Isolation and characterization of a lipoprotein receptor from the fat body of an insect, Manduca sexta

A lipoprotein receptor has been purified from the fat body of Manduca sexta larvae. The purification involves solubilization of membrane proteins in detergent, DEAE-, and hydroxyapatite chromatography, affinity chromatography on a concanavalin A column, and affinity chromatography on a lipoprotein-Sepharose column. An overall purification of 220-fold from the solubilized membranes was achieved. The receptor has an apparent molecular mass of 120 kDa. The receptor has an absolute requirement for Ca2+ and is inhibited by Suramin. The pH optimum of the receptor is 6.5, which is near the pH of the hemolymph. Binding data indicate a single high affinity binding site with a Kd = 4.1 +/- 0.19 x 10(-8) M as measured with the lipoprotein isolated from larval hemolymph. The major neutral lipid carried by insect lipoproteins is diacylglycerol, and it was shown that the affinity of the receptor for lipoprotein ligands correlates with their diacylglycerol content. It is proposed that the decrease in affinity of the receptor for lipoproteins depleted of diacylglycerol plays a key role in facilitating the transport of diacylglycerol from the midgut to the fat body during the larval feeding period. The insect receptor has some properties which are similar to those of vertebrate lipoprotein receptors, viz. molecular weight, requirement for Ca2+, and inhibition by Suramin. However, the insect receptor does not bind human low density lipoprotein.     

Isolation and identification of a peptide and its cDNA from the mosquito Aedes aegypti related to Manduca sexta allatotropin

Immunocytochemistry revealed that an allatotropin-immunoreactive peptide is produced by several neuroendocrince cells in the abdominal ganglia of the mosquito Aedes aegypti. The immunoreactive peptide was isolated and its structure determined to be Ala-Pro-Phe-Arg-Asn-Ser-Glu-Met-Met-Thr-Ala-Arg-Gly-Phe-amide. A cDNA clone encoding this novel neuropeptide was shown to encode a single copy of this peptide. The cDNA is unusual in that the first seven ATGs are not used for translation initiation.     

Biophysical characterization of an insect lysozyme from Manduca sexta

Insect lysozyme from Manduca sexta (MS-lys) was overexpressed in E. coli and refolded to obtain active protein. Recombinant MS-lys presented a globular structure, with an alpha-helical content of 57% as assessed by circular dichroism spectroscopy. Light scattering studies showed that in solution MS-lys has a quasi-monodisperse size distribution, with a rod-like structure similar to nucleation clusters reported in egg lysozyme pre-crystallization stages. These results show that MS-lys is an excellent candidate for crystallization, folding and denaturation studies.     

Interaction between Manduca sexta allatotropin and manduca sexta allatostatin in the fall armyworm Spodoptera frugiperda

A peptide that strongly stimulates juvenile hormone (JH) biosynthesis in vitro by the corpora allata (CA) was purified from methanolic brain extracts of adult Spodoptera frugiperda. Using HPLC separation followed by Edman degradation and mass spectrometry, the peptide was identified as Manduca sexta allatotropin (Mas-AT). Treating the CA from adult S. frugiperda with synthetic Mas-AT (at 10(-6) M) caused an up to sevenfold increase in JH biosynthesis. The stimulation of JH synthesis was dose-dependent and reversible. Synthetic M. sexta allatostatin (Mas-AS) (10(-6) M) did not affect the spontaneous rate of JH secretion from CA of adult S. frugiperda, nor did any of the allatostatins of the Phe-Gly-Leu-amide peptide family tested. However, when CA had been activated by Mas-AT (10(-6) M), addition of synthetic Mas-AS (10(-6) M) reduced JH synthesis by about 70%. This allatostatic effect of Mas-AS on allatotropin-activated glands was also reversible. When CA were incubated in the presence of both Mas-AT (10(-6) M) and various concentrations of Mas-AS (from 10(-8) to 10(-5) M), the stimulation of JH-biosynthesis observed was inhibited in a dose-dependent manner. The experiments demonstrate a novel mechanism of allatostatin action. In S. frugiperda JH synthesis was inhibited only in those glands which had previously been activated by an allatotropin.     

