Apolipophorin III Research Articles

Phospholipase A2 activity is required for immune defense of European (Apis mellifera) and Asian (Apis cerana) honeybees against American foulbrood pathogen, Paenibacillus larvae.

Honeybees require an efficient immune system to defend against microbial pathogens. The American foulbrood pathogen, Paenibacillus larvae, is lethal to honeybees and one of the main causes of colony collapse. This study investigated the immune responses of Apis mellifera and Apis cerana honeybees against the bacterial pathogen P. larvae. Both species of honeybee larvae exhibited significant mortality even at 102 103 cfu/mL of P. larvae by diet-feeding, although A. mellifera appeared to be more tolerant to the bacterial pathogen than A. cerana. Upon bacterial infection, the two honeybee species expressed both cellular and humoral immune responses. Hemocytes of both species exhibited characteristic spreading behaviors, accompanied by cytoskeletal extension along with F-actin growth, and formed nodules. Larvae of both species also expressed an antimicrobial peptide called apolipophorin III (ApoLpIII) in response to bacterial infection. However, these immune responses were significantly suppressed by a specific inhibitor to phospholipase A2 (PLA2). Each honeybee genome encodes four PLA2 genes (PLA2A ~ PLA2D), representing four orthologous combinations between the two species. In response to P. larvae infection, both species significantly up-regulated PLA2 enzyme activities and the expression of all four PLA2 genes. To determine the roles of the four PLA2s in the immune responses, RNA interference (RNAi) was performed by injecting gene-specific double stranded RNAs (dsRNAs). All four RNAi treatments significantly suppressed the immune responses, and specific inhibition of the two secretory PLA2s (PLA2A and PLA2B) potently suppressed nodule formation and ApoLpIII expression. These results demonstrate the cellular and humoral immune responses of A. mellifera and A. cerana against P. larvae. This study suggests that eicosanoids play a crucial role in mediating common immune responses in two closely related honeybees.

PH-Dependent Interactions of Apolipophorin-III with a Lipid Disk

Apolipophorin-III (ApoLp-III) is required for stabilization of molecular shuttles of lipid fuels in insects and is found to contribute to the insect immune reaction. Rearrangement of its five [Formula: see text]-helices enables ApoLp-III to reversibly associate with lipids. We investigate computationally the conformational changes of ApoLp-III and the pH-dependence of the binding free energy of ApoLp-III association with a lipid disk. A dominant binding mode along with several minor, low population, modes of the ApoLp-III binding to a lipid disk was identified. The pH-dependence of the binding energy for ApoLp-III with the lipid disk is predicted to be significant, with the pH-optimum at pH[Formula: see text]. The calculations suggest that there are no direct interactions between the lipid head groups and titratable residues of ApoLp-III. In the physiological pH range from 6.0 to 9.0, the binding free energy of ApoLp-III with the lipid disk decreases significantly with respect to its optimal value at pH 8.0 (at pH[Formula: see text], it is 1.02[Formula: see text]kcal/mol and at pH[Formula: see text] it is 0.23[Formula: see text]kcal/mol less favorable than at the optimal pH[Formula: see text]), indicating that the pH is an important regulator of ApoLp-III lipid disk association.

Molecular characterisation of Apolipophorin-III gene in Samia cynthia ricini and its roles in response to bacterial infection.

Apolipophorin-III (ApoLp-III) is an abundant hemolymph protein mainly involved in lipid transport and innate immunity in insects. In the present study, the gene Samia cynthia ricini ApoLp-III (ScApoLp-III) was identified from a transcriptome database, and contained 790 nucleotides with a putative open reading frame (ORF) of 561 bp encoding 186 amino acid residues. Phylogenetic analysis revealed that ScApoLp-III had significant homology with ApoLp-III protein from Antheraea pernyi. Higher ScApoLp-III expression levels were found in the fat body and silk gland by reverse transcription quantitative PCR (RT-qPCR). Injection of Staphylococcus aureus induced up-regulation of ScApoLp-III in the midgut, fat body and hemocytes. However, ScApoLp-III was down-regulated in the midgut and fat body after Pseudomonas aeruginosa injection, indicating that ScApoLp-III may contribute to the host's defense against invading pathogens. Additionally, recombinant ScApoLp-III was found to bind different bacteria, including E. coli, P. aeruginosa, S. aureus and B. subtilis. Bactericidal tests showed that recombinant ScApoLp-III strongly inhibited Gram-negative bacteria, including Escherichia coli and P. aeruginosa. However, it had no obvious influence on Gram-positive bacteria. Taken together, our results suggest that the ScApoLp-III might play an important role in the innate immunity of S. c. ricini.

