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.

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.

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.

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.

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.

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.

A Novel Adenosine Kinase from Bombyx mori: Enzymatic Activity, Structure, and Biological Function.

Adenosine kinase (ADK) is the first enzyme in the adenosine remediation pathway that catalyzes adenosine phosphorylation into adenosine monophosphate, thus regulating adenosine homeostasis in cells. To obtain new insights into ADK from Bombyx mori (BmADK), we obtained recombinant BmADK, and analyzed its activity, structure, and function. Gel-filtration showed BmADK was a monomer with molecular weight of approximately 38 kDa. Circular dichroism spectra indicated BmADK had 36.8% α-helix and 29.9% ß-strand structures, respectively. The structure of BmADK was stable in pH 5.0-11.0, and not affected under 30 °C. The melting temperature and the enthalpy and entropy changes in the thermal transition of BmADK were 46.51 ± 0.50 °C, 253.43 ± 0.20 KJ/mol, and 0.79 ± 0.01 KJ/(mol·K), respectively. Site-directed mutagenesis demonstrated G68, S201, E229, and D303 were key amino acids for BmADK structure and activity. In particular, S201A mutation significantly increased the α-helix content of BmADK and its activity. BmADK was located in the cytoplasm and highly expressed in the silk gland during the pre-pupal stage. RNA interference revealed the downregulation of BmADK decreased ATG-8, Caspase-9, Ec-R, E74A, and Br-C expression, indicating it was likely involved in 20E signaling, apoptosis, and autophagy to regulate silk gland degeneration and silkworm metamorphosis. Our study greatly expanded the knowledge on the activity, structure, and role of ADK.

Comparative analysis of seven types of superoxide dismutases for their ability to respond to oxidative stress in Bombyx mori.

Insects are well adapted to changing environmental conditions. They have unique systems for eliminating reactive oxygen species (ROS). Superoxide dismutase (SOD) is a key enzyme that plays a primary role in removing ROS. Bombyx mori is a lepidopteran insect, whose body size is larger than the model insect Drosophila melanogaster, which enabled us to more easily examine gene expression at the tissue level. We searched B. mori SOD (BmSOD) genes using genome database, and we analyzed their function under different type of oxidative stress. Consequently, we identified four new types of BmSODs in addition to the three types already known. Two of the seven types had a unique domain architecture that has not been discovered previously in the SOD family, and they were expressed in different tissues and developmental stages. Furthermore, these BmSODs responded differently to several kinds of stressors. Our results showed that the seven types of BmSODs are likely to play different roles in B. mori; therefore, B. mori could be used to distinguish the functions of each SOD for resistance to oxidative stress that changes with the environmental conditions.

A QM protein from Bombyx mori negatively regulates prophenoloxidase activation and melanization by interacting with Jun protein.

The QM gene that encodes for the ribosomal protein L10 was firstly identified from human tumour cells as a tumour suppressor. In this study, a QM gene was identified in silkworm Bombyx mori (BmQM) and its immunomodulatory function was explored. BmQM messenger RNA (mRNA) and protein were highly expressed in the silk gland and fat body, and expressed in all stages of silkworm growth. After challenged with four different microorganisms, the expression levels of BmQM mRNA in fat body or haemocytes were significantly upregulated compared with the control. After knock-down of BmQM gene, the expressions of some immune genes (PGRPS6, Gloverin0, Lysozyme and Moricin) were affected, and the transcripts of prophenoloxidase1 and prophenoloxidase2 have different degrees of change. The phenoloxidase activity was significantly reduced when the purified recombinant BmQM protein was injected. Recombinant BmQM protein inhibited systemic melanization and suppressed prophenoloxidase activation stimulated by Micrococcus luteus, but it did not affect phenoloxidase activity. Far-western blotting assays showed that the BmQM protein interacted with silkworm BmJun protein, which negatively regulates AP-1 expression. Our results indicated that BmQM protein could affect some immune gene expression and negatively regulate the prophenoloxidase-activating system, and it may play an important role in regulation of the innate immunity in insects.

Role of protein phosphatase 2A in PTTH-stimulated prothoracic glands of the silkworm, Bombyx mori.

