Phosphorylation of Rab proteins from the brain of Bombyx mori

Rab proteins play fundamental roles in the regulation of membrane traffic. Previously, from the brain of Bombyx mori we isolated two cDNA clones (BRab1 and BRab14), each of which encoded a different member of Rab-protein family and was expressed in Escherichia coli and purified using an affinity chromatography. In this study, one cDNA clone (BRab8) was isolated from a cDNA library from the brain of B. mori. The recombinant protein was expressed in E. coli and purified. Next, the phosphorylations of these three purified BRab proteins were examined, using mammalian protein kinases in vitro. Protein kinase C (PKC) phosphorylated BRab8 and BRab14 proteins. Protein kinase A faintly phosphorylated BRab8 and BRab14 proteins. Calcium/calmodulin-dependent protein kinase faintly phosphorylated BRab8 protein. Next, brains of B. mori were dissected and homogenized. The homogenate showed a calcium-dependent protein kinase activity of BRab8 and BRab14 proteins. So PKC from the brain of B. mori was partially purified by a sequence of chromatographies on DEAE-Cellulofine and affinity chromatography. This PKC phosphorylated BRab8 and BRab14 proteins. These results suggest that the function of Rab proteins in the brain of B. mori is regulated by calcium-dependent protein kinases.     

Determination of phosphorylated amino acid residues of Rab8 from Bombyx mori

The Rab family of small GTPases are key regulators of membrane trafficking. Partially purified Rab8 from Bombyx mori (BRab8) was phosphorylated by protein kinase C in mammalian cells in vitro. To determine which of the seven serines and four threonines are phosphorylated, we generated deletion and site-directed mutants of BRab8, inserted them in Escherichia coli, partially purified the encoded fusion proteins by affinity chromatography, and examined their phosphorylation by protein kinase C in vitro. We found that Ser-132 of BRab8 was specifically phosphorylated by protein kinase C. In addition, Western blotting using an antiserum against BRab8 and in-gel staining for phosphorylated proteins revealed that BRab8 is phosphorylated in vivo.     

Biochemical characterization of rab proteins from Bombyx mori

The small GTPases known as Rab proteins are key regulators of membrane trafficking. We used RT-PCR to isolate cDNA clones of insect-specific Rab proteins (BRabN1 and BRabN2) showing low homology with known Rab proteins from other animals, from mRNA of Bombyx mori. These 2 Rabs were produced in Escherichia coli and purified. BRabN1 bound [(3)H]-GDP and [(35)S]-GTPgammaS with dissociation constants of 0.087 x 10(-6) M and 1.02 x 10(-6) M, respectively, whereas those of BRabN2 were 0.546 x 10(-6) M and 1.02 x 10(-6) M, respectively. Binding of [(35)S]-GTPgammaS to BRabN1 and N2 was inhibited by GDP and GTP. The GTP-hydrolysis activities of BRabN1 and N2 were 154 and 35.5 mmol/min/mole, respectively, and bound [(35)S]-GTPgammaS was exchanged efficiently with GTP. BRabN1 also showed ATPase activity and exchange of [(35)S]-GTPgammaS with ATP. Monoclonal antibodies against BRabN1 and N2 did not recognize any other Rab proteins, and Western blotting using the anti-BRabN1 antibody revealed a single band in the testis of B. mori. These results suggest that BRabN1 and N2 of B. mori bind GTP, convert from the GTP-bound state to the GDP-bound state by intrinsic GTP hydrolysis activity, and return to the GTP-bound state with the exchange, and that BRabN1 is specifically expressed in testis. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.     

