori (bmPGES) and determined its crystal structure complexed with GSH. In the current study, we investigated the substrate-binding site of bmPGES by site-directed mutagenesis and X-ray crystallography. We found that the residues Tyr107, Val155, Met159, and Glu203 are located in the catalytic pockets of bmPGES, and mutagenesis of each residue reduced the bmPGES activity. Our results suggest that these four residues contribute to the catalytic activity of bmPGES. Overall, this structure-function study holds implications in controlling pests by designing rational and efficient pesticides.

Structural characterization and functional analysis of juvenile hormone diol kinase from the silkworm, Bombyx mori.

Juvenile hormone diol kinase (JHDK) is an important enzyme involved in the juvenile hormone metabolism pathway, which catalyzes the phosphorylation of juvenile hormone diol to form the polar metabolite JH diol phosphate. Here, we reported the first crystal structure of insect JHDK from Bombyx mori, BmJHDK-L2, determined at a resolution of 1.22 Å. The structure of BmJHDK-L2 mainly comprises of eight α-helical segments linked with loops, forming four helix-loop-helix motifs. In these four helix-loop-helix motifs with only one calcium ion bound in the first motif. Circular dichroism spectra indicated that BmJHDK-L2 has strong thermal stability, which is independent of the divalent cation. The structure of BmJHDK-L2 further allowed us to define an ATP-binding site using computational simulation and binding assays, providing a structural basis for development of inhibitor of JHDK. Moreover, the expression profile of BmJHDK-L2 indicated a predominant role in juvenile hormone metabolism in the Malpighian tubules of silkworm. Collectively, these findings expand our knowledge regarding the structural and biochemical features of insect JHDK proteins.

Characterization and potential application of an α-amylase (BmAmy1) selected during silkworm domestication.

Efficient resource utilization plays a central role in the high productivity of domesticated plants and animals. Whether artificial selection acts on digestive enzymes in the domesticated silkworm (Bombyx mori), which is larger than its wild ancestor, Bombyx mandarina (B. mandarina), remains unknown. In this study, we present the characteristics of a novel alpha-amylase, BmAmy1, in B. mori. The activity of recombinant BmAmy1 was maximal at 35 °C and pH 9.0, and could be suppressed by amylase inhibitors from mulberry, the exclusive food source of silkworms. Three different transposable element fragments, which were independently inserted in the 5'-upstream regulatory region, might be responsible for the enhanced expression of BmAmy1 in different domesticated silkworm strains as revealed by dual-luciferase reporter assay. The BmAmy1 overexpression increased the weight of female and male B. mori by 11.9% and 6.8%, respectively, compared with non-transgenic controls. Our results emphasize that, by exploring the genetic mechanisms of human-selected traits, the domestication process could be further accelerated through genetic engineering and targeted breeding.