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.     

Immulectin, an inducible C-type lectin from an insect, Manduca sexta, stimulates activation of plasma prophenol oxidase.

Immulectin, a C-type lectin from the tobacco hornworm, Manduca sexta, was cloned from a larval fat body cDNA library. The immulectin cDNA encodes a 309 residue polypeptide. Immulectin synthesis was induced by injection of killed gram-positive or gram-negative bacteria or yeast. After injection of bacteria, immulectin mRNA appeared in fat body and immulectin protein was detected in hemolymph. Immulectin contains two carbohydrate recognition domains. The carboxyl-terminal carbohydrate recognition domain is most similar (36% identity) to a lipopolysaccharide-binding protein from the American cockroach, Periplaneta americana. It also shares 26-35% identity to carbohydrate recognition domains of various mammalian C-type lectins. Two immulectin isoforms were identified in the hemolymph of bacteria-injected larvae. Recombinant immulectin agglutinated gram-positive and gram-negative bacteria and yeast. Addition of recombinant immulectin to M. sexta plasma stimulated activation of phenol oxidase. A combination of immulectin with lipopolysaccharide from E. coli activated phenol oxidase more rapidly and to a higher level than immulectin alone, whereas lipopolysaccharide by itself had little effect on phenol oxidase activation. Immulectin synthesized in response to bacterial or fungal infection may help to trigger protective responses in M. sexta in a manner similar to mannose-binding protein, a C-type lectin that functions in the mammalian innate immune system.     

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.     

Primary structure and comparative sequence analysis of an insect apolipoprotein. Apolipophorin-III from Manduca sexta

The amino acid sequence of an insect apolipoprotein, apolipophorin-III from Manduca sexta, was determined by a combination of cDNA and protein sequencing. The mature hemolymph protein consists of 166 amino acids. The cDNA also encodes for an amino-terminal extension of 23 amino acids which is not represented in the mature hemolymph protein. The existence of a precursor protein was confirmed by in vitro translation of fat body mRNA. Computer-assisted comparative sequence analysis revealed the following points: 1) the protein is composed of tandemly repeating tetradecapeptide units with a high potential for forming amphiphilic helical structures. Compared to mammalian apolipoproteins the repeat units in the insect apolipoprotein show considerable length variability; 2) the sequence has a striking resemblance to several human apolipoproteins including apoE, AIV, AI, and CI. However, the homology seems to be entirely functional since, although the insect and mammalian apoproteins contain very similar types of amino acid residues, the actual degree of sequence identity is quite low. Whether the mammalian and insect apoproteins are derived from a common ancestral amphiphilic helix forming, lipid-binding protein, or arose by convergent evolution can not be determined at present. This represents the first complete amino acid sequence for an insect apolipoprotein.     

Changes in lipoprotein composition during larval-pupal metamorphosis of an insect, Manduca sexta

During the transition from the last feeding larval stage to the pupal stage of the tobacco hornworm, Manduca sexta, significant changes occur in the properties of lipophorin, the major hemolymph lipoprotein. Within the first 24 h after cessation of feeding, the larval lipophorin (HDLp-L) is first converted to a higher density form (HDLp-W2) and then HDLp-W2 is converted to a lower density form (HDLp-W1). HDLp-W1 remains in the hemolymph until pupation, when another form, HDLp-P, with a density between HDLp-W1 and HDLp-L, is present. Although all the lipophorins contain identical apoproteins, they differ in lipid content and composition; the differences in density being primarily related to diacylglycerol content. The conversion of HDLp-L to HDLp-W1 is accompanied by a loss of hydrocarbon and uptake of carotenes. These latter changes in lipophorin composition reflect alterations in cuticular lipid composition. HDLp-L was radiolabeled in the apoproteins by injecting animals with 3H-amino acids early in the last larval stage. Subsequently HDLp-L was isolated at the end of the larval stage, HDLp-W2 and HDLp-W1 were isolated during the wandering stage, and HDLp-P was isolated after pupation. The specific activity of the apoproteins in the four lipophorins was not significantly different, suggesting that the observed alterations in lipophorin properties do not require synthesis of new apoproteins but result from retailoring the lipid composition of preexisting molecules. Examination of the hemolymph of individual animals during these transitions showed that only one species of lipoprotein was present, never a mixture of two or more species. These observations suggest that the lipoprotein conversions are precisely timed and that lipoprotein metabolism during larval development and pupation cannot be considered a static process. The unique finding of these studies was that synthesis of lipophorin apoproteins proceeds actively during the first part of the fifth instar but then ceases and does not recommence during the wandering or early pupal stages.     