Molecular Cloning of cDNA for BRab from the Brain of Bombyx mori and Biochemical Properties of BRab Expressed in Escherichia coli

From a brain cDNA library of Bombyx mori, we cloned cDNA for BRab, which encoded a 202-amino-acid polypeptide sharing 60-80% similarity with rab1 family members. To characterize its biochemical properties, cDNA for BRab was inserted into an expression vector (pGEX2T) and expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The purified GST-BRab bound [³⁵S]-GTPγS and [³H]-GDP with association constants of 1.5×10⁶ M^-1 and 0.58×10⁶ M^-1, respectively. The binding of [³⁵S]-GTPγS was inhibited with GTP and GDP, but with no other nucleotides. The GTP-hydrolysis activity was evaluated to be 5 m mole/min/mole of BRab. In the presence of 6 mM MgCl₂, bound [³⁵S]-GTPγS and [³H]-GDP were exchanged with GTPγS most efficiently. These results suggest that BRab, having a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolysis activity and returns to the GTP-bound state with the exchange of GDP with GTP.     

Purification and characterization of nucleoside diphosphate kinase from the brain of Bombyx mori

Nucleoside diphosphate kinase in the brain of Bombyx mori was purified by ammonium sulfate fractionation, and a sequence of chromatographies on DEAE-Cellulofine, hydroxyapatite, Mono-S, and Mono-Q column. The purified enzyme preparation was found to be electrophoretically homogeneous on SDS-PAGE, and its molecular mass was determined to be 18 kDa. The purified protein was digested and the amino acid sequences of resulting peptides were determined. The enzyme showed high similarity to the amino acid sequences of the Drosophila NDP kinase. The enzyme showed NDP kinase activity and mediated the phosphorylation of myelin basic protein. Gel filtration and Hill plot analysis indicate that the purified NDP kinase forms a tetramer and shows little interaction among substrates. Dephosphorylation of NDP kinase by bacterial alkaline phosphatase increased NDP kinase activity. This result indicates that phosphorylation of NDP kinase represses NDP kinase activity.     

家蚕信息素结合蛋白BmPBP2和BmPBP3基因的初步鉴定及表达分析

嗅觉对昆虫的生存、繁殖等起着重要的作用。依据家蚕Bombyx mori全基因组序列设计特异引物,扩增得到了两个信息素结合蛋白BmPBP2和BmPBP3基因的cDNA片段。结合已报道的家蚕信息素结合蛋白BmPBP1和两个普通气味结合蛋白BmGOBP的信息,对其基因结构分析表明,这5个基因均由3个外显子组成,具有保守的外显子/内含子边界和典型的6个Cys残基,且3个PBP基因在基因组上串联分布。序列同源性分析表明,BmPBP2和BmPBP3与烟草天蛾的PBP2和PBP3的同源性高达69%和63%。半定量RT-PCR分析结果显示,BmPBP2和BmPBP3基因在成虫触角中特异表达,且雌雄表达水平相当。这些结果表明BmPBP2和BmPBP3可能起着性信息素识别的作用。     

Localization and functional analysis of the insect‐specific RabX4 in the brain of Bombyx mori

Rab proteins are small monomeric GTPases/GTP‐binding proteins, which form the largest branch of the Ras superfamily. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane trafficking. RabX4 is an insect‐specific Rab protein that has no close homolog in vertebrates. There is little information about insect‐specific Rab proteins. RabX4 was expressed in Escherichia coli and subsequently purified. Antibodies against Bombyx mori RabX4 were produced in rabbits for western immunoblotting and immunohistochemistry. Western blotting of neural tissues revealed a single band, at approximately 26 kD. RabX4‐like immunohistochemical reactivity was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum in the brain. Further immunohistochemical analysis revealed that RabX4 colocalized with Rab6 and bombyxin in the corpus allatum, a neuronal organ that secretes neuropeptides synthesized in the brain into the hemolymph. RabX4 expression in the frontal ganglion, part of the insect stomatogastric nervous system that is found in most insect orders, was restricted to two neurons on the outer region and did not colocalize with allatotropin or Rab6. Furthermore, RNA interference of RabX4 decreased bombyxin expression levels in the brain. These findings suggest that RabX4 is involved in the neurosecretion of a secretory organ in Bombyx mori.     

