Characterization and cDNA cloning of the pheromone-binding protein from the tobacco hornworm, Manduca sexta: a tissue-specific developmentally regulated protein.
Ontology highlight
ABSTRACT:
SUBMITTER: Gyorgyi TK
PROVIDER: S-EPMC282879 | biostudies-other | 1988 Dec
REPOSITORIES: biostudies-other
Publications
Characterization and cDNA cloning of the pheromone-binding protein from the tobacco hornworm, Manduca sexta: a tissue-specific developmentally regulated protein.
Györgyi T K TK Roby-Shemkovitz A J AJ Lerner M R MR
Proceedings of the National Academy of Sciences of the United States of America 19881201 24
cDNA encoding pheromone-binding protein (PBP), the major soluble protein in olfactory sensilla of male moths, has been cloned from the tobacco hornworm, Manduca sexta. A study of the developmental time course of PBP reveals that it is first synthesized just prior to eclosion and that the percentage of antennal mRNA encoding PBP shifts from zero to about 20% at that time. PBP is also found in sensilla from female M. sexta antennae. No amino acid sequence homology is observed between PBP and the v ...[more]
Similar Datasets
Project description:Analogous to blood coagulation and complement activation in mammals, some insect defense responses (e.g. prophenoloxidase (proPO) activation and Toll pathway initiation) are mediated by serine proteinase cascades and regulated by serpins in hemolymph. We recently isolated Manduca sexta serpin-6 from hemolymph of the bacteria-challenged larvae, which selectively inhibited proPO-activating proteinase-3 (PAP-3) (Wang, Y., and Jiang, H. (2004) Insect Biochem. Mol. Biol. 34, 387-395). To further characterize its structure and function, we cloned serpin-6 from an induced fat body cDNA library using a PCR-derived probe. M. sexta serpin-6 is 55% similar in amino acid sequence to Drosophila melanogaster serpin-5, an immune-responsive protein. We produced serpin-6 in an Escherichia coli expression system and purified the soluble protein by nickel affinity and hydrophobic interaction chromatography. The recombinant protein specifically inhibited PAP-3 and blocked proPO activation in vitro in a concentration-dependent manner. Matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated that the cleavage site of serpin-6 is between Arg373 and Ser374. Serpin-6 is constitutively present in hemolymph of naive larvae, and its mRNA and protein levels significantly increase after a bacterial injection. The association rate constant of serpin-6 and PAP-3 is 2.6 x 10(4) m(-1) s(-1), indicating that serpin-6 may contribute to the inhibitory regulation of PAP-3 in the hemolymph. We also identified the covalent complex of serpin-6 and PAP-3 in induced hemolymph by immunoaffinity chromatography and mass spectrometry. Furthermore, immulectin-2, serine proteinase homologs, proPO, PO, attacin-2, and a complex of serpin-6 and hemolymph proteinase-8 were also detected in the proteins eluted from the immunoaffinity column using serpin-6 antibody. These results suggest that serpin-6 plays important roles in the regulation of immune proteinases in the hemolymph.
| S-EPMC2047605 | biostudies-literature
Project description:Fatty acids stored as triglycerides (TG) in the fat body serve as precursor in multiple processes including energy production and synthesis of cellular components. Mobilization of fatty acids from TG depends on the action of lipases. The fat body triglyceride lipase from Manduca sexta, MsTGL, is the only insect lipase that has been purified and characterized, so far. A TGL cDNA from M. sexta fat body encoding a 649 amino acid protein was cloned and its identity confirmed by mass spectrometry and Edman sequencing data of the purified protein. The protein sequence has conserved domains and residues of potential importance for the function and regulation of TGL activity. The expression of TGL and the lipase activity of fat body homogenates were studied in several developmental stages of M. sexta. TG-hydrolase activity of fat body increased as larva grew to the last instar and, then, decreased to minimal levels during pupa stage. Lipase activity was progressively restored in adult insects and reached maximum values at this stage. The fat body lipase activity from adult insects, 1-2 day after emergence, was 9-fold higher than that from 2 to 3 days old 5th-instar larvae. A good correlation was found between the abundance of TGL protein and the lipase activity of fat body homogenates. This correlation and the expression pattern of TGL throughout development are consistent with the notion that TGL is the main fat body TG lipase of M. sexta.