Isolation and characterization of bacteria-induced protein P4 from hemolymph of Manduca sexta

Insects synthesize several types of hemolymph proteins in response to bacterial infection. The objective of this study was to characterize a 48,000 dalton hemolymph protein induced in larvae of Manduca sexta after injection of bacteria. The protein, isolated by cation exchange and gel filtration chromatography from hemolymph of larvae injected with Micrococcus lysodeikticus, was found to be a glycoprotein with pI = 8.4. The molecular weight, isoelectric point, amino acid composition, and NH2 terminal sequence of the protein are similar to bacteria-induced protein P4 from Hyalophora cecropia, and the M. sexta protein is also designated P4. The hemolymph concentration of M. sexta P4 (35 +/- 7 micrograms/ml in day 3 fifth instar larvae) increases 30- to 45-fold by 48 h after injection of bacteria, but it does not increase in response to injection of distilled water. Lower levels of induction occur after injection of peptidoglycan fragments, zymosan, and lipopolysaccharide. The properties of M. sexta P4 are very similar to those of a previously characterized M. sexta hemolymph protein known as postlarval protein, and antibodies against P4 bind to post-larval protein.     

Isolation and characterization of a hemocyte aggregation inhibitor from hemolymph of Manduca sexta larvae

A protein that inhibits hemocyte aggregation has been isolated from hemolymph of Manduca sexta larvae and named hemocyte aggregation inhibitor protein (HAIP). HAIP has a M_r = 50,000, pI = 8.5, and contains 7% carbohydrate. It is present at 230 ± 20 μg/ml in hemolymph of day 3 fifth instar larvae. Antibodies to HAIP do not cross-react with M. sexta hemolin, which is similar in size and charge and also inhibits hemocyte aggregation. HAIP and hemolin have some similarity in amino acid composition and NH₂-terminal sequence, but are different in overall secondary structure, as determined by CD spectroscopy. The concentration of HAIP in hemolymph is not affected by injection of larvae with bacteria. A protein of approximately 50,000 daltons that reacts with antibody to M. sexta HAIP is present in hemolymph of Bombyx mori, Heliothis zea, and Galleria mellonella. Although the function of HAIP in vivo is not yet clear, it may have a role in modulating adhesion of hemocytes during defensive responses. © 1994 Wiley-Liss, Inc.     

Isolation and characterization of a larval lipoprotein from the hemolymph of Manduca sexta.

1. The larval high density lipoprotein (HDL) from the hemolymph of Manduca sexta, isolated by density gradient centrifugation, contains 61% protein, 37% lipid and 2% carbohydrate. 2. The molecular weight of HDL is 6 x 10(5), with two apoproteins of 2.85 x 10(5) and 8.1 x 10(4) daltons. 3. The large apoprotein is destroyed by trypsin treatment of the particle, while the small one is not. 4. Calculations based upon size and composition show that this particle is very different in structure from mammalian lipoproteins. It is proposed that a portion of the apoprotein occupies the central core region.     

Regulation of juvenile hormone biosynthesis in Heliothis virescens by Manduca sexta allatotropin

In Heliothis virescens, reproduction is strictly dependent on juvenile hormone (JH). In females, mating induces a sharp increase in JH titers, which stimulates increased vitellogenin biosynthesis and higher rates of egg production. JH biosynthesis is presumably stimulated by production and/or release of stimulatory neuropeptides such as allatotropins. There is evidence that allatotropin of H. virescens may be structurally related to Manduca sexta allatotropin (Manse-AT). In a radiochemical in vitro assay, synthetic Manse-AT stimulated JH biosynthesis by corpora allata (CA) of virgin H. virescens females in a dose-dependent manner, but had no effect on CA activity in H. virescens males. In females, the CA showed a transient increase in sensitivity to Manse-AT shortly after mating. Several structurally related peptides stimulated CA activity to a similar extent as Manse-AT. Corpora allata activity was stimulated by a Ca2+ ionophore, A23187. A membrane-permeable Ca2+ chelator, BAPTA/AM, antagonized the stimulatory effects of Manse-AT, suggesting that Manse-AT may enhance CA activity by increasing intracellular Ca2+ concentration.     

Isolation and characterization of four serine proteinase inhibitors (serpins) from hemolymph of Manduca sexta

Four serine proteinase inhibitors have been isolated from hemolymph of fifth instar larvae of Manduca sexta. One of these, an inhibitor specific for elastase, has been previously shown to be a member of the serpin family of serine proteinase inhibitors. Of the three remaining inhibitors, two are specific for chymotrypsin and one for trypsin. The four inhibitors have molecular weights of approx. 47,000 and isoelectric points between 4.4 and 4.8. The four proteins have very similar amino acid compositions, and NH₂-terminal sequence analysis suggests that they represent members of a gene family.     