Primary structure of a member of the serpin superfamily of proteinase inhibitors from an insect, Manduca sexta

A cDNA clone isolated from a fat body cDNA library from an insect, Manduca sexta, has been sequenced and shown to code for a member of the serpin family of proteinase inhibitors. The cDNA has an open reading frame which codes for a 392-residue polypeptide of Mr = 43,500 with a hydrophobic NH2-terminal sequence which appears to be a signal peptide. An alignment of this amino acid sequence with 11 members of the serpin superfamily reveals that the insect protein is 25-30% identical with most members of the superfamily. The alignment was used to construct an evolutionary tree of the serpin sequences analyzed, which indicates that the progenitor of the M. sexta serpin and the human serpins most closely related to it diverged from other serpin genes prior to the divergence of the vertebrates and invertebrates. The M. sexta serpin is predicted to inhibit elastase due to the presence of alanine at the P1 position of its reactive center and is classified as an alaserpin. A glycoprotein of Mr = 47,000 isolated from hemolymph of M. sexta larvae has an NH2-terminal sequence identical to that deduced from the alaserpin cDNA clone and inhibits porcine pancreatic elastase and bovine chymotrypsin.     

Expression of a glycosylphosphatidylinositol-linked Manduca sexta aminopeptidase N in insect cells.

Aminopeptidase N (APN; EC 3.4.11.2) is an exopeptidase that is attached to cell membranes by a hydrophobic amino-terminal stalk in vertebrates or a glycosylphosphatidylinositol (GPI) anchor in insects. In this study, we report the cloning, expression, and characterization of an aminopeptidase N from Manduca sexta midgut. The full-length aminopeptidase N cDNA (APN1a) encodes a 995-amino-acid protein. The predicted amino acid sequence differs by 8 amino acids from M. sexta APN1. These different amino acids do not modify any putative glycosylation or glycosylphosphatidylinositol anchor sites. The full-length cDNA was cloned into an expression plasmid, pHSP-HR5, and transiently expressed in an insect cell line derived from Spodoptera frugiperda (Sf21 cells). Immunoblot analysis with anti-APN antiserum showed that APN1a expressed in Sf21 cells is the same size (120 kDa) as APN found in midgut brush border membranes. After treatment with phosphatidylinositol-specific phospholipase C (PIPLC), anti-cross-reacting determinant antibody specific for PIPLC cleavage products recognized the expressed 120-kDa APN1a, but not endogenous Sf21 proteins, indicating that APN1a has an intact glycosylphosphatidylinositol anchor. These results are evidence that Sf21 cells synthesize few, if any, endogenous GPI-linked proteins. Immunofluorescence staining showed that the expressed APN1a was located on the surface of Sf21 cells.     

The lysozyme from insect (Manduca sexta) is a cold-adapted enzyme

Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of alpha-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5-30 degrees C. These results together with measured thermodynamic activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins.     

Isolation and identification of a new diuretic peptide from the tobacco hornworm, Manduca sexta.

A 30-amino acid diuretic peptide was isolated from the corpora cardiaca-corpora allata complexes and, separately, from medial neurosecretory cells of the Sphingid moth, Manduca sexta. The peptide was found to have the following sequence, determined by automated Edman degradation and mass spectrometry: SFSVNPAVDILQHRYMEKV AQNNRNFLNRV-NH2. We have named the peptide Mas-DP II. The peptide was synthesized and shown to possess diuretic activity in decapitated moths. Mas-DP II is related by sequence homology to a 41-amino acid diuretic peptide identified previously from M. sexta, and it belongs to the family of corticotropin releasing factor-like peptides.     