In the present study, the roles of a major serine/threonine protein phosphatase 2A (PP2A) in prothoracicotropic hormone (PTTH)-stimulated prothoracic glands (PGs) of Bombyx mori were evaluated. Immunoblotting analysis showed that Bombyx PGs contained a structural A subunit (A), a regulatory B subunit (B), and a catalytic C subunit (C), with each subunit undergoing development-specific changes. The protein levels of each subunit were not affected by PTTH treatment. However, the highly conserved tyrosine dephosphorylation of PP2A C subunit (PP2Ac), which appears to be related to activity, was increased by PTTH treatment in a time-dependent manner. We further demonstrated that phospholipase C (PLC), Ca2+, and reactive oxygen species (ROS) are upstream signaling for the PTTH-stimulated dephosphorylation of PP2Ac. The determination of PP2A enzymatic activity showed that PP2A enzymatic activity was stimulated by PTTH treatment both in vitro and in vivo. Okadaic acid (OA), a specific PP2A inhibitor, prevented the PTTH-stimulated dephosphorylation of PP2Ac and reduced both basal and PTTH-stimulated PP2A enzymatic activity. The determination of ecdysteroid secretion showed that treatment with OA did not affect basal ecdysteroid secretion but did significantly inhibit PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated PP2A activity is involved in ecdysteroidogenesis. Treatment with OA stimulated the basal phosphorylation of the extracellular signal-regulated kinase (ERK) and 4E-binding protein (4E-BP) without affecting PTTH-stimulated ERK and 4E-BP phosphorylation. From these results, we hypothesize that PTTH-regulated PP2A signaling is a necessary component for the stimulation of ecdysteroidogenesis, potentially by mediating the link between ERK and TOR signaling pathways.

A functional polypeptide N-acetylgalactosaminyltransferase (PGANT) initiates O-glycosylation in cultured silkworm BmN4 cells.

Mucin-type O-glycosylation is initiated by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts or PGANTs), attaching GalNAc to serine or threonine residue of a protein substrate. In the insect model from Lepidoptera, silkworm (Bombyx mori), however, O-glycosylation pathway is totally unexplored and remains largely unknown. In this study, as the first report regarding protein O-glycosylation analysis in silkworms, we verified the O-glycan profile that a common core 1 Gal (ß1-3) GalNAc disaccharide branch without terminally sialylated structure is mainly formed for a baculovirus-produced human proteoglycan 4 (PRG4) protein. Intriguingly, functional screenings in cultured silkworm BmN4 cells for nine Bmpgants reveal that Bmpgant2 is the solo functional BmPGANT for PRG4, implying that Bmpgants may have unique cell/tissue or protein substrate preferences. Furthermore, a recombinant BmPGANT2 protein was successfully purified from silkworm-BEVS and exhibited a high ability to transfer GalNAc for both peptide and protein substrates. Taken together, the present results clarified the functional BmPGANT2 in cultured silkworm cells, providing crucial fundamental insights for future studies dissecting the detailed silkworm O-glycosylation pathways and productions of glycoproteins with O-glycans.

Aluminum toxicity related to SOD and expression of presenilin and CREB in Bombyx mori.

Aluminum (Al) is an important environmental metal factor that can be potentially associated with pathological changes leading to neurotoxicity. The silkworm, Bombyx mori, is an important economic insect and has also been used as a model organism in various research areas. However, the toxicity of Al on silkworm physiology has not been reported. Here, we comprehensively investigate the toxic effects of Al on the silkworm, focusing on its effects on viability and development, superoxide dismutase (SOD) activity, and the expression of presenilin and cAMP response element-binding protein (CREB) in BmE cells and silkworm larvae. BmE cell viability decreased after treatment with aluminum chloride (AlCl3 ) in both dose- and time-dependent manners. When AlCl3 solution was injected into newly hatched fifth instar larvae, both larval weight gain and survival rate were significantly decreased in a manner correlating with AlCl3 dose and developmental stage. Furthermore, when BmE cells and silkworm larvae were exposed to AlCl3 , SOD activity decreased significantly relative to the control group, whereas presenilin expression increased more than twofold. Additionally, CREB and phosphorylated CREB (p-CREB) expression in the heads of fifth instar larvae decreased by 28.0% and 50.0%, respectively. These results indicate that Al inhibits the growth and development of silkworms in vitro and in vivo, altering SOD activity and the expressions of presenilin, CREB, and p-CREB. Our data suggest that B. mori can serve as a model animal for studying Al-induced neurotoxicity or neurodegeneration.

A rapid and sensitive colorimetric assay for the determination of adenosine kinase activity.

