Enzymatic properties of a bee venom serine protease from the bumblebee Bombus ignitus

We previously reported that bumblebee (Bombus ignitus) venom serine protease (Bi-VSP) acts as a prophenoloxidase-activating factor in arthropods and a fibrin(ogen)olytic enzyme in mammals. In the present study, we characterized the enzymatic properties of Bi-VSP purified from B. ignitus venom. The 34-kDa active form of Bi-VSP was purified from the venom of B. ignitus worker bees. Glycoprotein staining showed that approximately 20% of the total molecular mass of Bi-VSP is due to carbohydrate moieties. Bi-VSP had an optimal pH and temperature of pH 9.0 and 40 °C, respectively, and was stable at 50 °C for at least 10 min. Bi-VSP activity decreased abruptly below pH 6.0, indicating that Bi-VSP activity is almost completely inhibited at pH 5.4 of B. ignitus venom. The protease activity of Bi-VSP was strongly inhibited by typical serine protease inhibitors such as phenylmethanesulfonyl fluoride, leupeptin, and soybean trypsin inhibitor.     

Molecular cloning and fibrin(ogen)olytic activity of a bumblebee (Bombus hypocrita sapporoensis) venom serine protease

Although several bee venom serine protease genes have been previously described, fibrin(ogen)olytic activity of these serine proteases has been reported for only two bumblebees to date, Bombus ignitus and B. terrestris. Here, we cloned venom serine proteases from the other bumblebee species, B. hypocrita sapporoensis and B. ardens ardens. The venom serine protease genes of B. h. sapporoensis and B. a. ardens consist of 358 amino acids and 357 amino acids, respectively. We compared the predicted mature protein sequences of these serine protease genes to those previously reported for other bees. A phylogenetic analysis shows that B. h. sapporoensis venom serine protease is further immediately close to B. ignitus and B. terrestris venom serine proteases, excluding the venom serine protease of B. a. ardens. Using B. h. sapporoensis venom serine protease (Bs-VSP), we identified that Bs-VSP acts as a fibrin(ogen)olytic enzyme. We also found that Bs-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. Our results further define roles for bumblebee venom serine proteases as fibrin(ogen)olytic agents.     

A novel serine protease inhibitor from Bungarus fasciatus venom

By Sephadex G-50 gel filtration, cation-exchange CM-Sephadex C-25 chromatography and reversed phase high-performance liquid chromatography (HPLC), a novel serine protease inhibitor named bungaruskunin was purified and characterized from venom of Bungarus fasciatus. Its cDNA was also cloned from the cDNA library of B. fasciatus venomous glands. The predicted precursor is composed of 83 amino acid (aa) residues including a 24-aa signal peptide and a 59-aa mature bungaruskunin. Bungaruskunin showed maximal similarity (64%) with the predicted serine protease inhibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin, chymotrypsin, and elastase. By screening the cDNA library, two new B chains of beta-bungarotoxin are also identified. The overall structures of bungaruskunin and beta-bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.     

Expressed sequence tag analysis of the diapausing queen of the bumblebee Bombus ignitus

We constructed a full‐length cDNA library from diapausing queens of the bumblebee Bombus ignitus. A total of 480 randomly selected clones was sequenced by single‐run 5′‐end sequencing. Of these, there were 437 high quality clones, 23 poor quality clones and 20 read‐fail clones. Each high quality clone sequence was searched against a public protein database. The most frequently found matching genes were ribosomal proteins (12.5%), p10 (3.58%), cytochrome P450 monooxygenase (3.13%) and sensory appendage protein (2.9%). Sequence similarity analysis between bumblebees and other insect species showed that 72 out of 437 (16.5%) bumblebee expressed sequence tags (EST) matched sequences of Apis mellifera, with matches to Drosophila melanogaster (6.6%), Caenorhabditis briggsae (6.2%), Lysiphlebus testaceipes (4.8%), Periplaneta americana (3.7%) and Anopheles gambiae (3.4%) following, suggesting that sequence similarity of bumblebee EST is closest to that of A. mellifera. Functional classification of EST based on Gene Ontology showed that most genes found by sequencing are associated with physiological processes in the bumblebee. The results of sequencing and analysis of our 437 cDNA demonstrated that high‐throughput EST sequencing and data analysis are powerful means for identifying novel genes and for expression profiling. Our bumblebee EST collection could be a useful platform for further studies of gene expression in diapausing bumblebees.     

