Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putativelow-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identifiedfrom honeybee (Apis mellifera) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated.In this study, we identified an Asiatic honeybee (A. cerana) venom serine protease inhibitor (AcVSPI) that was shownto act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-likedomain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putativelow-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide.Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin,but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however,it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPIinhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial andfungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. Thesefindings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor.

Serine proteases and serine protease homologs are involved in the prophenoloxidase (proPO)-activating system leadingto melanization.The Bombyx mori serine protease homolog BmSPH-1 regulates nodule melanization. Here, we show the dualrole of BmSPH-1 in the development and immunity of B. mori. BmSPH-1 was expressed in hemocytes after molting andduring the larval-pupal transformation in normal development. In contrast, following infection, BmSPH-1 was expressed inhemocytes and activated in the hemolymph, which resulted in the induction of PO activity. Moreover, BmSPH-1 was activatedin the cuticle during the larval-pupal transformation and early pupal stages. In BmSPH-1 RNAi-treated silkworms, the reducedBmSPH-1 mRNA levels during the spinning stage or the prepupal stage resulted in the arrest of pupation or pupal cuticularmelanization, respectively. The binding assays revealed that BmSPH-1 interacts with B. mori immulectin, proPO, andproPO-activating enzyme. Our findings demonstrate that BmSPH-1 is responsible for the larval-pupal transformation, pupalcuticular melanization and innate immunity of silkworms, illustrating the dual role of BmSPH-1 in development and immunity.

In insects, serine proteases are involved in a variety of physiological processes including digestion, development, and immunity. Bombyx mori serine protease homolog BmSPH-1 regulates nodule melanization and is recruited into nodules from the hemolymph by B. mori lipopolysaccharide-binding protein. Here, we show the dual role of BmSPH-1 in development and immunity of B. mori. BmSPH-1 was expressed in the hemocytes during larval-pupal transformation and localized to the cuticle of silkworms, which indicates that BmSPH-1 is secreted from hemocytes and then transported to the cuticle via the hemolymph. BmSPH-1 was proteolytically activated in the cuticle during larval-pupal transformation and the early pupal stage. BmSPH-1 RNAi resulted in the arrest of larval-pupal transformation and pupal cuticular melanization. Furthermore, the expression of BmSPH-1 was up-regulated in the hemocytes during infection. Taken together, we found that BmSPH-1 is involved in larval-pupal transformation and pupal cuticular melanization as well as the innate immunity of silkworms, which indicates that BmSPH-1 is responsible for either development or immunity.

Serine protease inhibitors play a critical role in physiological processes and immune responses by regulating serine protease activities. Here we describe the molecular cloning and antimicrobial activities of a serine protease inhibitor from the mason bee, Osmia cornifrons (OcSPI). OcSPI consists of 405 amino acid residues and contains a potential reactive center loop (RCL) region in its C-terminus. Recombinant OcSPI was produced as a 64-kDa glycoprotein in baculovirus-infected insect cells and exhibited inhibitory activity against chymotrypsin. Additionally, OcSPI demonstrated inhibitory activity against microbial serine proteases, such as subtilisin A and proteinase K, but not against tissue plasminogen activator, thrombin, or plasmin. Recombinant OcSPI bound directly to Escherichia coli, Bacillus subtilis, and Beauveria bassiana and exhibited antimicrobial activity against both bacteria and fungi. Our results demonstrated the antimicrobial functions of OcSPI and suggest a role for OcSPI in the immune response of O. cornifrons.

Serine protease는 병원체의 표면 melanization, hemolymph coagulation, antimicrobial peptide synthesis 등을 통해 여러 무척추동물의 방어기작을 조절하는것으로 알려 져 있다. 곤충의 경우 Tribolium을 대상으로 이와 같은 연구가 이루어져 왔지만, 혈 액의 량이 그리 많지 않아 연구자들은 최근 갈색거저리(Tenebrio)를 이용하기 시작 하였다. 하지만 아직 유전체(자) 서열정보가 충분하지 않은 상황이다. 본 연구에서 는 이러한 갈색거저리 유충을 이용하여 세포벽이 없으며 사람에서 pneumonia나 다른 호흡기 질환을 일으키는 mycoplasma 와 유사한 acholeplasma lysate를 처리 한 후 접종 전과 후의 전사체의 비교를 통하여 무척추 동물에서의 선천성 면역 관련 유전자들을 동정하고자 하였다. Acholeplasma lysate를 처리하기 전과 후의 각 샘 플들로부터 cDNA library를 구축한 후 random sequencing 을 통해 염기서열을 분 석하였고, 얻어진 서열들로부터 NCBI nr 데이터베이스에 Blastx 분석을 하여 획득 한 서열들을 comparative transcriptomic 방법을 이용하여 분석한 결과, 여러 종류 의 Serine protease 관련 유전자들이 동정되었다. Serine protease (XP_970766.1)의 경우에는 acholeplasma를 처리한 샘플에서 2배 정도 발현이 증가하였고, serine protease P66 (EFA09207.1)의 경우 증감의 변화는 보이지 않았다. 또한 serine protease P146 (EFA04636.1), serine protease H1 (EEZ99180.1) 등의 유전자들도 동정되어 연구하고 있다.