Immulectin-2, a lipopolysaccharide-specific lectin from an insect, Manduca sexta, is induced in response to gram-negative bacteria

A lipopolysaccharide-specific lectin, immulectin-2, was isolated from plasma of the tobacco hornworm, Manduca sexta. Immulectin-2 has specificity for xylose, glucose, lipopolysaccharide, and mannan. A cDNA clone encoding immulectin-2 was isolated from an Escherichia coli-induced M. sexta larval fat body cDNA library. The cDNA is 1253 base pairs long, with an open reading frame of 981 base pairs, encoding a 327-residue polypeptide. Immulectin-2 is a member of the C-type lectin superfamily. It consists of two carbohydrate recognition domains, which is similar to the organization of M. sexta immulectin-1. Immulectin-2 was present at a constitutively low level in plasma of control larvae and increased 3-4-fold after injection of Gram-negative bacteria or lipopolysaccharide. Immulectin-2 mRNA was detected in fat body of control larvae, and its level increased dramatically after injection of E. coli. The concentration of immulectin-2 in plasma did not change significantly after injection of Gram-positive bacteria or yeast, even though its mRNA level was increased by these treatments. Compared with immulectin-1, immulectin-2 has a more restricted specificity for binding to Gram-negative bacteria. Immulectin-2 at low physiological concentrations agglutinated E. coli in a calcium-dependent manner. It also bound to immobilized lipopolysaccharide from E. coli. Binding of immulectin-2 to lipopolysaccharide stimulated phenol oxidase activation in plasma. The properties of immulectin-2 are consistent with its function as a pattern recognition receptor for detection and defense against Gram-negative bacterial infection in M. sexta.     

Lipoprotein interconversions in an insect, Manduca sexta. Evidence for a lipid transfer factor in the hemolymph

Hemolymph lipoproteins (lipophorins) of adult Manduca sexta are disinct from larval forms in density, lipid content, composition, and the presence of a third, low molecular weight apoprotein. Generally, only one lipoprotein species exists in M. sexta hemolymph during any given life stage. Progression through the life cycle results in alterations of existing lipoproteins to produce new forms, without new protein synthesis. The observed alterations in lipoprotein density could result from facilitated lipid transfer in insect hemolymph. An in vitro assay of facilitated lipid transfer was developed which employs a high density lipophorin from the wandering larva (density = 1.18 g/ml) as acceptor and adult low density lipophorin (density = 1.03 g/ml) as donor. Adult lipophorin-deficient hemolymph was shown to catalyze a time-dependent equilibration of the starting lipoproteins to produce a new intermediate lipophorin, Lp-I. Hydrodynamic experiments on the donor, acceptor, and product lipoproteins excluded fusion as the mechanism whereby Lp-I is produced. Thus, it is concluded that Lp-I results from facilitated net lipid transfer from low to high density lipoprotein. Furthermore, experiments conducted with radioiodinated donor and radioiodinated acceptor lipoproteins demonstrated that apoprotein exchange does not occur during the lipid transfer reaction. When donor lipoprotein was labeled in the lipid moiety with carbon-14, evidence of diacylglycerol and phospholipid exchange was obtained. Partial characterization of the lipid transfer factor revealed a relationship between incubation time, donor concentration, acceptor concentration, lipophorin-deficient hemolymph concentration, and transfer activity, as measured by Lp-I production. It is concluded that lipophorin-deficient hemolymph contains one or more factor(s) that catalyze net lipid transfer as well as diacylglycerol and phospholipid exchange between lipophorins to produce a single form at equilibrium.     