Adenosine kinase (ADK) plays an important role in the growth and development of organisms. A convenient, quick, reliable, sensitive and low-cost assay for ADK activity is of great significance. Here, we found the reaction system with bromothymol blue as the pH indicator had a maximum absorption peak at 614 nm. The absorbance change in 614 nm was positively correlated with the generated hydrogen ions in the reaction catalyzed by ADK. Then, we demonstrated this assay was feasible for ADK activity. Further, we analyzed the effects of buffer, bromothymol blue concentrations on the sensitivity of the assay, and investigated the sensitivity of ADK contents and adenosine concentration on the assay. Finally, we calculated the Km and Vmax of ADK from Bombyx mori with this assay. Our results suggested this assay was quick, convenient, reliable, sensitive and economic for the activity of ADK. It is an excellent alternative for the conventional ADK assays.

SUMOylation regulates the localization and activity of Polo-like kinase 1 during cell cycle in the silkworm, Bombyx mori.

Polo-like kinase 1 (Plk1) is a crucial cell cycle regulator by its specific localization and activity during cell cycle. It has been shown that the phosphorylation and ubiquitylation of Plk1 are required for its own activation and localization. Here, we report that SUMOylation regulates the activity of Plk1 in the lepidopteran insect of Bombyx mori. In the absence of SUMOylation, it causes the lost localization of Plk1 on centrosomes and kinetochores, as well as an uneven distribution in midzone. We further identify that the putative SUMOylation site of Bombyx Plk1 at lysine 466 is required for its localization on centrosomes, and K466 mutation in Plk1 could influence its interaction with Smt3/Ubc9 complex. These findings are also confirmed by Drosophila Polo and human Plk1, which together reveals a conserved role of Plk1 SUMOylation in mammals. Moreover, conjugation of Smt3 to Plk1 SUMOylation mutant promotes its localization on centrosomes and kinetochores, and rescues functional defects of chromosome alignment in cells depleted of endogenous Plk1. Altogether, the present data indicate that the SUMOylation of Plk1 could participate in proper chromosome alignment and segregation during mitosis, and provides a novel layer for the regulation of Plk1 localization and activity throughout cell cycle.

A possible mechanism for low affinity of silkworm Na+/K+-ATPase for K.

The affinity for K+ of silkworm nerve Na+/K+-ATPase is markedly lower than that of mammalian Na+/K+-ATPase (Homareda 2010). In order to obtain clues on the molecular basis of the difference in K+ affinities, we cloned cDNAs of silkworm (Bombyx mori) nerve Na+/K+-ATPase α and ß subunits, and analyzed the deduced amino acid sequences. The molecular masses of the α and ß subunits were presumed to be 111.5 kDa with ten transmembrane segments and 37.7 kDa with a single transmembrane segment, respectively. The α subunit showed 75% identity and 93% homology with the pig Na+/K+-ATPase α1 subunit. On the other hand, the amino acid identity of the ß subunit with mammalian counterparts was as low as 30%. Cloned α and ß cDNAs were co-expressed in cultured silkworm ovary-derived cells, BM-N cells, which lack endogenous Na+/K+-ATPase. Na+/K+-ATPase expressed in the cultured cells showed a low affinity for K+ and a high affinity for Na+, characteristic of the silkworm nerve Na+/K+-ATPase. These results suggest that the ß subunit is responsible for the affinity for K+ of Na+/K+-ATPase.

Localization of an Aldo-Keto Reductase (AKR2E4) in the Silkworm Bombyx mori (Lepidoptera: Bombycidae).

The aldo-keto reductase AKR2E4 reduces 3-dehydroecdysone to ecdysone in the silkworm Bombyx mori L. In this study, a quantitative polymerase chain reaction analysis revealed that the level of AKR2E4 mRNA was higher in the testes than in other tissues, and a western immunoblot analysis revealed that the AKR2E4 content in the testes was stage-specific from the fifth larval instar to the pupal stage. Immunohistochemical analysis showed that the AKR2E4 protein was present in cyst cells associated with sperm cells and spermatocytes. These results indicate that AKR2E4 plays an important role in 3-dehydroecdysone conversion to ecdysone and spermatogenesis in silkworm testes.

MTase Domain of Dendrolimus punctatus cypovirus VP3 Mediates Virion Attachment and Interacts with Host ALP Protein.