Screening of Bombyx mori brain proteins interacting with protein tyrosine phosphatase of BmNPV

In this study, glutathione-S-transferase pull-down combined with mass spectrometry techniques were used to identify the candidate proteins interacting with protein tyrosine phosphatase of the Bombyx Mori nucleopolyhedrovirus in the B. mori (BmNPV-PTP) brain. A total of 36 proteins were identified from BmNPV-PTP coprecipitate samples by searching the NCBI_Bombyx Mori database with the original mass spectrum data. Among those proteins, the interaction between BmNPV-PTP and B. mori cyclophilin A may accelerate the apoptosis of certain nerve cells involved in regulating behavior, and thus may be an inducer of enhanced locomotor activity (ELA). After the BmNPV invasion, BmNPV-PTP binding to peripheral-type benzodiazepine receptors may initiate a series of abnormal cascades of the nervous system, which results in abnormal hyperactive behavior in B. mori. Besides this, vacuolar ATP synthase catalytic subunit A, annexin, and several enzymes for energy conversion were identified, which may play a role in enhancing viral entry and infectivity and provide energy for enhancing the locomotor activity of B. mori. In general, the results of this study will facilitate the understanding of the molecular mechanisms underlying the ELA of B. mori larva induced by BmNPV.     

Small GTPases of the Rab family in the brain of Bombyx mori

Small GTPases of the Rab family are key regulators of membrane trafficking. We produced antibodies against the Rab7 protein of Bombyx mori (BRab7) in rabbits, and against the Rab11 protein of B. mori (BRab11) in mice. The antibodies recognized BRab7 and BRab11 proteins, but did not recognize other Rab proteins. Immunoblotting of samples from brain tissue of B. mori revealed a single band for each antibody. Rab11 was expressed in most tissues, whereas Rab7 was expressed in the brain, ovary, and testis. Immunohistochemical reactivity of Rab7 and Rab11 in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Double-labeling experiments demonstrated that immunohistochemical reactivity of Rab7 co-localized with that of Rab11 and partially with that of Rab8. Immunohistochemical reactivity of Rab11 and Rab8 co-localized with that of PERIOD, one of the proteins associated with circadian rhythm. These findings suggest that Rab7, Rab8, and Rab11 are involved in protein transport in the neurons of the brain of B. mori and might play a role in the control of circadian rhythm.     

家蚕保幼激素结合蛋白Bmtol基因的克隆及功能分析

家蚕头部是一个神经中枢和感受的器官,其头部含有触角和感觉毛,感受外界的信号,并将外界信号传送到大脑进行反应。保幼激素主要是由咽侧体合成和分泌的,而保幼激素结合蛋白是保幼激素转运和发挥功能的载体,在昆虫体内具有极其重要的功能。文中通过Silk DB和NCBI数据库筛选并鉴定到一个新的具有保幼激素结合蛋白家族保守结构的蛋白Bm TOL,其编码基因编号为BGIBMGA003404(Gen Bank登录号:KY681053)。利用原核表达系统成功表达了该蛋白,通过Ni-NTA亲和层析的方法获得了Bm TOL的重组蛋白并制备了多克隆抗体。组织表达分析发现无论是转录水平还是蛋白水平Bm TOL在头部都是高量表达,且Bmtol基因在起蚕时表达量较高,在5龄和蛹期表达量较低,而在化蛾后表达量又开始上调。免疫组化结果显示Bm TOL蛋白定位在头部的皮层、触角和脑中,推测其可能与头部信息传递有关,为家蚕的生长发育和行为调控提供重要的信息来源。     