Insecticidal activity of recombinant baculovirus co-expressing Bacillus thuringiensis crystal protein and Kunitz-type toxin isolated from the venom of bumblebee Bombus ignitus

To improve the insecticidal activity of Autographa californica nucleopolyhedrovirus (AcMNPV), using co-expression of Bacillus thuringiensis crystal protein and a Kunitz-type toxin isolated from bumblebee Bombus ignitus venom, a recombinant AcMNPV, ApPolh5-3006BiKTI, expressing Bi-KTI under the control of early promoter from Cotesia plutellae bracovirus (CpBV) was constructed. In this recombinant virus, B. thuringiensis cry1-5 crystal protein gene was introduced into the genome by the fusion of polyhedrin-cry1-5 under the control of polyhedrin gene promoter. RT-PCR analysis indicated that both Bi-KTI and polyhedrin-cry1-5 fusion protein were successfully expressed from the infected cells. In addition, SDS-PAGE revealed that polyhedrin-cry1-5 fusion protein expressed by recombinant viruses was occluded into the polyhedra. ApPolh5-3006BiKTI showed an improved insecticidal activity against larvae of Plutella xylostella and Spodoptera exigua. At low dosage rates, it was more effective against S. exigua than on P. xylostella, but more rapid insecticidal activity was shown in P. xylostella. These results strongly suggest that co-expression of Bt toxin and Kunitz-type toxins could be successfully applied to improve the insecticidal activity of baculoviruses.     

Nest takeover by queen and its positive impact on colony development in the Japanese bumblebee Bombus ignitus (Apidae: Hymenoptera)

Lone Bombus ignitus queens are known to start nests in small underground cavities. To examine the nest‐site preference of post‐hibernating queens, choice tests were carried out by providing queens with orphan colonies and empty cavities as possible nesting sites within an experimental box. Our results showed that the queens had a strong preference for takeover of an orphan colony, suggesting that nest takeover (usurpation) could occur in nature even with the presence of possible empty cavities for nesting. We compared the colony‐growth process and final production of sexuals between non‐takeover and takeover colonies. The increase in the number of egg cups was faster in the takeover colonies, suggesting that orphan broods elicit earlier oviposition by the usurping queen. Reproductives emerged earlier (significant for new queens) and in greater numbers (males) from takeover colonies than from non‐takeover colonies. Thus, post‐hibernating B. ignitus queens would search for and take over small orphan colonies to increase their fitness.     

红光熊蜂卵黄原蛋白基因的cDNA全长序列克隆和表达分析

红光熊蜂Bombus ignitus Smith是许多经济作物和野生植物的重要授粉昆虫之一。卵黄原蛋白基因(vitellogenin,Vg)在昆虫的生殖调控中和行为方面起到重要的作用,本试验对Vg基因全长cDNA的克隆和测序及在蜂王、工蜂和雄性蜂三型蜂中的表达分析得出:Vg基因的全长cDNA为5 481 bp,GenBank中的登录号为FJ913883,有一个完整的开放阅读框(ORF),编码1 772个氨基酸,N-末端的前16个氨基酸为一个信号肽。接近C-末端区域存在保守的GL/ICG基元,其后含有9个半胱氨酸,而且DGXR位于GL/ICG基元上游18个氨基酸残基处。其氨基酸序列与韩国的熊蜂B.ignitus和B.hypocrita相似性高达95%,与西方蜜蜂Apis mellifera的相似性达到51%。Vg的mRNA首先在蜂王蛹期的白眼蛹(Pw)时期出现,其表达量在蜂王整个蛹期发育过程中呈上升趋势,且在黑眼蛹(Pbd)时期达到最高,在成年蜂的脂肪体中的表达量仍在升高,甚至更高。Vg也在工蜂蛹期的白眼蛹(Pw)时期被检测到,然后在整个蛹期发育过程中呈现上升趋势,在刚羽化出房时达到高峰,Vg的mRNA水平随着成年蜂日龄的增加而增加,到15日龄时达到最高,然后呈现下降趋势。对于雄性蜂,Vg的mRNA虽然卵黄原蛋白基因的mRNA水平几乎在整个蛹期发育阶段都表达,但是表达水平非常低,只有在刚羽化出房时期表达水平较高。     

Molecular cloning and oxidative stress response of a sigma-class glutathione S-transferase of the bumblebee Bombus ignitus