Apolipophorin-III Acts as a Positive Regulator of Plasmodium Development in Anopheles stephensi.

Apolipophorin III (ApoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune responses of insects. Here we report the molecular and functional characterization of Anopheles stephensi Apolipophorin-III (AsApoLp-III) gene. This gene consists of 679 nucleotides arranged into two exons of 45 and 540 bp that give an ORF encoding 194 amino acid residues. Excluding a putative signal peptide of the first 19 amino acid residues, the 175-residues in mature AsApoLp-III protein has a calculated molecular mass of 22 kDa. Phylogenetic analysis revealed the divergence of mosquitoes (Order Diptera) ApoLp-III from their counterparts in moths (Order: Lepidoptera). Also, it revealed a close relatedness of AsApoLp-III to ApoLp-III of An. gambiae. AsApoLp-III mRNA expression is strongly induced in Plasmodium berghei infected mosquito midguts suggesting its crucial role in parasite development. AsApoLp-III silencing decreased P. berghei oocysts numbers by 7.7 fold against controls. These effects might be due to the interruption of AsApoLp-III mediated lipid delivery to the developing oocysts. In addition, nitric oxide synthase (NOS), an antiplasmodial gene, is also highly induced in AsApoLp-III silenced midguts suggesting that this gene acts like an agonist and protects Plasmodium against the mosquito immunity.

Identification and characterization of an Apolipophorin-III gene from Actias selene Hübner (Lepidoptera: Saturniidae)

Apolipophorin-III (ApoLp-III) is involved in lipid transport and innate immunity in insects. In this study, we isolated ApoLp-III gene (As-ApoLp-III) cDNA from Actias selene Hübner (Lepidoptera: Saturniidae). It contained 660 nucleotides with a putative open reading frame (ORF) of 561bp encoding 186 amino acid residues. It showed similarities with ApoLp-III proteins from other insect species, especially Antheraea pernyi. The recombinant protein of As-ApoLp-III was expressed in Escherichia coli, and anti-As-ApoLp-III antibodies were prepared. The As-ApoLp-III gene was expressed at high levels in the fat body and integument, and at low levels in the hemolymph, testis, silk gland, malpighian tubule, ovary, and midgut. As-ApoLp-III gene can be upregulated by E. coli, Micrococcus luteus, Beauveria bassiana, and nuclear polyhedrosis virus (NPV) and showed a greater sensitivity for gram-negative bacteria and fungi than for gram-positive bacteria and virus. These data indicated that As-ApoLp-III may play an important role in innate immune responses of Actias selene.

Molecular characterization of an Apolipophorin-III gene from the Chinese oak silkworm, Antheraea pernyi (Lepidoptera: Saturniidae).

Apolipophorin-III (ApoLp-III) acts in lipid transport, lipoprotein metabolism, and innate immunity in insects. In this study, an ApoLp-III gene of Antheraea pernyi pupae (Ap-ApoLp-III) was isolated and characterized. The full-length cDNA of Ap-ApoLp-III is 687 bp, including a 5'-untranslated region (UTR) of 40 bp, 3'-UTR of 86 bp and an open reading frame of 561 bp encoding a polypeptide of 186 amino acids that contains an Apolipophorin-III precursor domain (PF07464). The deduced Ap-apoLp-III protein sequence has 68, 59, and 23% identity with its orthologs of Manduca sexta, Bombyx mori, and Aedes aegypti, respectively. Phylogenetic analysis showed that the Ap-apoLp-III was close to that of Bombycoidea. qPCR analysis revealed that Ap-ApoLp-III expressed during the four developmental stages and in integument, fat body, and ovaries. After six types of microorganism infections, expression levels of the Ap-ApoLp-III gene were upregulated significantly at different time points compared with control. RNA interference (RNAi) of Ap-ApoLp-III showed that the expression of Ap-ApoLp-III was significantly downregulated using qPCR after injection of E. coli. We infer that the Ap-ApoLp-III gene acts in the innate immunity of A. pernyi.