Candidate pheromone binding proteins of the silkmoth Bombyx mori

Pheromone reception is thought to be mediated by pheromone binding proteins (PBPs) in the aqueous lymph of the antennal sensilla. Recent studies have shown that the only known PBP of Bombyx mori (BmorPBP1) appears to be specifically tuned to bombykol but not to bombykal, raising the question of whether additional subtypes may exist. We have identified two novel genes, which encode candidate PBPs (BmorPBP2, BmorPBP3). Comparison with PBPs from various moth species have revealed a high degree of sequence identity and the three BmorPBP-subtypes can be assigned to distinct groups within the moth PBP family. In situ hybridization revealed that BmorPBP2 and BmorPBP3 are expressed only in relatively few cells compared to the number of cells expressing BmorPBP1. Double-labeling experiments have shown that the two novel BmorPBPs are expressed in the same cells but are not co-expressed with BmorPBP1. Furthermore, unlike BmorPBP1, cells expressing the newly identified PBPs did not surround neurons containing the BmOR-1 receptor. The results indicate that BmorPBP2 and BmorPBP3 are located in sensilla types, which are different from the long sensilla trichodea.Data deposition: The sequences reported in this paper have been deposited in the EMBL database under accession nos. AM403100 (BmorPBP2) and AM403101 (BmorPBP3).     

Expression analysis of chlorophyllid a binding protein a secretory,red fluorescence protein in the midgut of silkworm,Bombyx mori

Chlorophyllid a binding protein （ chbp） was recently characterized by its ability to bind the prosthetic group of chlorophylls and little information is known regarding its expression. In the present study, we found that chpb was expressed highly and exclusively in the midgut of silkworm, Bombyx mori. The expression level of chbp was very high in the newly molted fifth instar larvae followed by gradual decline in the same instar. Our results demonstrated that CHBP was a secretory protein and located mainly in the apical of midgut epithelial cells. Real-time polymerase chain reaction analysis results showed that chpb highly expressed in the anterior midgut, threefold and sixfold higher compared with that of the middle midgut and posterior midgut, respectively, and chpb expression declined in darkness. In addition, the expression of chbp was affected by high-dose virus or bacterium infection.     

家蚕化学感受蛋白CSP16的表达及结合特性分析

【目的】化学感受蛋白(chemosensory proteins,CSPs)在昆虫化学信号的感受识别和生长发育调节等生理过程具有重要作用。本研究旨在探索CSP16在家蚕Bombyx mori中的功能。【方法】利用RT-q PCR分析csp16在家蚕不同发育时期和不同组织的表达特征,用原核表达系统对家蚕CSP16进行表达纯化,并通过荧光结合实验检测该蛋白与不同配体化合物的结合特性。【结果】RT-q PCR结果表明,csp16在家蚕1-5龄幼虫中呈规律性表达,各龄期眠蚕中表达量最高,5龄第3天幼虫中主要表达于头、表皮、精巢和卵巢等。蜕皮激素(20E)处理后csp16在不同龄期幼虫和5龄幼虫不同组织中的表达量上调。纯化后CSP16的荧光结合实验表明,CSP16与醇类、酯类、醛类、酚类和苯环类等化合物的亲和力都较弱。【结论】csp16在家蚕不同龄期幼虫眠蚕中表达量最高,蜕皮激素使csp16在家蚕取食幼虫中的表达量出现上调,提示其可能参与家蚕幼虫的蜕皮过程。     

Rab14 from Bombyx mori (Lepidoptera: Bombycidae) shows ATPase activity

Rab GTPases are essential for vesicular transport, whereas adenosine triphosphate (ATP) is the most important and versatile of the activated carriers in the cell. But there are little reports to clarify the connection between ATP and Rab GTPases. A cDNA clone (Rab14) from Bombyx mori was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified. The protein bound to [³H]-GDP and [³⁵S]-GTPγS. Binding of [³⁵S]-GTPγS was inhibited by guanosine diphosphate (GDP), guanosine triphosphate (GTP) and ATP. Rab14 showed GTP- and ATP-hydrolysis activity. The Km value of Rab14 for ATP was lower than that for GTP. Human Rab14 also showed an ATPase activity. Furthermore, bound [³H]-GDP was exchanged efficiently with GTP and ATP. These results suggest that Rab14 is an ATPase as well as GTPase and gives Rab14 an exciting integrative function between cell metabolic status and membrane trafficking.     