Bumblebee venom serine protease inhibitors have been shown to inhibit plasmin activity. In this study, a bumblebee (Bombus ignitus) venom serine protease inhibitor (BiVSPI) that acts as an antimicrobial factor was identified. BiVSPI is a 55-amino acid mature peptide with ten conserved cysteine residues and a P1 methionine residue. BiVSPI was expressed in the venom gland and was present as an 8-kDa peptide in venom. Recombinant BiVSPI expressed in baculovirusinfected insect cells exhibited inhibitory activity against chymotrypsin, but not trypsin. BiVSPI also exhibited inhibitory activity against microbial serine proteases, such as subtilisin A (Ki 6.57 nM) and proteinase K (Ki 7.11 nM), indicating that BiVSPI acts as a microbial serine protease inhibitor. In addition, BiVSPI was also shown to bind directly to Bacillus subtilis, B. thuringiensis, and Beauveria bassiana, but not to Escherichia coli. Consistent with these results, BiVSPI exhibited antimicrobial activity against Gram-positive bacteria and fungi. These findings provide novel evidence for the antimicrobial function of this bumblebee venom serine protease inhibitor.

Insect-derived Kazal-type serine protease inhibitors exhibit thrombin, elastase, plasmin, proteinase K, or subtilisin A inhibition activity, but so far, no functional roles for bee-derived Kazal-type serine protease inhibitors have been identified. In this study, a bee (Apis cerana) venom Kazal-type serine protease inhibitor (AcKTSPI) that acts as a microbial serine protease inhibitor was identified. AcKTSPI contained a single Kazal domain that displayed six conserved cysteine residues and a P1 threonine residue. AcKTSPI was expressed in the venom gland and was present as a 10-kDa peptide in bee venom. Recombinant AcKTSPI Kazal domain (AcKTSPI-Kd) expressed in baculovirus-infected insect cells demonstrated inhibitory activity against subtilisin A (Ki 67.03 nM) and proteinase K (Ki 91.53 nM), but not against α-chymotrypsin or typsin, which implies a role for AcKTSPI as a microbial serine protease inhibitor. However, AcKTSPI-Kd exhibited no detectable inhibitory effects on factor Xa, thrombin, tissue plasminogen activator, or elastase. Additionally, AcKTSPI-Kd bound directly to Bacillus subtilis, B. thuringiensis, Beauveria bassiana, and Fusarium graminearum but not to Escherichia coli. Consistent with these findings, AcKTSPI-Kd showed antibacterial activity against Gram-positive bacteria and antifungal activity against both plant-pathogenic and entomopathogenic fungi. These findings constitute molecular evidence that AcKTSPI acts as an inhibitor of microbial serine proteases. This paper provides a novel view of the antimicrobial functions of a bee venom Kazal-type serine protease inhibitor.

Insect-killing fungi have high potential for controlling agriculturally harmful pests. However, their pathogenicity is slow and this is one reason for their poor acceptance as a fungal insecticide. The expression of bumblebee, Bombus ignitus, venom serine protease (VSP) by Beauveria bassiana ERL1170 induced melanization of yellow spotted longicorn beetles, Psacothea hilaris as an over-reactive immune response, and caused substantially earlier mortality in beet armyworm, Spodopetra exigua larvae when compared to the wild type. No fungal outgrowth or sporulation was observed on the melanized insects, thus suggesting a self-restriction of the dispersal of the genetically modified fungus in the environment. The research is the first use of a multi-functional bumblebee VSP to significantly increase the speed of fungal pathogenicity, while minimizing the dispersal of the fungal transformant in the environment