Functional study of the gene encoding apolipophorin III in development and immune responses in the beet armyworm, Spodoptera exigua

Apolipophorin III (ApoLpIII) plays a crucial role in lipid transport in insects. Its property of binding to lipid moieties is also used in pathogen recognition, such as its binding to lipopolysaccharides of Gram-negative bacteria. This study identified the cDNA sequence of ApoLpIII from the beet armyworm, Spodoptera exigua. Its cDNA was cloned to be 692 bp and it encoded 188 amino acid residues. The predicted amino acid sequence contained a signal peptide (17 residues) at the amino terminal region and one N-glycosylation site. Its predicted molecular weight was 19,042.18 Da after cleavage of the signal peptide. Secondary structure analysis of ApoLpIII predicted that 85.38% of the protein was composed of five α helix domains. RT-PCR analysis showed that ApoLpIII transcript was expressed in all developmental stages, but its expression was restricted to fat body and hemocyte in S. exigua. The expression of ApoLpIII was highly upregulated by bacterial or fungal challenges based on quantitative RT-PCR analysis. When the expression of ApoLpIII was suppressed by injection of its specific dsRNA, the treated larvae showed significant immunosuppression, which was analyzed by hemocyte nodule formation against bacterial challenge in S. exigua. In addition, larvae treated with the specific dsRNA showed high mortality during development. These results suggest that ApoLpIII is associated with development and immune responses in S. exigua.

Apolipophorin-III Mediates Antiplasmodial Epithelial Responses in Anopheles gambiae (G3) Mosquitoes

BackgroundApolipophorin-III (ApoLp-III) is known to play an important role in lipid transport and innate immunity in lepidopteran insects. However, there is no evidence of involvement of ApoLp-IIIs in the immune responses of dipteran insects such as Drosophila and mosquitoes.Methodology/Principal FindingsWe report the molecular and functional characterization of An. gambiae apolipophorin-III (AgApoLp-III). Mosquito ApoLp-IIIs have diverged extensively from those of lepidopteran insects; however, the predicted tertiary structure of AgApoLp-III is similar to that of Manduca sexta (tobacco hornworm). We found that AgApoLp-III mRNA expression is strongly induced in the midgut of An. gambiae (G3 strain) mosquitoes in response to Plasmodium berghei infection. Furthermore, immunofluorescence stainings revealed that high levels of AgApoLp-III protein accumulate in the cytoplasm of Plasmodium-invaded cells and AgApoLp-III silencing increases the intensity of P. berghei infection by five fold.ConclusionThere are broad differences in the midgut epithelial responses to Plasmodium invasion between An. gambiae strains. In the G3 strain of An. gambiae AgApoLp-III participates in midgut epithelial defense responses that limit Plasmodium infection.

Immunosuppression induced by entomopathogens is rescued by addition of apolipophorin III in the diamondback moth, Plutella xylostella

Apolipophorin III (ApoLpIII) has been known to play critical roles in lipid transport and immune activation in insects. This study reports a partial ApoLpIII gene cloned from the diamondback moth, Plutella xylostella. It showed that the gene was expressed in all developmental stages of P. xylostella. In larval stage, it was expressed in all tested tissues of hemocyte, fat body, gut, and epidermis. In response to bacterial challenge, the larvae showed an enhanced level of ApoLpIII expression by a quantitative real-time RT-PCR. RNA interference of ApoLpIII by its specific double stranded RNA (dsRNA) caused significant knockdown of its expression level and resulted in significant suppression in hemocyte nodule formation in response to bacterial challenge. However, larvae treated with the dsRNA exhibited a significant recovery in the cellular immune response by addition of a recombinant ApoLpIII. Parasitization by an endoparasitoid wasp, Cotesia plutellae, suppressed expression of ApoLpIII and resulted in a significant suppression in the hemocyte nodule formation. The addition of the recombinant ApoLpIII to the parasitized larvae significantly restored the hemocyte activity. Infection of an entomopathogenic bacterium, Xenorhabdus nematophila, caused potent pathogenicity of P. xylostella. However, the addition of the recombinant ApoLpIII to the infected larvae significantly prevented the lethal pathogenicity. This study suggests that ApoLpIII limits pathogenicity induced by parasitization or bacterial infection in P. xylostella.