| S-EPMC2829384 | biostudies-literature
Project description:Odorant-binding proteins (OBPs) are small abundant extracellular proteins thought to participate in perireceptor events of odor-pheromone detection by carrying, deactivating, and/or selecting odor stimuli. The honeybee queen pheromone is known to play a crucial role in colony organization, in addition to drone sex attraction. We identified, for the first time in a social insect, a binding protein called antennal-specific protein 1 (ASP1), which binds at least one of the major queen pheromone components. ASP1 was characterized by cDNA cloning, expression in Pichia pastoris, and pheromone binding. In situ hybridization showed that it is specifically expressed in the auxiliary cell layer of the antennal olfactory sensilla. The ASP1 sequence revealed it as a divergent member of the insect OBP family. The recombinant protein presented the exact characteristics of the native protein, as shown by mass spectrometry, and N-terminal sequencing and exclusion-diffusion chromatography showed that recombinant ASP1 is dimeric. ASP1 interacts with queen pheromone major components, opposite to another putative honeybee OBP, called ASP2. ASP1 biosynthetic accumulation, followed by nondenaturing electrophoresis during development, starts at day 1 before emergence, in concomitance with the functional maturation of olfactory neurons. The isobar ASP1b isoform appears simultaneously to ASP1a in workers, but only at approximately 2 weeks after emergence in drones. Comparison of in vivo and heterologous expressions suggests that the difference between ASP1 isoforms might be because of dimerization, which might play a physiological role in relation with mate attraction.
| S-EPMC6782524 | biostudies-literature
Project description:Male Manduca sexta moths are attracted to a mixture of two components of the female's sex pheromone at the natural concentration ratio. Deviation from this ratio results in reduced attraction. Projection neurons innervating prominent male-specific glomeruli in the male's antennal lobe produce maximal synchronized spiking activity in response to synthetic mixtures of the two components centering around the natural ratio, suggesting that behaviorally effective mixture ratios are encoded by synchronous neuronal activity. We investigated the physiological activity and morphology of downstream protocerebral neurons that responded to antennal stimulation with single pheromone components and their mixtures at various concentration ratios. Among the tested neurons, only a few gave stronger responses to the mixture at the natural ratio whereas most did not distinguish among the mixtures that were tested. We also found that the population response distinguished among the two pheromone components and their mixtures, prior to the peak population response. This observation is consistent with our previous finding that synchronous firing of antennal-lobe projection neurons reaches its maximum before the firing rate reaches its peak. Moreover, the response patterns of protocerebral neurons are diverse, suggesting that the representation of olfactory stimuli at the level of protocerebrum is complex.
| S-EPMC3805782 | biostudies-literature
Project description:Insect odorant receptors (ORs) are 7-transmembrane receptors with inverse membrane topology. They associate with the conserved ion channel Orco. As chaperon, Orco maintains ORs in cilia and, as pacemaker channel, Orco controls spontaneous activity in olfactory receptor neurons. Odorant binding to ORs opens OR-Orco receptor ion channel complexes in heterologous expression systems. It is unknown, whether this also occurs in vivo. As an alternative to this ionotropic transduction, experimental evidence is accumulating for metabotropic odor transduction, implicating that insect ORs couple to G-proteins. Resulting second messengers gate various ion channels. They generate the sensillum potential that elicits phasic-tonic action potentials (APs) followed by late, long-lasting pheromone responses. Because it is still unclear how and when Orco opens after odor-OR-binding, we used tip recordings to examine in vivo the effects of the Orco antagonist OLC15 and the amilorides MIA and HMA on bombykal transduction in the hawkmoth Manduca sexta. In contrast to OLC15 both amilorides decreased the pheromone-dependent sensillum potential amplitude and the frequency of the phasic AP response. Instead, OLC15 decreased spontaneous activity, increased latencies of phasic-, and decreased frequencies of late, long-lasting pheromone responses Zeitgebertime-dependently. Our results suggest no involvement for Orco in the primary transduction events, in contrast to amiloride-sensitive channels. Instead of an odor-gated ionotropic receptor, Orco rather acts as a voltage- and apparently second messenger-gated pacemaker channel controlling the membrane potential and hence threshold and kinetics of the pheromone response.
