RESUMO
Genetic engineering of Bacillus thuringiensis (Bt) Cry proteins has resulted in the synthesis of various novel toxin proteins with enhanced insecticidal activity and specificity towards different insect pests. In this study, a fusion protein consisting of the DI-DII domains of Cry1Ac and garlic lectin (ASAL) has been designed in silico by replacing the DIII domain of Cry1Ac with ASAL. The binding interface between the DI-DII domains of Cry1Ac and lectin has been identified using protein-protein docking studies. Free energy of binding calculations and interaction profiles between the Cry1Ac and lectin domains confirmed the stability of fusion protein. A total of 18 hydrogen bonds was observed in the DI-DII-lectin fusion protein compared to 11 hydrogen bonds in the Cry1Ac (DI-DII-DIII) protein. Molecular mechanics/Poisson-Boltzmann (generalized-Born) surface area [MM/PB (GB) SA] methods were used for predicting free energy of interactions of the fusion proteins. Protein-protein docking studies based on the number of hydrogen bonds, hydrophobic interactions, aromatic-aromatic, aromatic-sulphur, cation-pi interactions and binding energy of Cry1Ac/fusion proteins with the aminopeptidase N (APN) of Manduca sexta rationalised the higher binding affinity of the fusion protein with the APN receptor compared to that of the Cry1Ac-APN complex, as predicted by ZDOCK, Rosetta and ClusPro analysis. The molecular binding interface between the fusion protein and the APN receptor is well packed, analogously to that of the Cry1Ac-APN complex. These findings offer scope for the design and development of customized fusion molecules for improved pest management in crop plants.
Assuntos
Proteínas de Bactérias/química , Antígenos CD13/química , Endotoxinas/química , Proteínas Hemolisinas/química , Lectinas/química , Manduca/enzimologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Antígenos CD13/metabolismo , Endotoxinas/metabolismo , Alho/química , Proteínas Hemolisinas/metabolismo , Ligação de Hidrogênio , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Lectinas/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Conformação Proteica , Engenharia de Proteínas , Estrutura Terciária de ProteínaRESUMO
In insects, 3,4-dihydroxyphenylalanine (DOPA) is required for tanning of newly formed cuticle and the production of melanin during some types of immune responses. DOPA is produced by the hydroxylation of tyrosine, and this reaction can be catalyzed by two types of enzymes: tyrosine hydroxylase (TH) and phenoloxidase (PO). TH is required for cuticle tanning in Drosophila melanogaster and for cuticle pigmentation in other insect species, but additional functions of TH have been uncertain. In contrast, an immune function for PO has been well documented. The goal of this study was to characterize TH from Manduca sexta with a focus on its possible contribution to cuticle tanning and immune-associated melanization. We cloned a full-length TH cDNA, purified recombinant TH, and confirmed that MsTH and MsPO have tyrosine hydroxylating activity. To determine possible functions, we analyzed TH expression profiles. TH mRNA and protein were present in eggs at the stage when the pharate larval cuticle begins to tan and also in the integument of molting larvae. The amount of TH in the integument was correlated with the degree of cuticle tanning. Unlike PO, which was found to be constitutively expressed by hemocytes and was present in plasma, TH was upregulated in hemocytes and the fat body in response to an immune challenge and remained intracellular. These data suggest that TH is required for cuticle tanning and immunity in M. sexta. Based on the collective information from many studies, we propose a model in which TH is a major producer of the DOPA required for both cuticle tanning and immune-associated melanization.