Expression and functional analysis of the cellular retinoic acid binding protein from silkworm pupae (Bombyx mori)

Cellular retinoic acid binding protein (CRABP) is a member of intracellular lipid-binding protein (iLBP), and closely associated with retinoic acid (RA) activity. We have cloned the CRABP gene from silkworm pupae and studied the interaction between Bombyx mori CRABP (BmCRABP) and all-trans retinoic acid (atRA). The MTT assay data indicated that when BmCRABP is overexpressed in Bm5 cells, the cells dramatically resisted to atRA-induced growth inhibition. Conversely, the cells were sensitive to atRA treatment upon knocking down the BmCRABP expression. Subcellular localization revealed that BmCRABP is a cytoplasm protein, even when treated with atRA, the CRABP still remained in the cytoplasm. These data demonstrated that the function of BmCRABP have an effect on the physiological function of atRA.     

Small GTP-binding proteins in the brain-corpus cardiacum-corpus allatum complex of the silkworm,Bombyx mori: Involvement in the secretion of prothoracicotropic hormone

At least three GTP-binding proteins (G-proteins), 28, 25, and 21 kDa, were found in the brain-corpus cardiacum-corpus allatum complex (BR-CC-CA) of the silkworm, Bombyx mori. They bound to GTP and GDP specifically among nucleotides tested, indicating that these proteins are small G-proteins. The 25 kDa G-protein showed a cross-reactivity to anti-rab3A antibody, while it did not cross-react with anti-rhoA, rab3B, and anti-ras antibodies. On the other hand, the 28 and 21 kDa G-proteins showed no cross-reactivity to any of those antibodies tested. Immunoblot analysis using the anti-rab3A antibody demonstrated that the 25 kDa G-protein was detected preferentially in the BR-CC-CA, and to some extent in the suboesophageal ganglion, but not in the salivary gland, fat body, prothoracic gland, and oesophagus. These results suggested that the 25 kDa G-protein was a member of the rab family of G-proteins. Furthermore, 1 mM GTPγS capable of activating G-proteins induced BR-CC-CA to release PTTH under the conditions that stimulation of the PTTH release with hetero-trimeric G-protein was suppressed. These results indicated that the small G-proteins may possibly contribute to PTTH release in Bombyx mori. Arch. Insect Biochem. Physiol. 38:177–184, 1998. © 1998 Wiley-Liss, Inc.     

家蚕化学感受蛋白BmCSP4表达谱及结合特性分析

化学感受蛋白(chemosensory proteins, CSPs)是昆虫体内存在的一类主要识别和运载非挥发性的气味分子和化学刺激物的可溶性蛋白。本研究运用半定量RT-PCR方法分析了BmCSP4的时空及组织表达谱。结果表明： BmCSP4在家蚕Bombyx mori各发育阶段均表达, 但表达量从4龄到蛹期逐渐减少, 且在雌成虫头部、 胸部和腹部表达量较少。用1-NPN作为荧光探针, 测定了15种外源配基与BmCSP4蛋白的结合特性, 结果显示： 仅芳香醛类和芳香酮类化合物在浓度10 μmol/L能将1-NPN从BmCSP4中替换50%, 苯甲醛解离常数为3.20 μmol/L, 对甲氧基苯甲醛解离常数为2.24 μmol/L, 2-戊基-3-苯丙基-烯醛解离常数为2.88 μmol/L, 1-苯基-1-丁酮解离常数为2.04 μmol/L, 苯乙酮解离常数为2.52 μmol/L。据此推测, BmCSP4在不同的发育阶段执行不同的生理功能, 并可能参与对芳香醛、 芳香酮类气味识别过程。