| S-EPMC5102459 | biostudies-literature
Project description:Food digestion is vital for the survival and prosperity of insects. Research on insect digestive enzymes yields knowledge of their structure and function, and potential targets of antifeedants to control agricultural pests. While such enzymes from pest species are more relevant for inhibitor screening, a systematic analysis of their counterparts in a model insect has broader impacts. In this context, we identified a set of 122 digestive enzyme genes from the genome of Manduca sexta, a lepidopteran model related to some major agricultural pests. These genes encode hydrolases of proteins (85), lipids (20), carbohydrates (16), and nucleic acids (1). Gut serine proteases (62) and their noncatalytic homologs (11) in the S1A subfamily are encoded by abundant transcripts whose levels correlate well with larval feeding stages. Aminopeptidases (10), carboxypeptidases (10), and other proteases (3) also participate in dietary protein digestion. A large group of 11 lipases as well as 9 esterases are probably responsible for digesting lipids in diets. The repertoire of carbohydrate hydrolases (16) is relatively small, including two amylases, three maltases, two sucrases, two α-glucosidases, and others. Lysozymes, peptidoglycan amidases, and β-1,3-glucanase may hydrolyze peptidoglycans and glucans to harvest energy and defend the host from microbes on plant leaves. One alkaline nuclease is associated with larval feeding, which is likely responsible for hydrolyzing denatured DNA and RNA undergoing autolysis at a high pH of midgut. Proteomic analysis of the ectoperitrophic fluid from feeding larvae validated at least 131 or 89% of the digestive enzymes and their homologs. In summary, this study provides for the first time a holistic view of the digestion-related proteins in a lepidopteran model insect and clues for comparative research in lepidopteran pests and beyond.
| S-EPMC7554134 | biostudies-literature
Project description:Serotonin and octopamine (OA) are biogenic amines that are active throughout the nervous systems of insects, affecting sensory processing, information coding and behavior. As an initial step towards understanding the modulatory roles of these amines in olfactory processing we cloned two putative serotonin receptors (Ms5HT1A and Ms5HT1B) and one putative OA (MsOAR) receptor from the moth Manduca sexta. Ms5HT1A and Ms5HT1B were both similar to 5HT1-type receptors but differed from each other in their N-terminus and 3rd cytoplasmic loop. Ms5HT1A was nearly identical to a serotonin receptor from Heliothis virescens and Ms5HT1B was almost identical to a serotonin receptor from Bombyx mori. The sequences for homologs of Ms5HT1A from B. mori and Ms5HT1B from H. virescens were also obtained, suggesting that the Lepidoptera likely have at least two serotonin receptors. The MsOAR shares significant sequence homology with pharmacologically characterized OA receptors, but less similarity to putative OA/tyramine receptors from the moths B. mori and H. virescens. Using the MsOAR sequence, fragments encoding putative OA receptors were obtained from B. mori and H. virescens, suggesting that MsOAR is the first OA receptor cloned from a lepidopteran.