Molecular characterisation of cDNAs from the fall armyworm Spodoptera frugiperda encoding Manduca sexta allatotropin and allatostatin preprohormone peptides

Allatotropin (AT) is a 13-residue amidated neuropeptide, first isolated from pharate adult heads of the tobacco hornworm, Manduca sexta (Manse-AT), which strongly stimulates the biosynthesis of juvenile hormones (JH) in the corpora allata (CA) of adult moths. In Spodoptera frugiperda, a cDNA that encodes 134 amino acids, including an AT peptide, has been cloned. The S. frugiperda allatotropin mature peptide (Spofr-AT) [GFKNVEMMTARGFa] is identical to that isolated from M. sexta. The basic organization of the Spofr-AT precursor is similar to that of Agrius convolvuli, M. sexta, Pseudaletia unipuncta, and Bombyx mori with 83-93% amino acid sequence identity. The Spofr-AT gene is expressed in at least three mRNA isoforms with 134, 171 and 200 amino acids, differing from each other by alternative splicing.All allatostatins (AS) have an inhibitory action on the JH biosynthesis in the CA. A cDNA that encodes 125 amino acid residues including one copy of the Manse-AS peptide has been cloned from S. frugiperda (Spofr-AS; QVRFRQCYFNPISCF). The basic organization of the Spofr-AS precursor is similar to that of P. unipuncta with 85% amino acid sequence identity.Using one step RT-PCR for semi-quantification of the gene expression, we showed that the three mRNAs of the Spofr-AT gene and the Spofr-AS gene are expressed in brains of last instar larvae, prepupae, pupae, and adults of both sexes of S. frugiperda with variable intensity.     

Cardioacceleratory effects of Manduca sexta allatotropin in the true armyworm moth, Pseudaletia unipuncta

Manduca sexta allatotropin (Manse-AT), a peptide originally isolated on the basis of its ability to stimulate juvenile hormone (JH) biosynthesis in the tobacco hornworm, is a potent in vitro stimulator of the corpora allata (CA) in Pseudaletia unipuncta (Lepidoptera: Noctuidae). At 10(-6)M, Manse-AT stimulated in vitro rates of JH biosynthesis by CA of day 0 and 6 adult females 15- and 10-fold respectively. Both Manse-AT and serotonin were also shown to be dose-dependent stimulators of heart rate in day 0, 3 and 6 adult males and females. Furthermore, analysis suggests that there are differences in both resting and Manse-AT-stimulated heart rates depending on age and rearing conditions.     

Isolation and maintenance-free culture of contractile myotubes from Manduca sexta embryos

Skeletal muscle tissue engineering has the potential to treat tissue loss and degenerative diseases. However, these systems are also applicable for a variety of devices where actuation is needed, such as microelectromechanical systems (MEMS) and robotics. Most current efforts to generate muscle bioactuators are focused on using mammalian cells, which require exacting conditions for survival and function. In contrast, invertebrate cells are more environmentally robust, metabolically adaptable and relatively autonomous. Our hypothesis is that the use of invertebrate muscle cells will obviate many of the limitations encountered when mammalian cells are used for bioactuation. We focus on the tobacco hornworm, Manduca sexta, due to its easy availability, large size and well-characterized muscle contractile properties. Using isolated embryonic cells, we have developed culture conditions to grow and characterize contractile M. sexta muscles. The insect hormone 20-hydroxyecdysone was used to induce differentiation in the system, resulting in cells that stained positive for myosin, contract spontaneously for the duration of the culture, and do not require media changes over periods of more than a month. These cells proliferate under normal conditions, but the application of juvenile hormone induced further proliferation and inhibited differentiation. Cellular metabolism under normal and low glucose conditions was compared for C2C12 mouse and M. sexta myoblast cells. While differentiated C2C12 cells consumed glucose and produced lactate over one week as expected, M. sexta muscle did not consume significant glucose, and lactate production exceeded mammalian muscle production on a per cell basis. Contractile properties were evaluated using index of movement analysis, which demonstrated the potential of these cells to perform mechanical work. The ability of cultured M. sexta muscle to continuously function at ambient conditions without medium replenishment, combined with the interesting metabolic properties, suggests that this cell source is a promising candidate for further investigation toward bioactuator applications.     

Isolation and functional characterization of an inositol trisphosphate receptor from brain

We have identified an IP3 receptor protein in brain membranes through the binding of radiolabelled IP3. Autoradiographic studies localize the receptor to various areas of the brain with highest densities in Purkinje cells of the cerebellum. IP3 binding is inhibited by physiologic intracellular concentrations of calcium. Purification of the IP3 receptor to homogeneity reveals it to be comprised of four identical subunits of 260 kD each. Antisera to the purified receptor protein have been employed for immunohistochemical studies which, at the electron microscopic level, localize the IP3 receptor to a subdivision of the rough endoplasmic reticulum occurring in synaptic areas and in close association with the nuclear membrane. The IP3 receptor protein is selectively phosphorylated by cyclic AMP (cAMP) dependent protein kinase. This phosphorylation decreases 10-fold the potency of IP3 in releasing calcium from brain membranes.     