Assuntos
Di-Hidroxifenilalanina/biossíntese , Manduca/enzimologia , Melaninas/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Sequência de Aminoácidos , Animais , DNA Complementar , Expressão Gênica , Manduca/genética , Manduca/imunologia , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/isolamento & purificação , Monofenol Mono-Oxigenase/metabolismo , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de DNA , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/isolamento & purificaçãoRESUMO
The main triglyceride-lipase (TG-lipase) from the fat body of Manduca sexta has been identified as the homolog of Drosophila melanogaster CG8552. This protein is conserved among insects and also shares significant sequence similarity with vertebrate phospholipases (PLs) from the phosphatidic acid preferring-phospholipase A1 (PA-PLA(1)) family. It is shown here that the TG-lipase is also a PL. TG-lipase and PL activities copurify and are inhibited by, or resistant to, the same lipase inhibitors, indicating that both activities are catalyzed by the same enzyme and active site. The PL activity of TG-lipase corresponded to PL type A(1). The concentration dependence of lipase activity with TG and PL micellar substrates showed saturation kinetics, with apparent K(m) values of 152 +/- 11 and 7.8 +/- 1.1 muM, respectively. TG-lipase was able to hydrolyze the major phospholipid components of the lipid droplets, phosphatidylcholine and phosphatidylethanolamine. The enzyme hydrolyzes 77 molecules of TG for every molecule of PL contained in the lipid droplets. It was observed that the activation of lipolysis in vivo is accompanied by activation of the hydrolysis of phospholipids of the lipid droplets. These results suggest that the PL activity of the insect TG-lipase could be required to allow access of the lipase to TG molecules contained in the core of the lipid droplets.
Assuntos
Corpo Adiposo/enzimologia , Lipase/metabolismo , Manduca/enzimologia , Fosfolipases A/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Inibidores Enzimáticos/farmacologia , Hormônios de Inseto/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/isolamento & purificação , Proteínas de Insetos/metabolismo , Lipase/genética , Lipase/isolamento & purificação , Lipólise , Manduca/genética , Modelos Biológicos , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Fosfolipases A/genética , Fosfolipases A/isolamento & purificação , Ácido Pirrolidonocarboxílico/análogos & derivados , Ácido Pirrolidonocarboxílico/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
The response of V(1) ATPase of the tobacco hornworm Manduca sexta to Mg(2+) and nucleotide binding in the presence of the enhancer methanol has been studied by CuCl(2)-induced disulfide formation, fluorescence spectroscopy, and small-angle X-ray scattering. When the V(1) complex was supplemented with CuCl(2) nucleotide-dependence of A-B-E and A-B-E-D cross-linking products was observed in absence of nucleotides and presence of MgADP+Pi but not when MgAMP.PNP or MgADP were added. A zero-length cross-linking product of subunits D and E was formed, supporting their close proximity in the V(1) complex. The catalytic subunit A was reacted with N-4[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide (CM) and spectral shifts and changes in fluorescence intensity were detected upon addition of MgAMP.PNP, -ATP, -ADP+Pi, or -ADP. Differences in the fluorescence emission of these nucleotide-binding states were monitored using the intrinsic tryptophan fluorescence. The structural composition of the V(1) ATPase from M. sexta and conformational alterations in this enzyme due to Mg(2+) and nucleotide binding are discussed on the basis of these and previous observations.
Assuntos
Manduca/enzimologia , ATPases Vacuolares Próton-Translocadoras/isolamento & purificação , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Cobre , Reagentes de Ligações Cruzadas/metabolismo , Ligantes , Magnésio/farmacologia , Metanol/metabolismo , Nucleotídeos/metabolismo , Conformação Proteica/efeitos dos fármacos , Espalhamento de Radiação , Espectrometria de Fluorescência , Raios XRESUMO
Laccase (EC 1.10.3.2) is an enzyme with p-diphenol oxidase activity that is a member of a group of proteins collectively known as multicopper, or blue copper, oxidases. Laccase is hypothesized to play an important role in insect cuticle sclerotization by oxidizing catechols in the cuticle to their corresponding quinones, which then catalyze protein cross-linking reactions. To facilitate studies of the structure, function and regulation of insect laccases, we have cloned two cDNAs for laccases from the tobacco hornworm, Manduca sexta (MsLac1 and 2), and one from the malaria mosquito, Anopheles gambiae (AgLac1). The MsLac1 and 2 cDNAs encode proteins of 801 amino acids (aa) and 760 aa, respectively, while the AgLac1 cDNA encodes a protein of 1009 aa. All three cDNAs contain putative secretion signal sequences, and the 10 histidines and one cysteine that form the copper-binding centers, as well as a methionine in the T1 copper center. Novel to the insect laccases, relative to both fungal and plant laccases, is a longer amino-terminal sequence characterized by a unique domain consisting of several conserved cysteine, aromatic, and charged residues. Northern blot analyses identified single transcripts of approximately 3.6, 3.5, and 4.4 kb for MsLac1, MsLac2, and AgLac1, respectively, and also showed that AgLac1 was expressed in all life stages of the mosquito. RT-PCR revealed that the MsLac1 transcript was most abundant in the midgut, Malpighian tubules, and epidermis, whereas the MsLac2 transcript was most abundant in the epidermis. MsLac2 showed strong expression in the pharate pupal and reduced expression in the early pupal epidermis, consistent with the laccases' presumed role in cuticle sclerotization.
