Bacteria-induced protein P4 (hemolin) from Manduca sexta: a member of the immunoglobulin superfamily which can inhibit hemocyte aggregation.

The synthesis of a number of hemolymph proteins is induced in insects in response to bacterial infections. The major induced hemolymph protein in larvae of Manduca sexta is a glycoprotein of Mr = 48,000 known as P4. We have isolated a clone for P4 from a fat body cDNA library constructed from RNA isolated from larvae injected with bacteria. The cDNA has an open reading frame encoding a 411 residue polypeptide with a hydrophobic NH2-terminal sequence, which appears to be a signal peptide. Analysis of the deduced amino acid sequence shows that P4 is a member of the immunoglobulin (Ig) gene superfamily, and is composed largely of four C2 type Ig domains. The M. sexta P4 amino acid sequence is 60% identical with hemolin (P4) from Hyalophora cecropia. The name "hemolin" has also been adopted for the M. sexta P4 protein. Hemolin mRNA levels in fat body begin to increase within 1 h after injection of bacteria into fifth instar larvae and within 4 h after injection of adults. Hemolin associates with the surface of hemocytes and inhibits hemocyte aggregation responses, suggesting a role for the protein in modulating hemocyte adhesion during recognition and response to bacterial infections in insects.     

Cloning of Immune Protein Hemolin cDNA from the Indianmeal Moth, Plodia interpunctella, and its High Induction During Development and by Bacterial Challenge

Partial cDNA of hemolin, an insect immune protein, was cloned from indianmeal moth, Plodia interpunctella, and the rates of hemolin mRNA expression were demonstrated in each stage of development and also by bacterial injections. A deduced amino acid sequence from the cloned hemolin cDNA was approximately 44‐54% similar to hemolins of 5 other moths. During development the level of hemolin mRNA was the highest at the time of the larval‐pupal matamorphosis. Hemolin was also rapidly induced by the injection of bacteria into the 4^th or the 5^th instar larvae. Hemolin induction rate by bacterial challenge was higher in the 5^th instar larvae than in 4^th instars. Our results suggest that hemolin could have multiple roles that act both on cellular processes during development and on the immune reactions for the resistance to pathogen invasion.     

Binding of hemolin to bacterial lipopolysaccharide and lipoteichoic acid. An immunoglobulin superfamily member from insects as a pattern-recognition receptor.

Hemolin, a plasma protein from lepidopteran insects, is composed of four immunoglobulin domains. Its synthesis is induced by microbial challenge. We investigated the biological functions of hemolin in Manduca sexta. It was found to bind to the surface of bacteria and yeast, and caused these micro-organisms to aggregate. Hemolin was demonstrated to bind to lipopolysaccharide (LPS) from Gram-negative bacteria and to lipoteichoic acid from Gram-positive bacteria. Binding of hemolin to smooth-type forms of LPS was competed for efficiently by lipoteichoic acid and by rough mutant (Ra and Rc) forms of LPS, which differ in polysaccharide length. Binding of hemolin to LPS was partially inhibited by calcium and phosphate. Hemolin bound to the lipid A component of LPS, and this binding was completely blocked by free phosphate. Our results suggest that hemolin has two binding sites for LPS, one that interacts with the phosphate groups of lipid A and one that interacts with the O-specific antigen and the outer-core carbohydrates of LPS. The binding properties of M. sexta hemolin suggest that it functions as a pattern-recognition protein with broad specificity in the defense against micro-organisms.     

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.     