Isolation and characterization of an apically sorted 41-kDa protein from the midgut of tobacco hornworm (Manduca sexta)

Immunocytochemical localization and sorting properties of a newly purified 41-kDa protein (MsM41) were investigated in an insect, the tobacco hornworm Manduca sexta. The protein purified from midgut homogenates of feeding fifth-stadium larvae was found exclusively in this tissue on Western blots. Presence of MsM41 protein was indicated in both anterior and posterior regions of the midgut during the whole fifth stadium. However, in the posterior region an additional 39-kDa protein was also detected during the feeding period of the last larval stage. Upon light-microscopic examination immunoreactivity was localized in the columnar cells, while the goblet, endocrine and regenerative cells remained unlabeled. Distribution of the label during the feeding period was different in the anterior and posterior regions. In the anterior region immunoreactivity was localized only to the brush border membrane of columnar cells, while in the posterior region some cytoplasmic structures identified as large trans-Golgi vesicles, endoplasmic reticulum and small secretory vesicles were also labeled. Large, apical extrusions remained immunonegative. In vitro translation confirmed that our protein was expressed only in the posterior region of the midgut. The primary translation product was a 39-kDa protein. Putative post-translational modifications yielded the 41-kDa form, which was then secreted apically. Its presence in the region of the anterior part microvilli was probably due to the countercurrent flux of the ectoperitrophic fluid.     

Isolation and embryonic expression of an abdominal-A-like gene from the lepidopteran, Manduca sexta.

Using sequence homology to the Drosophila Antennapedia gene, we isolated a homeobox-containing gene from the lepidopteran, Manduca sexta. Sequence analysis and in situ hybridizations to tissue sections suggest that the Manduca gene encodes a lepidopteran homologue of the Drosophila Bithorax complex gene abdominal-A. The predicted amino acid sequence of a 76 amino acid region that includes the homeobox and the regions immediately flanking it are identical between the Manduca and Drosophila genes. Northern blots reveal that the manduca abd-A gene is expressed first in the early embryo and continues to be expressed throughout later embryonic and larval stages. In situ hybridizations show that the posterior half of the first abdominal segment marks the anterior border of the Manduca abd-A expression. This expression pattern demonstrates the conservation of parasegments as domains of gene activity in the lepidopteran embryo. The Manduca abd-A expression extends from the posterior half of the first abdominal segment through the tenth abdominal segment, a domain that is greater than that of the Drosophila abd-A expression, and reflects the difference in visible segment number between the two insects.     

Endopeptidase activity of larval Lacanobia oleracea corpus allatum: metabolism of Manduca sexta allatostatin and allatotropin

The degradation of synthetic Manduca sexta allatostatin (Manse-AS) and allatotropin (Manse-AT) by enzymes associated with the corpus allatum (CA) of larvae of the tomato moth, Lacanobia oleracea, was investigated using reversed-phase high performance liquid chromatography and matrix-assisted laser desorption ionisation-time of flight mass spectrometry. Manduca sexta allatostatin was metabolised by CA extract to Manse-AS5-15, Manse-AS6-15, and Manse-AS7-15, which indicates enzymic cleavage at the C-terminal side of arginine residues R3 and R5 and the N-terminal side of R5, suggesting this is due to a trypsin-like enzyme. In support of this, the same degradation products were identified after Manse-AS was incubated with trypsin, and CA enzymic activity could be inhibited up to 79% by aprotinin. Degradation of Manse-AT by CA extract was also trypsin-like, cleaving at the C-terminal side of the basic residues K3 and R11 to produce Manse-AT4-13 and Manse-AT1-11. Metabolism by trypsin produced the same deletion peptides, but the major product due to this enzyme was Manse-AT4-11. Hydrolysis of Manse-AT by CA could only be partially inhibited by high doses of aprotinin (36%), and the CA extract also cleaved Manse-AT between M8 and T9 to produce Manse-AT1-8. A trypsin-like peptidase appears to be the major enzyme present in the CA of larval L. oleracea that acts to metabolise Manse-AS and Manse-AT. In addition, an unidentified enzyme that cleaves between M and T residues degraded Manse-AT.     

Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta

The identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [³⁵S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of M_r ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ～8.6). Electrophoresis under denaturing conditions reveals a subunit M_r ≈ 50,000 while the native protein has an apparent M_r ～ 85,000 by gel permeation chromatography. Anti-PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.