Glutathione S-transferases (EC 2.5.1.18; GSTs) are multifunctional enzymes that are mainly involved in xenobiotic metabolism and protection against oxidative damage. Most studies of GSTs in insects have been focused on their role in detoxifying exogenous compounds in particular insecticides. Here, we show the expression profiles of GSTs of the bumblebee Bombus ignitus in response to oxidative stress. We identified a sigma-class GST from B. ignitus (BiGSTS). The BiGSTS gene consists of 4 exons that encode 201 amino acids. Comparative analysis indicates that the predicted amino acid sequence of BiGSTS shares a high identity with the sigma-class GSTs of hymenopteran insects such as Apis mellifera (70% protein sequence identity) and Solenopsis invicta (59% protein sequence identity). Tissue distribution analyses showed the presence of BiGSTS in all tissues examined, including the fat body, midgut, muscle and epidermis. The oxidative stress responses analyzed by quantitative real-time PCR showed that under H(2)O(2) overload, BiGSTS and BiGSTD (identified in our previous study) were upregulated in all tissues examined, including the fat body and midgut of B. ignitus worker bees. Under uniform conditions of H(2)O(2) overload, the expression profile of GSTs and other antioxidant enzyme genes, such as phospholipid-hydroperoxide glutathione peroxidase (Bi-PHGPx) and peroxiredoxins (BiPrx1 and BiTPx1), showed that other antioxidant enzyme genes are acutely induced at 3h after H(2)O(2) exposure, whereas BiGSTS and BiGSTD are highly induced at 9h after H(2)O(2) exposure in the fat body of B. ignitus worker bees. These findings indicate that GSTs and other antioxidant enzyme genes in B. ignitus are differentially expressed in response to oxidative stress. Taken together, our findings indicate that BiGSTS and BiGSTD are oxidative stress-inducible antioxidant enzymes that may play a role in oxidative stress response.     

The complete nucleotide sequence and gene organization of the mitochondrial genome of the bumblebee, Bombus ignitus (Hymenoptera: Apidae)

The complete 16,434-bp nucleotide sequence of the mitogenome of the bumble bee, Bombus ignitus (Hymenoptera: Apidae), was determined. The genome contains the base composition and codon usage typical of metazoan mitogenomes. An unusual feature of the B. ignitus mitogenome is the presence of five tRNA-like structures: two each of the tRNALeu(UUR)-like and tRNASer(AGN)-like sequences and one tRNAPhe-like sequence. These tRNA-like sequences have proper folding structures and anticodon sequences, but their functionality in their respective amino acid transfers remained uncertain. Among these sequences, the tRNALeu(UUR)-like sequence and the tRNASer(AGN)-like sequence are seemingly located within the A+T-rich region. This tRNASer(AGN)-like sequence is highly unusual in that its sequence homology is very high compared to the tRNAMet of other insects, including Apis mellifera, but it contains the anticodon ACT, which designates it as tRNASer(AGN). All PCG and rRNAs are conserved in positions observed most frequently in insect mitogenome structures, but the positions of the tRNAs are highly variable, presenting a new arrangement for an insect mitogenome. As a whole, the B. ignitus mitogenome contains the highest A+T content (86.9%) found in any of the complete insects mt sequences determined to date. All protein-coding sequences started with a typical ATN codon. Nine of the 13 PCGs have a complete termination codon (all TAA), but the remaining four genes terminate with the incomplete TA or T. All tRNAs have the typical clover-leaf structures of mt tRNAs, except for tRNASer(AGN), in which the DHU arm forms a simple loop. All anticodons of B. ignitus tRNAs are identical to those of A. mellifera. In the A+T-rich region, a highly conserved sequence block that was previously described in Orthoptera and Diptera was also present. The stem-and-loop structures that may play a role in the initiation of mtDNA replication were also found in this region. Phylogenetic analysis among three corbiculate tribes, represented by Melipona bicolor (Meliponini), A. mellifera (Apini), and B. ignitus (Bombini), showed the closest relationship between M. bicolor and B. ignitus.     

Cobra venom phospholipase A2 inhibition by manoalide. A novel type of phospholipase inhibitor

Manoalide, an unusual nonsteroidal sesterterpenoid recently isolated from sponge, antagonizes phorbol-induced inflammation but not that induced by arachidonic acid, suggesting that manoalide acts prior to the cyclooxygenase step in prostaglandin synthesis, possibly by inhibiting phospholipase A2. We have now studied the inhibitory effect of manoalide on a homogeneous preparation of phospholipase A2 from cobra venom. For a given concentration of manoalide, the inhibition of phospholipase A2 activity toward dipalmitoylphosphatidylcholine/Triton X-100 mixed micelles is time-dependent and plateaus at about 85% inhibition of the initial velocity even after extensive preincubation. Metal ions (Ca2+, Ba2+, Mn2+) increase the inhibition, while lysophosphatidylcholine and substrate micelles protect. Increasing manoalide concentration shows increasing inhibition of the initial velocity until a plateau is reached, giving a typical saturation curve with a linear double-reciprocal plot. Under typical conditions (20-min preincubation, 40 degrees C, pH 7.1), 50% inhibition is achieved at a manoalide concentration of about 2 X 10(-6) M. The data indicate that manoalide is a potent inhibitor of the cobra venom phospholipase A2. Manoalide is now shown to react irreversibly with lysine residues in the enzyme. Surprisingly, the cobra venom phospholipase normally acts poorly on phosphatidylethanolamine as substrate, but after reaction with manoalide, the enzyme is somewhat more active toward this substrate rather than being inhibited. This suggests that a lysine residue may be important in understanding the substrate specificity of phospholipase A2.     