Molecular characterization of the insect immune protein hemolin and its high induction during embryonic diapause in the gypsy moth,Lymantria dispar

During the embryonic (pharate first instar) diapause of the gypsy moth, Lymantria dispar, a 55 kDa protein is highly up-regulated in the gut. We now identify that protein as hemolin, an immune protein in the immunoglobulin superfamily. We isolated a gypsy moth hemolin cDNA and demonstrated a high degree of similarity with hemolins from three other moth species. Hemolin mRNA levels increased at the time of diapause initiation and remained high throughout the mandatory period of chilling required to terminate diapause in this species, and then dropped in late diapause. This mRNA pattern reflects the pattern of protein synthesis. These results suggest that hemolin is developmentally up-regulated in the gut during diapause. Diapause in this species can be prevented using KK-42, an imidazole derivative known to inhibit ecdysteroid biosynthesis, and gypsy moths treated in this manner failed to elevate hemolin mRNA. Conversely, this diapause appears to be initiated and maintained by the steroid hormone, 20-hydroxyecdysone, and the addition of 20-hydroxyecdysone to the culture medium elevated hemolin mRNA in the gut. Our results thus indicate a role for 20-hydroxyecdysone in the elevation of hemolin mRNA during diapause. Presumably, hemolin functions to protect the gypsy moth from microbial infection during its long, overwintering diapause.     

Upregulation of the immune protein gene hemolin in the epidermis during the wandering larval stage of the Indian meal moth, Plodia interpunctella

Expression of hemolin, which generates an immune protein, was up-regulated in wandering fifth instar larval stage of Plodia interpunctella. The mRNA level peaked in the middle of the wandering stage. Major expression was in the epidermis, rather than in the fat body or gut. To test a possible ecdysteroid effect on hemolin induction we treated with RH-5992, an ecdysteroid agonist, and KK-42, which inhibits ecdysteroid biosynthesis in both feeding and wandering fifth instar larvae. When feeding larvae were treated with RH-5992 the hemolin mRNA level was increased. When wandering larvae were treated with KK-42 its level was reduced. In addition, when KK-42-treated larvae were subsequently treated with RH-5992 the hemolin mRNA level was recovered. These results strongly suggest that ecdysteroid up-regulates the expression of hemolin mRNA. Hormonal and bacterial effects on hemolin induction were further analyzed at the tissue level. Major induction of hemolin mRNA was detected following both RH-5992 treatment and bacterial injection in the epidermis of both feeding and wandering larvae. Minor induction of hemolin was detected in the fat body following a bacterial injection, but not RH-5992 treatment. We infer that in P. interpunctella larvae, the epidermis is the major tissue for hemolin induction in na?ve insects and in insects manipulated with bacterial and hormonal treatments.     

Hemolin expression in the silk glands of Galleria mellonella in response to bacterial challenge and prior to cell disintegration

Hemolin, a member of the immunoglobulin protein superfamily, functions in Lepidoptera as an opsonin in defence against potential pathogens and seems to play a role in tissue morphogenesis. We show that hemolin gene is expressed in several organs of Galleria mellonella larvae, including the nervous system and the silk glands. The expression in the silk glands of the wandering larvae and their isolated abdomens is enhanced within 6 h after an injection of bacteria, lipopolysaccharides, or peptidoglycans. The magnitude of silk gland response to bacterial challenge is similar to that seen in the fat body. A profound rise of hemolin expression without bacterial inoculation occurs in the silk glands of isolated abdomens when they are induced to pupate by a topical application of 20-hydroxyecdysone (20E). The induction of pupation is associated with silk gland programming for disintegration by apoptosis and phagocytosis. Administration of a juvenile hormone agonist prevents pupation and abolishes the stimulatory 20E effect on the hemolin expression. Hemolin protein can be immunodetected in the silk glands as well as in the spun-out cocoon silk. The results suggest that silk glands are a component of the insect immune system and that hemolin may mark the apoptic cells for the elimination by hemocytes.     

Effects of azadirachtin on insect cytochrome P-450 dependent ecdysone 20-monooxygenase activity

The effects of the insect growth and ecdysis inhibitor azadirachtin on ecdysone 20-monooxygenase activity were examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female Aedes aegypti, or fat body or midgut from last instar larvae of Manduca sexta were incubated with radiolabelled ecdysone and increasing concentrations of azadirachtin and the ecdysone 20-monoxygenase activity quantified by radioassay. Azadirachtin was found to inhibit in a dose-response fashion the ecdysone 20-monooxygenase activity associated with all the insect preparations. The concentration of azadirachtin required to elicit approximately 50% inhibition of the ecdysone 20-monooxygenase activity ranged from a low of 1 x 10(-4) M for Drosophila to a high of 4 x 10(-4) M for Manduca midgut.     