Insect-killing (entomopathogenic) fungi have high potential for controlling agriculturally harmful pests. However, their pathogenicity is slow and this is one reason for their poor acceptance as a fungal insecticide. The expression of bumblebee, Bombus ignitus, venom serine protease (VSP) by Beauveria bassiana (ERL1170) induced melanization of yellow spotted longicorn beetles (Psacothea hilaris) as an over-reactive immune response, and caused substantially earlier mortality in beet armyworm (Spodopetra exigua) larvae when compared to the wild type. No fungal outgrowth or sporulation was observed on the melanized insects, thus suggesting a self-restriction of the dispersal of the genetically modified fungus in the environment. The fungal transformant also shows mammal fibrinolytic activity, by which the transformant can be used pharmaceutically. The research is the first use of a multi-functional bumblebee VSP to significantly increase the speed of fungal pathogenicity, while minimizing the dispersal of the fungal transformant in the environment.

Bee venom is a rich source of pharmacologically active substances. In this study, we characterized a B. terrestris venom Kunitz-type serine protease inhibitor (Bt-KTI). Bt-KTI consists of two exons encoding 82-amino acids (aa), including a predicted 24-aa signal peptide and a 58-aa mature peptide. Recombinant Bt-KTI was expressed as a 6.5-kDa peptide in baculovirus-infected insect cells. Bt-KTI showed no detectable inhibitory effect on factor Xa, thrombin, or tissue plasminogen activator. In contrast, Bt-KTI strongly inhibited plasmin, indicating that it acts as a plasmin inhibitor. The electrophoretic mobility shift assay showed that Bt-KTI binds to plasmin, indicating the formation of a plasmin-Bt-KTI complex. These results demonstrate that Bt-KTI acts as an antifibrinolytic agent, suggesting a role for Bt-KTI as an anti-bleeding agent.

Bee venom contains serine proteases and serine protease inhibitors. In this study, we identified a bumblebee (Bombus ignitus) venom Kunitz-type serine protease inhibitor (Bi-KTI) that acts as a plasmin inhibitor. Bi-KTI showed no detectable inhibitory effect on factor Xa, thrombin, or tissue plasminogen activator. In contrast, Bi-KTI strongly inhibited plasmin, indicating that it acts as an antifibrinolytic agent. The fibrin(ogen)olytic activities of B. ignitus venom serine protease (Bi-VSP) and plasmin in the presence of Bi-KTI indicate that Bi-KTI targets plasmin more specifically than Bi-VSP. These findings demonstrate a novel mechanism by which bumblebee venom affects the hemostatic system through the antifibrinolytic activity of Bi-KTI and through Bi-VSP-mediated fibrin(ogen) olytic activities, raising interest in Bi-KTI and Bi-VSP as potential clinical agents.

We cloned venom serine proteases from two bumblebee species, Bombus hypocrita sapporoensis and B. ardens ardens. We compared the predicted mature protein sequences of these serine proteasegenes to those previously reported from other bees. Using B. h. sapporoensis venom serine protease(Bs-VSP), we identify that Bs-VSP acts as a fibrin(ogen)olytic enzyme. Bs-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, as demonstrated for B. ignitus and B. terrestrisvenom serine proteases. Our results further define roles for bumblebee venom serine proteases as fibrin(ogen)olytic enzyme, providing strong evidence that bumblebee venom serine proteases are hemostatically active proteins that are potentially promising therapeutic agents.

Bee venom is a rich source of pharmacologically active substances. In this study, we identified a bumblebee (Bombus ignitus) venom Kunitz-type serine protease inhibitor (Bi-KTI) that acts as a plasmin inhibitor. Bi-KTI showed no detectable inhibitory effect on factor Xa, thrombin, or tPA. However, it strongly inhibited plasmin, although this inhibitory ability was two-fold weaker than that of aprotinin. The activities of B. ignitusvenom serine protease (Bi-VSP) and plasmin in the presence of Bi-KTI indicate that Bi-KTI targets plasmin more specifically than Bi-VSP. These findings demonstrate a novel mechanism for bee venom by which Bi-KTI acts as an antifibrinolytic agent, raising interest in Bi-KTI as a potential clinical agent.