Morphometric and molecular identification of the female castes of Bombus ignitus and B. ardens (Apidae: Hymenoptera)

Among Korean bumblebees, Bombus ignitus and B. ardens are relatively abundant and important for pollination of wildflowers and agricultural crops. Although the males are easily distinguishable phenotypically, the female castes are difficult to identify from each other. Here we evaluated the value of some morphometric characters in species identification. Also, we developed a polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) to discriminate these similar species. In spite of statistically significant differences of some morphological characters between two species, overlapping quantitative traits hindered accurate identification of the species. However, using 435 bp of COI gene and AluI, BspHI and Earl restriction enzymes allowed molecular identifications of these two species with unique profiles from the digestion by these restriction enzymes. This method can also be applied for older specimens with some morphological characters damaged. We also developed species-specific primers for fast and cost-effective identification of these species.     

叉角厉蝽丝氨酸蛋白酶及抑制剂基因的克隆与表达分析

为明确叉角厉蝽Eocanthecona furcellata (Wolff)丝氨酸蛋白酶基因EfSP1及抑制剂基因EfSPI20的基因序列特征和时空转录特征,为其生理功能研究奠定基础。利用PCR克隆技术获得叉角厉蝽唾液腺EfSPI20和EfSP1的完整开放阅读框(Open reading frame, ORF)序列,使用生物信息学软件进行序列分析以及系统进化分析,采用实时荧光定量PCR (Real time quantitativate PCR,RT-qPCR)分析两个基因分别在叉角厉蝽不同发育时期和组织中的表达特征。结果表明,EfSPI20与EfSP1基因完整开放阅读框长度分别为378 bp和921 bp,分别编码125个氨基酸和306个氨基酸,预测均为亲水蛋白质,理论分子量分别为13.48 kDa和33.82 kDa,等电点分别为6.68和5.80,分别有30个和23个氨基酸残基的信号肽序列,EfSPI20有跨膜结构域,EfSP1无跨膜结构域。序列比对显示叉角厉蝽EfSPI20与茶翅蝽Halyomorpha halys PPI同源性最高,氨基酸序列一致性达58%;EfSP1与稻绿蝽Nezara viridula SP同源性最高,氨基酸序列一致性达66%;系统发育树显示叉角厉蝽与同为蝽科的茶翅蝽和稻绿蝽物种亲缘关系近。EfSPI20基因在雌雄成虫和唾液腺中高表达,推测EfSPI20可能具有抑制胰蛋白酶活性的功能和与叉角厉蝽的捕食消化相关;EfSP1基因在卵期、卵巢和肠道中高表达,推测EfSP1可能与叉角厉蝽的生殖功能和蛋白消化相关。     

小峰熊蜂蜂毒磷脂酶A2基因的克隆及表达分析

磷脂酶A2 （phospholipase A2, PLA2）是蜂毒主要成分, 也是蜂毒的主要过敏原, 在熊蜂个体和群体防御方面具有重要功能。为了探究熊蜂A2基因的生物学功能, 本研究以小峰熊蜂Bombus hypocrita为材料进行了蜂毒PLA2基因的克隆、 鉴定与表达特性分析。结果表明： 该基因全长为2 272 bp, GenBank登录号为KF214771, 由4个外显子和3个内含子组成, 编码区（CDS）长为543 bp, 共编码180个氨基酸残基。氨基酸序列相似性分析显示, 成熟的小峰熊蜂PLA2（含有136个氨基酸）与其他蜂类PLA2的氨基酸序列相似性较高, 均包含10个保守的半胱氨酸残基、 1个保守的Ca2+结合位点和1个酶活性中心。基于PLA2氨基酸序列的系统进化树分析表明, 熊蜂属Bombus与蜜蜂属Apis在不同分支上, 属单系群, 且蜜蜂属分化较早。荧光定量PCR结果表明, PLA2基因在小峰熊蜂各日龄均有表达, 且随日龄增长, 表达量呈先上升后下降的趋势, 10日龄时出现峰值, 其表达量显著高于其他日龄（P<0.05）。半定量PCR结果表明, PLA2基因在毒腺、 卵巢、 中肠中表达量较高, 在足、 触角、 食道腺中表达量较低, 在脂肪体、 肌肉、 神经、 气管、 复眼、 脑中未表达。本研究探明了小峰熊蜂PLA2的基因结构及其表达特性, 丰富了熊蜂PLA2的生物学基础, 为进一步深入研究熊蜂PLA2生物学功能和作用机制以及开发蜂毒生物制剂等鉴定了基础。     