Lipid-triggered Conformational Switch of Apolipophorin III Helix Bundle to an Extended Helix Organization

Apolipophorin III (ApoLp-III) from the Sphinx moth, Manduca sexta, is an 18 kDa protein that binds reversibly to hydrophobic surfaces generated on metabolizing lipoprotein particles. It is comprised of amphipathic α-helices (H1–H5) organized in an up-and-down topology forming a helix bundle in the lipid-free state. Upon interaction with lipids, apoLp-III has been proposed to undergo a dramatic conformational change, involving helix bundle opening about putative hinge loops such that H1, H2 and H5 move away from H3 and H4. In the present study, we examine the relative spatial disposition of H1 and H5 on discoidal phospholipid complexes and spherical lipoproteins. Cysteine residues were engineered at position 8 in H1 and/or at position 138 in H5 in apoLp-III (which otherwise lacks Cys) yielding A8C-, A138C- and A8C/A138C–apoLp-III. Tethering of H1 and H5 by a disulfide bond between A8C and A138C abolished the ability of apoLp-III to transform phospholipid vesicles to discoidal particles, or to interact with lipoproteins, demonstrating that these helices are required to reposition during lipid interaction. Site-specific labeling of A8C/A138C–apoLp-III with N-(1-pyrene)maleimide in the lipid-free state resulted in intramolecular pyrene “excimer” fluorescence emission indicative of spatial proximity between these sites. Upon association with dimyristoylphosphatidylcholine (DMPC) discoidal complexes, the intramolecular excimer was replaced by intermolecular excimer fluorescence due to proximity between pyrene moieties on A8C and A138C in neighboring apoLp-III molecules on the discoidal particle. No excimer emission was observed in the case of pyrene–A8C–apoLp-III/DMPC or pyrene–A138C–apoLp-III/DMPC complexes. However, equimolar mixing of the two labeled single-cysteine mutants prior to disc formation resulted in excimer emission. In addition, intramolecular pyrene excimer formation was diminished upon binding of pyrene–A8C/A138C–apoLp-III to spherical lipoproteins. The data are consistent with repositioning of H1 away from H5 upon encountering a lipid surface, resulting in an extended conformation of apoLp-III that circumscribes the discoidal bilayer particle.

Purification and characterization of apolipophorin III from immune hemolymph of Heliothis virescens pupae

Apolipophorin III (ApoLp-III) from Heliothis virescens pupae was purified by heat-treatment followed by Sephadex G-50 filtration and reverse phase-HPLC. The molecular mass of the purified ApoLp-III was determined as 17 965.9±5 Da by mass spectrometry. The N-terminal sequence confirmed the protein as ApoLp-III with homology of 56–83% to other insect ApoLp-III molecules. The amino acid spatial arrangement of the predicted α-helix 1 of Heliothis ApoLp-III was nearly identical to that of the amphipatic α-helix 1 of Manduca sexta ApoLp-III. The absorption spectrum from 240–340 nm of the Heliothis ApoLp-III was the same as the UV spectra of ApoLp-III from Manduca sexta and Galleria mellonella, showing absorption maxima at 280, 268, 264 and 259 nm. These results indicated that the primary structure of ApoLp-III is conserved in lepidopterans. The Heliothis ApoLp-III was not a glycoprotein and showed hemagglutination activity against rabbit red blood cells. This hemagglutination activity was abolished by Tween 80, but not by six different carbohydrates. Hydrophobic interaction of ApoLp-III with red blood cells agreed with structural studies since ApoLp-III binds lipid through hydrophobic interaction after conformational change. Bacterial injection apparently increased the amount of ApoLp-III in immune hemolymph when compared with normal hemolymph, and may indicate that ApoLp-III plays a role in insect immunity.