| S-EPMC1794002 | biostudies-literature
Project description:The prophenoloxidase (proPO) activation system is an important defense mechanism in arthropods, and activation of proPO to active phenoloxidase (PO) involves a serine proteinase cascade. Here, we report the purification and characterization of a small cationic protein CP8 from the tobacco hornworm, Manduca sexta, which can stimulate proPO activation. BLAST search showed that Manduca CP8 is similar to a fungal proteinase inhibitor-1 (AmFPI-1), an inducible serine proteinase inhibitor-1 (ISPI-1), and other small cationic proteins with unknown functions. However, we showed that Manduca CP8 did not inhibit proteinase activity, but stimulated proPO activation in plasma. When small amount (0.1 microg) of purified native CP8 or BSA was added to cell-free plasma samples and incubated for 20 min, low PO activity was observed in both groups. But significantly higher PO activity was observed in the CP8-group than in the BSA-group when more proteins (0.5 microg) were added and incubated for 20 min. However, when the plasma samples were incubated with proteins for 30 min, high PO activity was observed in both the CP8 and BSA groups regardless of the amount of proteins added. Moreover, when PO in the plasma was pre-activated with Micrococcus luteus, addition of CP8 did not have an effect on PO activity, and CP8/bacteria mixture did not stimulate PO activity to a higher level than did BSA/bacteria. These results suggest that CP8 helps activate proPO more rapidly at the initial stage. CP8 mRNA was specifically expressed in fat body and its mRNA level decreased when larvae were injected with saline or bacteria. However, CP8 protein concentration in hemolymph did not change significantly in larvae injected with saline or microorganisms.
| S-EPMC2659724 | biostudies-literature
Project description:Insects synthesize a battery of antimicrobial peptides (AMPs) and expression of AMP genes is regulated by the Toll and Imd (immune deficiency) pathways in Drosophila melanogaster. Drosophila Toll pathway is activated after Spätzle (Spz) is cleaved by Spätzle processing enzyme (SPE) to release the active C-terminal C106 domain (DmSpz-C106), which then binds to the Toll receptor to initiate the signaling pathway and regulate expression of AMP genes such as drosomycin. Toll and Spz genes have been identified in other insects, but interaction between Toll and Spz and direct evidence for a Toll-Spz pathway in other insect species have not been demonstrated. Our aim is to investigate a Toll-Spz pathway in Manduca sexta, and compare M. sexta and D. melanogaster Toll-Spz pathways. Co-immunoprecipitation (Co-IP) assays showed that MsToll(ecto) (the ecto-domain of M. sexta Toll) could interact with MsSpz-C108 (the active C-terminal C108 domain of M. sexta Spz) but not with full-length MsSpz, and DmToll(ecto) could interact with DmSpz-C106 but not DmSpz, suggesting that Toll receptor only binds to the active C-terminal domain of Spz. Co-expression of MsToll-MsSpz-C108, but not MsToll-MsSpz, could up-regulate expression of drosomycin gene in Drosophila S2 cells, indicating that MsToll-MsSpz-C108 complex can activate the Toll signaling pathway. In vivo assays showed that activation of AMP genes, including cecropin, attacin, moricin and lebocin, in M. sexta larvae by purified recombinant MsSpz-C108 could be blocked by pre-injection of antibody to MsToll, further confirming a Toll-Spz pathway in M. sexta, a lepidopteran insect.
| S-EPMC3361650 | biostudies-literature
Project description:The tobacco hornworm, Manduca sexta, is a lepidopteran insect that is used extensively as a model system for studying insect biology, development, neuroscience, and immunity. However, current studies rely on the highly fragmented reference genome Msex_1.0, which was created using now-outdated technologies and is hindered by a variety of deficiencies and inaccuracies. We present a new reference genome for M. sexta, JHU_Msex_v1.0, applying a combination of modern technologies in a de novo assembly to increase continuity, accuracy, and completeness. The assembly is 470 Mb and is ∼20× more continuous than the original assembly, with scaffold N50 > 14 Mb. We annotated the assembly by lifting over existing annotations and supplementing with additional supporting RNA-based data for a total of 25,256 genes. The new reference assembly is accessible in annotated form for public use. We demonstrate that improved continuity of the M. sexta genome improves resequencing studies and benefits future research on M. sexta as a model organism.
| S-EPMC8022704 | biostudies-literature