Purification of a Class B1 platelet aggregation inhibitor phospholipase A2 from Indian cobra (Naja Naja) venom

A platelet aggregation inhibitor phospholipase A(2) (NND-IV-PLA(2)) was isolated from Naja naja (Eastern India) venom by a combination of cation and anion exchange chromatography. NND-IV-PLA(2) is the most catalytically active enzyme isolated from the Indian cobra venom. The acidic PLA(2) profile of Eastern regional Indian cobra venom is distinctly different from that of the western regional venom. However the acidic PLA(2)s from both the regions follow the pattern of increasing catalytic activity with increase in acidic nature of the PLA(2) isoform. NND-IV-PLA(2) is a Class B1 platelet aggregation inhibitor and inhibits platelet aggregation induced by ADP, collagen and epinephrine. Modification of active site histidine abolishes both catalytic activity and platelet aggregation inhibition activities while aristolochic acid, a phospholipase A(2) inhibitor has only partial effect on the two activities.     

Molecular characterization of iron binding proteins, transferrin and ferritin heavy chain subunit, from the bumblebee Bombus ignitus

Transferrin and ferritin are iron-binding proteins involved in transport and storage of iron as part of iron metabolism. Here, we describe the cDNA cloning and characterization of transferrin (Bi-Tf) and the ferritin heavy chain subunit (Bi-FerHCH), from the bumblebee Bombus ignitus. Bi-Tf cDNA spans 2340 bp and encodes a protein of 706 amino acids and Bi-FerHCH cDNA spans 1393 bp and encodes a protein of 217 amino acids. Comparative analysis revealed that Bi-Tf appears to have residues comprising iron-binding sites in the N-terminal lobe, and Bi-FerHCH contains a 5'UTR iron-responsive element and seven conserved amino acid residues associated with a ferroxidase center. The Bi-Tf and Bi-FerHCH cDNAs were expressed as 79 kDa and 27 kDa polypeptides, respectively, in baculovirus-infected insect Sf9 cells. Northern blot analysis revealed that Bi-Tf exhibits fat body-specific expression and Bi-FerHCH shows ubiquitous expression. The expression profiles of the Bi-Tf and Bi-FerHCH in the fat body of B. ignitus worker bees revealed that Bi-Tf and Bi-FerHCH are differentially induced in a time-dependent manner in a single insect by wounding, bacterial challenge, and iron overload.     

Antifibrinolytic role of a bee venom serine protease inhibitor that acts as a plasmin inhibitor

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 tissue plasminogen activator. In contrast, Bi-KTI strongly inhibited plasmin, indicating that it acts as an antifibrinolytic agent; however, this inhibitory ability was two-fold weaker than that of aprotinin. 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.     

草乌花蜜产量的梯度分布及熊蜂自下而上的访花行为

收益降低假说(declining reward hypothesis)认为熊蜂自下而上的访花顺序是对花蜜产量的直接响应,先访问下部花蜜产量高的花可以获得更多的收益;花开口方向假说认为自下而上访花是因为熊蜂更容易看见其上部的花朵。为了验证上述两个假说,我们于2008年8月在北京小龙门国家森林公园调查了红光熊蜂(Bombus ignitus)访问草乌(Aconitum kusnezoffii)直立和倒立顶生花序的访问顺序,测量了直立花序下部雌性阶段花和上部雄性阶段花花蜜的糖浓度、体积,计算了花蜜中的糖含量。结果表明,红光熊蜂在直立花序和倒立花序内均以向上运动为主,分别占总运动次数的62.77%和68.35%;直立花序下部雌性阶段花花蜜糖浓度比上部雄性阶段花低1.44%,但是花蜜体积和花蜜中的糖含量都显著高于雄性阶段的花。由于熊蜂访问倒立花序时先访问的是下部的低回报雄性阶段的花,然后再访问上部高回报的雌性阶段的花,这与收益降低假说矛盾,表明红光熊蜂自下而上访问草乌直立花序可能不是受到花蜜产量的调节。