Bee venom contains a variety of protein allergens, including serine proteases. Additionally, bee venom has been used in therapeutic application through immunotherapy for bee venom hypersensitivity and venom therapy as an alternative medicine. Here we present a novel view of the application of bee venom through which bee venom serine protease exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a larger amount of a serine protease as one of its major components. Immunologically, venom serine proteases from bumblebees did not show cross-reactivity with the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. However, Bt-VSP did not activate plasminogen and the fibrinolytic activity of Bt-VSP is less than plasmin. These findings offer insight into the allergic reaction sequence of bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis.

Bee venom contains a variety of peptides and enzymes, including serine proteases. While the presence of serine proteases in bee venom has been demonstrated, the role of these proteins in bee venom has not been elucidated. Furthermore, there is currently no information available regarding the melanization response or the fibrin(ogen)olytic activity of bee venom serine protease, and the molecular mechanism of its action remains unknown. Here we show that bee venom serine protease (Bi-VSP) is a multifunctional enzyme. In insects, Bi-VSP acts as an arthropod prophenoloxidase (proPO)-activating factor (PPAF), thereby triggering the phenoloxidase (PO) cascade. Bi-VSP injected through the stinger induces a lethal melanization response in target insects by modulating the innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease, which exhibits fibrin(ogen)olytic activity. Bi-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles forBi-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings provide a novel view of the mechanism of bee venom in which the bee venom serine protease kills target insects via a melanization strategy and exhibits fibrin(ogen)olytic activity.

Bee venom contains a variety of peptides and enzymes, including serine proteases. Here we describe the molecular cloning and characterization of a serine protease (Bt-VSP) isolated from the venom of the bumblebee Bombus terrestris. The Bt-VSP gene consists of six exons encoding a 358-amino acid protein. The form of Bt-VSP detected in bee venom was the 34-kDa mature protein, which is created by cleavage of the catalytic domain of Bt-proVSP between Arg111 and Val112. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. The finding that Bt-VSP acts as a fibrin(ogen)olytic enzyme is similar to a previous finding that Bi-VSP, a venom serine protease of B. ignitus, exhibits fibrin(ogen)olytic activity. We also compared major venom components in honeybee and bumblebee, and found that bumblebee venom contains a larger amount of serine protease. Furthermore, unlike bumblebee venom, which exhibits fibrin(ogen)olytic activity owing to the presence of a serine protease, it is likely that honeybee venom lacks fibrin(ogen)olytic activity.

Protease from various sources have been studied biotecnologically. For biotechnological applications, one highly preferred enzyme is protease. There have been no reports of cloned genes encoding digestive proteases in the Laccotrephes japonenis, Ranatra unicolor, Muljarus japonicus. These insects are considered to be a predator of aquatic insects. RT-PCR was used to amplify cDNA fragments for digestive proteases from total RNA the hole body of the insects. The flanking sequences of the 5'- and 3'- end of the these genes were characterized by RACE-PCR. Sequence analysis showed that these genes contained complete ORF. The deduced amino acid sequences of these protease showed 62% identity to the serine protease of Creontiades dilutus, 58% to Lygus loneolaris trypsin-like serine proteinase, 54% to Triatonatoma infestans salivary trypsin. To generate Laccotrephes japonensis serine protease, the DNA fragment coding for serine protease is cloning into suttle vector pBACⅠ, named pBAC1-JG and infected to Spodoptera frugiperda (sf9) insect cell. The cDNA encoding JG was expressed as a 32-kDa polypeptide in baculovirus infected insect cells and the recombinant protein showed activity in the protease enzyme assay using gelatin as a substrate.

The morecular cloning, gene structure, expression and enzyme activity of a serine-like proteas frome Laccotrephes Japonensis were examined. In this study, RT-PCR was used to amplify cDNA fragments for serine-like proteases from total RNA the hole body of Laccotrephes japonensis. The flanking sequences of the 5'- and 3'- end of the this gene were characterized by RACE-PCR. Sequence analysis showed that this gene contained an 963bp ORF encoding 321 amino acids. The deduced amino acid sequence of this protease showed 62% identity to the serine protease of Creontiades dilutus, 58% to Lygus loneolaris trypsin precuror LlsgP4, 54% to Triatonatoma infestans salivary trypsin. To generate Laccotrephes japonensis serine-like protease, the DNA fragment coding for serine protease is cloning into suttle vector pBACⅠ, named pBAC1-JG and infected to Spodoptera frugiperda (sf9) insect cell. The cDNA encoding JG was expressed as a 32-kDa polypeptide in baculovirus infected insect cells and the recombinant JG showed activity in the protease enzyme assay using gelatin as a substrate.