Absorption and tissue distribution of cholesterol in Manduca sexta

In Manduca sexta larvae, radioactive free cholesterol is absorbed directly from the midgut into mucosal cells where it is stored both in the free form (87% in males and 93% in females) and esterified form (13% in males and 7% in females). Subsequently, cholesterol is transported to fat body via lipophorin in the hemolymph exclusively in the free form. In fat body, the distribution of cholesterol between the free and esterified form varied significantly between genders and developmental stages. Except for the larval stage, males and females were able to store cholesterol in both free and esterified forms in the fat body and in the adult stage cholesterol ester accounted for more than 75% of the stored cholesterol. Placement of radioactive cholesterol at different locations in the gut-foregut, midgut, and hindgut-demonstrated that the midgut is the site where cholesterol is absorbed and released into the hemolymph. Cholesterol-labeled lipophorin injected into larval hemolymph was cleared from the hemolymph with a half-life of 10.2 h. After 17 h, most of the cleared radioactivity was recovered in the fat body (38%). Arch.     

Purification and properties of microvitellogenin of Manduca sexta role of juvenile hormone in appearance and uptake

Microvitellogenin, a female specific protein found in hemolymph and eggs of adult female Manduca sexta (tobacco hornworm moth) has been purified to homogeneity. It is a 31,000 dalton protein lacking covalently bound carbohydrate. It appears in the hemolymph near the time of adult eclosion. The appearance of microvitellogenin in adult hemolymph is not dependent upon the presence of juvenile hormone. Uptake of both vitellogenin and microvitellogenin are greatly reduced in the absence of juvenile hormone.     

Regulation of cytochrome P-450 dependent steroid hydroxylase activity in Manduca sexta: effects of the ecdysone agonist RH 5849 on ecdysone 20-monooxygenase activity

The non-steroidal ecdysone agonist RH 5849 (1,2-dibenzoyl-1-tert-butylhydrazine) was found to inhibit in a dose-response and apparently competitive fashion the cytochrome P-450 dependent ecdysone 20-monooxygenase activity in the midgut of wandering stage last instar larvae of the tobacco hornworn, Manduca sexta. More effectively on a per molar basis than the naturally occurring molting hormones ecdysone and 20-hydroxyecdysone, RH 5849 was also found to elicit the dramatic 50-fold increase in midgut steroid hydroxylase activity (which normally occurs with the onset of the wandering stage) when injected into competent head or thoracic ligated pre-wandering last instar larvae. These data support and extend the potential usefulness of RH 5849 as a pharmacological probe for further investigating the actions of ecdysteroids and their role(s) in the regulation of ecdysteroid monooxygenases.     

Transepithelial flux of an allatostatin and analogs across the anterior midgut of Manduca sexta larvae in vitro

The transepithelial flux of cydiastatin 4 and analogs across flat sheet preparations of the anterior midgut of larvae of the tobacco hawkmoth moth, Manduca sexta, was investigated using a combination of reversed-phase high-performance liquid chromatography (RP-HPLC), enzyme-linked immunosorbent assay (ELISA) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The lumen to hemolymph (L-H) flux of cydiastatin 4 was dose and time-dependent, with a maximum rate of flux of c. 178 pmol/cm2/h) measured after a 60-min incubation with 100 micromol/l of peptide in the lumen bathing fluid. The rates of flux, L-H and H-L, across the isolated gut preparations were not significantly different. These data suggest that uptake across the anterior midgut of larval M. sexta is via a paracellular route. Cydiastatin 4 was modified to incorporate a hexanoic acid (Hex) moiety at the N-terminus, the N-terminus extended with 5 P residues and/or the substitution of G7 with Fmoc-1-amino-cyclopropylcarboxylic acid (Acpc). The incorporation of hexanoic acid enhanced the uptake of these amphiphilic analogs compared to the native peptide. Analogs were also more resistant to enzymes in hemolymph and gut preparations from larval M. sexta. A modified N-terminus gave protection against aminopeptidase-like activity and incorporation of Acpc inhibited endopeptidase-like activity. Although analogs were stable in the hemolymph, they were susceptible to amidase-like activity in the gut, which appears to convert the C-terminal amide group to a free carboxylic acid, identified by an increase in 1 mass unit of the peptide analog.     