Dendrolimus punctatus cypovirus (DpCPV) is an important pathogen of D. punctatus, but little is known about the mechanisms of DpCPV infection. Here, we investigated the effects of VP3, VP4 and VP5 structural proteins on the viral invasion. Both the C-terminal of VP3 (methyltransferase (MTase) domain) and VP4 (A-spike) bound to Spodoptera exigua midgut brush border membrane vesicles (BBMVs) in a dose-dependent manner, and the binding was inhibited by purified DpCPV virions. Importantly, anti-MTase and anti-VP4 antibodies inhibited viral binding to S. exigua BBMVs. Using far-Western blots, a 65 kDa protein in Bombyx mori BBMVs, identified as alkaline phosphatase protein (BmALP) by mass spectrometry, specifically interacted with DpCPV MTase. The interaction between MTase and BmALP was verified by co-immunoprecipitation in vitro. Pretreatment of B. mori BBMVs with an anti-ALP antibody or incubation of DpCPV virions with prokaryotically expressed BmALP reduced viral attachment. Additionally, BmALP inhibited DpCPV infection in S. exigua larvae. Our data provide evidence that the MTase domain and A-spike function as viral attachment proteins during the DpCPV infection process, and ALP is the ligand that interacts with DpCPV via the MTase domain. These results augment our understanding of the mechanisms used by cypoviruses to enter their hosts.

Identification and characterization of a pyridoxal 5'-phosphate phosphatase in the silkworm (Bombyx mori).

Vitamin B6 comprises six interconvertible pyridine compounds, among which pyridoxal 5'-phosphate (PLP) is a coenzyme for over 140 enzymes. PLP is also a very reactive aldehyde. The most well established mechanism for maintaining low levels of free PLP is its dephosphorylation by phosphatases. A human PLP-specific phosphatase has been identified and characterized. However, very little is known about the phosphatase in other living organisms. In this study, a cDNA clone of putative PLP phosphatase was identified from B. mori and characterized. The cDNA encodes a polypeptide of 343 amino acid residues, and the recombinant enzyme purified from E. coli exhibited properties similar to that of human PLP phosphatase. B. mori has a single copy of the PLPP gene, which is located on 11th chromosome, spans a 5.7kb region and contains five exons and four introns. PLP phosphatase transcript was detected in every larva tissue except hemolymph, and was most highly represented in Malpighian tube. We further down-regulated the gene expression of the PLP phosphatase in 5th instar larvae with the RNA interference. However, no significant changes in the gene expression of PLP biosynthetic enzymes and composition of B6 vitamers were detected as compared with the control.

Aurora B kinase is required for cell cycle progression in silkworm.

Aurora B kinase, a member of serine/threonine kinase family, is the catalytic subunit of the chromosomal passenger complex and is essential for chromosome alignment, chromosome segregation, and cytokinesis during mitosis. Here, we cloned the full-length cDNA sequence of silkworm Aurora B (BmAurB) gene and predicted that BmAurB protein contains a conserved S_TKc domain. Phylogenetic analysis between BmAurB and other Aurora kinases indicates that Aurora kinases may have evolved after separation between mammalian and insect, and prior to radiation of either mammalian or insects. RT-PCR examination revealed that the expression of the BmAurB gene was high in mitotic cycling gonads, moderate in mitotic cycling brain, and undetectable in endocycling silk gland during silkworm larval development. RNAi or inhibitor-mediated inhibition of the BmAurB gene in silkworm ovary-derived BmN4-SID1 cells disrupted cell cycle progression during mitosis and induced an accumulation of polyploid cells, cell cycle arrest at G2/M phase, chromosome misalignment, chromosome bridge, and bi-nucleation. Taken together, our results suggest that the BmAurB gene is required for cell cycle progression in silkworm.

Identification of Tyrosine Kinase Src Responsible for Antimicrobial Peptides Production in Bombyx mori.

Src is a non-receptor protein tyrosine kinase ubiquitously expressed in animals. It is involved in various cellular processes, including the innate immune response in mammals. However, less is known about the function of insect Src. Here we presented a homologue of Src in silkworm (Bombyx mori), named as BmSrc by phylogenetic analysis, homologous comparison and domain prediction. BmSrc contains the conserved phosphorylation residues and possesses tyrosine kinase activity. The expression pattern of BmSrc mRNA was specific in developmental stages and tissues. The highest expression of BmSrc was detected in moth stage, and the gonads showed the highest expression during larval stage. We then found over-expression of BmSrc in BmE cell resulted in an increase of p38 mitogen-activated protein kinase (p38 MAPK) and Akt phosphorylation but a decrease in extracellular signal-regulated kinase (ERK) phosphorylation. Finally, we demonstrated that BmSrc promoted the production of antimicrobial peptides (AMPs). These results implied that BmSrc is involved in immune response of silkworm possibly through activating p38 MAPK and Akt signaling pathway. Our study may provide reference for further investigation of the biological function of BmSrc in Bombyx mori.