Assuntos
Anopheles/enzimologia , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , DNA Complementar/genética , Lacase/biossíntese , Lacase/genética , Manduca/enzimologia , Sequência de Aminoácidos , Animais , Anopheles/genética , Anopheles/crescimento & desenvolvimento , Proteínas de Transporte/química , Clonagem Molecular , Epiderme/enzimologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Lacase/química , Larva/enzimologia , Masculino , Manduca/genética , Manduca/crescimento & desenvolvimento , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Pupa/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
The gene sequence of Manduca sexta juvenile hormone diol kinase (JHDK) codes for an enzyme that has 59% sequence identity to Drosophila melanogaster sarcoplasmic calcium-binding protein-2 (dSCP2). JHDK and dSCP2 are similar to G-proteins with three conserved sequence elements involved in purine nucleotide binding. Both proteins contain two pairs of EF-hand motifs. Characterization and partial purification of the D. melanogaster homolog of M. sexta JHDK from adult D. melanogaster gave material with JHDK activity. This activity has an experimental pI and molecular mass that are nearly identical to those of dSCP2. Moreover, D. melanogaster phosphotransferase activity has very similar chromatographic retention in three systems compared with M. sexta JHDK. Substrate docking to three-dimensional models of JHDK has shown that the three conserved nucleotide-binding elements surround the putative substrate-binding site and align with conserved sequence elements of p21(Ras) and adenylate kinase. D. melanogaster dSCP2 is a homolog of M. sexta JHDK, and these proteins constitute a novel kinase family that binds nucleotides using the scaffold of an SCP (Protein Data Bank code ).
Assuntos
Hormônios Juvenis/metabolismo , Manduca/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/metabolismo , Clonagem Molecular , DNA Complementar/metabolismo , Dimerização , Drosophila melanogaster , Biblioteca Gênica , Lisina/química , Modelos Moleculares , Dados de Sequência Molecular , Fosfotransferases/química , Ligação Proteica , Dobramento de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Retículo Sarcoplasmático/metabolismoRESUMO
An aminopeptidase N (APN) with a molecular weight of 110kDa was released from the midgut membrane of Bombyx mori by phosphatidylinositol-specific phospholipase C (PI-PLC), and purified to a homogeneous state. This 110-kDa APN was different from the 100-kDa APN that we previously reported, in chromatographic behaviors, substrate specificity, and N-terminal and internal amino acid sequences. However, the N-terminal sequence of 110-kDa APN, DPAFRLPTTTRPRHYQVTLT, was highly homologous with those of Manduca sexta and Heliothis virescens APNs, which were identified as a receptor for an insecticidal toxin of Bacillus thuringiensis. From a B. mori midgut cDNA library, we cloned the 110-kDa APN cDNA that possessed a 2958-bp open reading frame encoding a 111573-Da polypeptide of 986 residues. The sequence of the eicosa-peptide Asp42Thr61 deduced from the cDNA was completely matched with the N-terminal sequence of the mature 110-kDa APN. One potential N-glycosylation site, HEXXHXW zinc-binding motif and characteristic proline-rich repeats were observed in the ORF. Moreover, the primary sequence contained two hydrophobic peptides on N- and C-termini. The N-terminal peptide sequence showed characteristics of leader peptide for secretion and the C-terminal peptide contained a possible glycosylphosphatidylinositol (GPI) anchoring site. Taken together, the deduced amino acid sequence suggests that the 110-kDa APN is a GPI-anchored protein and a specific receptor protein for B. thuringiensis CryIA delta-endotoxin.