Effect of sublethal Bacillus thuringiensis crystal endotoxin treatment on the larval midgut of a moth, Manduca: SEM study

The effect of a single, sublethal dose of B. thuringiensis crystal endotoxin on the midgut of the moth Manduca sexta larvae was monitored during acute and recovery stages. Initially both goblet and columnar cells swelled. Many columnar cells produced membrane extrusions. In some cases the affected cells ruptured, extruding cellular debris into the midgut lumen. Following the acute stage, the midgut tissue recovered, the damaged cells being extruded into the midgut lumen apparently as newly regenerated cells rose to take their place. The insects appeared to recover completely and continue normal development.     

Implications of hemolin glycosylation and Ca2+-binding on homophilic and cellular interactions.

Insects are useful models for the study of innate immune mechanisms because of their lack of antibodies and receptors involved in adaptive immune response. Nevertheless, hemolin cloned from moths is a soluble and membrane associated Ig-related molecule that is up-regulated during immune response [Lanz-Mendoza, H. & Faye, I. (1999) Dev. Comp. Immunol. 23, 359-374]. The hemolin monomeric form has four, pair-wise, interacting Ig-domains, forming a strongly bent horseshoe structure [Su, X.-D., Gastinel, L.N., Vaughn, D.E., Faye, I., Poon, P. & Bjorkman, P. (1998) Science 281, 991-995]. To elucidate the nature of its homophilic and cellular interactions, the glycosylation and Ca2+-binding properties of hemolin were investigated. We used Hyalophora cecropia hemolin isolated from hemolymph of bacteria-injected pupae, or produced as a recombinant protein in a baculovirus/insect cell system. Both types of hemolin contain N-acetylglucosamine and probably sialic acid, as indicated by peptide:N-glycosidase F and neuraminidase digestion and glycosylation detection by Western-blotting analysis. The N-acetylglucosamine residues on hemolin were confirmed with the use of specific lectins. In addition, hemolin was shown to specifically bind calcium when spotted onto nitrocellulose and treated as for 45Ca2+ autoradiography. Earlier studies demonstrated that hemolin can bind to hemocytes and this was tested for its dependence on calcium and carbohydrates, using hemolin-coated fluorescent microspheres. A greater level of attachment of microspheres occurred in the presence of calcium than if calcium was absent. Furthermore, this binding was inhibited by EGTA and N-acetylglucosamine or N-acetylneuraminic acid, implying that carbohydrates and calcium are crucial factors in homophilic binding and cell-adhesion events mediated by this Ig-superfamily molecule.     

The insect immune protein hemolin is expressed during oogenesis and embryogenesis

Hemolin is the most abundant bacteria-induced proteins in Hyalophora cecropia hemolymph. Its structural features, both at the protein and gene level, ascribe this molecule to the immunoglobulin gene superfamily (IgSF) with particular homology to neural cell adhesion molecules. An increasing number of evidence suggest a role in immune recognition and in cell adhesion events. Hemolin is also developmentally regulated as suggested by changes in its concentration during larval and pupal ecdysis (Trenczek, T., 1998. Endogenous defense mechanisms of insects. Zoology 101, 298-315; Lanz-Mendoza, H., Faye, I., 1999. Physiological aspects of the immunoglobulin superfamily in invertebrates. Dev. Comp. Immunol. 23, 359-374). In the present study the expression of hemolin was investigated in oogenesis and in early embryogenesis. Our results reveal that hemolin is expressed in follicles and in epidermal and neural tissues of embryos.