Biological activity of Manduca sexta paralytic and plasmatocyte spreading peptide and primary structure of its hemolymph precursor

A family of hemolymph peptides was previously identified in several lepidopteran insects, which exhibited multiple biological activities including rapid paralysis, blockage of growth and development, or stimulation of plasmatocyte spreading and aggregation. We synthesized Manduca sexta paralytic peptide 1 (PP1) and found that after it was injected into larvae, bleeding from wounds was dramatically reduced. PP1 also stimulated spreading and aggregation behavior of M. sexta plasmatocytes in vitro. Stimulation of plasmatocyte aggregation and adherence to the body wall may explain a decrease observed in the number of circulating plasmatocytes after injection of PP1. Such aggregates might rapidly form plugs in wounds to prevent bleeding. We cloned a cDNA for a Manduca paralytic peptide precursor, using polymerase chain reactions and cDNA library screening. The active 23-residue PP2 peptide encoded by this clone is at the carboxyl-terminal end of a precursor protein predicted to be 107 amino acid residues long after cleavage of a secretion signal peptide. Active PP2 was produced by processing of recombinant proPP2 by bovine factor X_a. A single proPP2 mRNA was present in fat body but not in hemocytes. The level of this mRNA was not affected by injection of bacteria into larvae. We produced recombinant proPP2 in Escherichia coli and used this protein to produce an antiserum. The antiserum detected proPP2 in plasma and was used to observe rapid proteolytic processing of proPP2 after hemolymph collection.     

Structure and development of antennae in a moth,Manduca sexta

The antenna of the moth, Manduca sexta, comprises two small basal segments and a long (2 cm) flagellum, which is divided into nearly 80 annuli. The annuli bear cuticular scales and small sensory organs, sensilla. A trachea, a blood vessel, and two nerve trunks run through the lumen of the antenna and into the head. Sensilla are arranged in an orderly pattern that is repeated on each flagellar annulus. Each flagellum bears about 10⁵ sensilla, which contain about 2.5 × 10⁵ primary sensory neurons. Clumps of undifferentiated cells (imaginal disks), present in the larva, form pupal antennae during the larval-pupal molt. During the subsequent metamorphic development of the adult, cell divisions, changes in cell shape, and cellular differentiation transform pupal into adult antennae. Sensilla and scales arise and differentiate in the antenna during metamorphosis; regions in which sensilla and scales will arise can be recognized before overt differentiation occurs. All of the flagellar annuli develop synchronously. The dense innervation and neuronal simplicity of antennal flagella, as well as their synchronous development at a late and accessible stage in the animal's life cycle, suit them for studies of neuronal differentiation.     

An integrin-tetraspanin interaction required for cellular innate immune responses of an insect, Manduca sexta

In their encounters with foreign intruders, the cells of the insect innate immune system, like those of the mammalian immune system, exhibit both humoral and cell-mediated responses. Some intruders can be dispatched by the humoral immune system alone, but many must be phagocytosed by individual hemocytes or encapsulated by interacting hemocytes. Surface proteins of hemocytes control the abrupt transition of hemocytes from resting, nonadherent cells to activated, adherent cells during these cell-mediated responses. Two of these surface proteins, an integrin and a tetraspanin, interact during this adhesive transition. As demonstrated with a hemocyte adhesion assay and a surface plasmon resonance assay, the large extracellular loop of tetraspanin D76 binds to a hemocyte-specific integrin of Manduca sexta. The interaction between the large extracellular loop domain and hemocyte-specific integrin is interrupted not only by a monoclonal antibody (MS13) that binds to a domain of beta-integrin known to be a ligand-binding site for cell adhesion but also by double-stranded beta-integrin RNA. Transfected S2 cells expressing tetraspanin mediate adhesion of hemocytes. A monoclonal antibody to tetraspanin D76 perturbs the cell-mediated immune response of encapsulation. These studies involving antibody blocking, RNA interference, and binding assays imply a trans interaction of integrin and tetraspanin on hemocyte surfaces.     

Central nervous system features of a nicotine-resistant insect, the tobacco hornworm Manduca sexta

The purpose of this study is to look for structural correlates of the demonstrated nicotine-insensitivity of larval Manduca sexta CNS, an insensitivity which is only slightly perturbed by desheathing (a technique used to disrupt perineurial diffusion barriers). The general organization of the hornworm ganglion is found to conform to the conventional insect pattern, but the following points are noted and discussed in terms of their potential relationship to nicotine-insensitivity: the damage caused to perineurial cells by desheathing is extremely localized, with cells immediately adjacent to the torn region showing good ultrastructural integrity; ionic lanthanum does not gain access to the subperineurial extracellular space following desheathing; lanthanum penetrates the ganglion in the cytoplasm of tracheal cells damaged peripherally during desheathing, but is excluded from the extracellular space surrounding such tracheal cells; smooth endoplasmic reticulum is much in evidence in perineurial cells and tracheal cells, sites where it might be implicated in nicotine detoxification; individual basal perineurial cells appear to cover extensive regions of the ganglion, thereby limiting intercellular diffusion.     

Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta

Invertebrates, including insects, are being developed as model systems for the study of bacterial virulence. However, we understand little of the interaction between bacteria and specific invertebrate tissues or the immune system. To establish an infection model for Photorhabdus, which is released directly into the insect blood system by its nematode symbiont, we document the number and location of recoverable bacteria found during infection of Manduca sexta. After injection into the insect larva, P. luminescens multiplies in both the midgut and haemolymph, only later colonizing the fat body and the remaining tissues of the cadaver. Bacteria persist by suppressing haemocyte-mediated phagocytosis and culture supernatants grown in vitro, as well as plasma from infected insects, suppress phagocytosis of P. luminescens. Using GFP-labelled bacteria, we show that colonization of the gut begins at the anterior of the midgut and proceeds posteriorly. Within the midgut, P. luminescens occupies a specific niche between the extracellular matrix and basal membrane (lamina) of the folded midgut epithelium. Here, the bacteria express the gut-active Toxin complex A (Tca) and an RTX-like metalloprotease PrtA. This close association of the bacteria with the gut, and the production of toxins and protease, triggers a massive programmed cell death of the midgut epithelium.     

A beta1,3-glucan recognition protein from an insect, Manduca sexta, agglutinates microorganisms and activates the phenoloxidase cascade

Pattern recognition proteins function in innate immune responses by binding to molecules on the surface of invading pathogens and initiating host defense reactions. We report the purification and molecular cloning of a cDNA for a 53-kDa beta1,3-glucan-recognition protein from the tobacco hornworm, Manduca sexta. This protein is constitutively expressed in fat body and secreted into hemolymph. The protein contains a region with sequence similarity to several glucanases, but it lacks glucanase activity. It binds to the surface of and agglutinates yeast, as well as gram-negative and gram-positive bacteria. Beta1,3-glucan-recognition protein in the presence of laminarin, a soluble glucan, stimulated activation of prophenoloxidase in plasma, whereas laminarin alone did not. These results suggest that beta1,3-glucan-recognition protein serves as a pattern recognition molecule for beta1,3-glucan on the surface of fungal cell walls. After binding to beta1,3-glucan, the protein may interact with a serine protease, leading to the activation of the prophenoloxidase cascade, a pathway in insects for defense against microbial infection.     

Antisense inhibition of neuronal nicotinic receptors in the tobacco-feeding insect,Manduca sexta

Acetylcholine is the predominant excitatory transmitter in the insect central nervous system with many of its effects mediated by nicotinic acetylcholine receptors. These receptors are present at very high density and are structurally heterogeneous, although little is known about functional distinctions between them. An interesting system for examining these receptors is the larval stage of Manduca sexta, a nicotine-resistant tobacco-feeding insect. The nicotinic responses of cultured neurons were found to be blocked by mecamylamine and curare but highly resistant to alpha-bungarotoxin. The responses were also unaffected by the reducing agent dithiothreitol and the alkylating agent bromoacetylcholine suggesting that the alpha-subunit dicysteine agonist binding site is protected. To begin determining the functional roles of different subunits in these receptors, cultured neurons were treated with oligonucleotides based on the gene sequence of the alpha subunit, MARA1. Antisense DNA caused a significant downward shift in the amplitude distribution of nicotinic responses compared to sense or reverse antisense treatments. These treatments did not affect currents mediated by the application of GABA. The reduction in the nicotinic depolarization and inward currents did not affect the rate of current onset or recovery, suggesting that antisense MARA1 causes a partial block of all nicotinic responses in these neurons. These results demonstrate that receptor gene expression in insect neurons can be manipulated in a sequence-specific manner by antisense treatment and they provide evidence that MARA1 is important for normal nicotinic responses in Manduca.