Assuntos
Toxinas Bacterianas , Bombyx/enzimologia , Bombyx/genética , Antígenos CD13/genética , Proteínas de Insetos , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Animais , Bacillus thuringiensis , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Sequência de Bases , Antígenos CD13/isolamento & purificação , Antígenos CD13/metabolismo , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Sistema Digestório/enzimologia , Endotoxinas/metabolismo , Genes de Insetos , Glicosilfosfatidilinositóis/metabolismo , Proteínas Hemolisinas , Humanos , Manduca/enzimologia , Manduca/genética , Dados de Sequência Molecular , Peso Molecular , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
The Bacillus thuringiensis CryIA(c) insecticidal delta-endotoxin binds to a 120-kDa glycoprotein receptor in the larval midgut epithelia of the susceptible insect Manduca sexta. This glycoprotein has recently been purified and identified as aminopeptidase N. We now report the cloning of aminopeptidase N from a M. sexta midgut cDNA library. Two overlapping clones were isolated, and their combined 3095-nucleotide sequence contains an open reading frame encoding a 990-residue pre-pro-protein. The N-terminal amino acid sequence derived from the glycoprotein is present in the open reading frame, immediately following a predicted cleavable signal peptide and a pro-peptide. There are four potential N-linked glycosylation sites. The C-terminal sequence contains a possible glycosylphosphatidylinositol (GPI) anchor signal peptide, which suggests that, unlike most other characterized aminopeptidases, the lepidopteran enzyme is anchored in the membrane by a GPI anchor. This was confirmed by partial release of aminopeptidase N activity from M. sexta midgut brush border membranes by phosphatidylinositol-specific phospholipase C. The deduced amino acid sequence shows significant similarity to the zinc-dependent aminopeptidase gene family, particularly in the region surrounding the consensus zinc-binding motif characteristic of these enzymes.
Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas , Antígenos CD13/genética , Endotoxinas/metabolismo , Proteínas de Insetos , Manduca/enzimologia , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Animais , Toxinas de Bacillus thuringiensis , Sequência de Bases , Antígenos CD13/metabolismo , Clonagem Molecular , DNA Complementar , Proteínas Hemolisinas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Receptores de Superfície Celular/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The proteasome plays a central role in ubiquitin-dependent and -independent proteolysis in eukaryotic cells. The hawkmoth proteasome was purified from larval body wall and characterized with respect to substrate specificity, sensitivity to protease inhibitors, and cross-reactivity with monoclonal antibodies (mAbs) raised against human placenta proteasome. Leupeptin selectively inhibited the trypsin-like activity (T-L) and N-ethylmaleimide inhibited both T-L and chymotrypsin-like activities, whereas 0.02% sodium dodecyl sulfate stimulated the peptidylglutamyl peptide hydrolase, branched-chain amino acid preferring, and caseinolytic activities 20-, 18-, and 3.8-fold, respectively. All four peptidase activities were inhibited by 3,4-dichloroisocoumarin. One-dimensional immunoblot analysis showed that the level and subunit composition of the proteasome varied between tissues. The relative levels of proteasome were high in intersegmental muscle and ovary, lower in Malpighian tubule, male accessory gland, and ventral nerve cord, and lowest in flight muscle and fat body. The tissues differed in the relative amount of a 41-kDa doublet; a 22-kDa subunit was present only in the male accessory gland. Two-dimensional polyacrylamide gel electrophoresis showed that the hawkmoth proteasome contained at least 26 subunits, compared with 28 subunits in lobster. Immunological analysis using four subunit-specific mAbs identified the putative homologs of the human zeta, C2, C3, and C8 alpha-type subunits in the hawkmoth and lobster enzymes. Two of the four mAbs reacted with three or more of the hawkmoth subunits and three of the mAbs reacted with two or more of the lobster subunits. In addition, two other mAbs that recognize epitopes shared by a number of alpha-type subunits indicated that at least 15 (lobster) or 16 (hawkmoth) subunits were alpha-type. These results suggest that much of the subunit complexity of the arthropod proteasomes is a consequence of extensive post-translational modifications.