RESUMO
Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein. Its function, as currently understood, lies in the stabilization of low-density lipophorin particles (LDLp) crossing the hemocoel in phases of high energy consumption to deliver lipids from the fat body to the flight muscle cells. Recent studies with native Galleria mellonella-apoLp-III gave first indications of an unexpected role of that protein in insect immune activation. Here we report the immune activation by the recombinant protein, documenting a newly discovered correlation between lipid physiology and immune defense in insects. The complete cDNA sequence of G. mellonella-apoLp-III was identified by mixed oligonucleotide-primed amplification of cDNA (MOPAC), 3'-RACE-PCR, and cRACE-PCR. The sequence coding for the native protein was ligated into a pET-vector; this construct was transfected into Escherichia coli and overexpressed in the bacteria. Photometric turbidity assays with human low density lipoprotein (LDL) and transmission electron microscopy studies on apoLp-III-stabilized lipid discs revealed the full functionality of the isolated recombinant apoLp-III with regard to its lipid-association ability. For proving its immune-stimulating capacity, apoLp-III was injected into the hemocoel of last instar G. mellonella larvae and the antibacterial activity in cell-free hemolymph was determined 24 h later. As a result, the hemolymph samples of injected insects contained strongly increased antibacterial activities against E. coli as well as clearly enhanced lysozyme-like activities. From Northern blot analysis of total RNA from insects injected with apoLp-III or the bacterial immune provocator lipopolysaccharide, it could be concluded that the transcription rate of apoLp-III mRNA does not vary in comparison to untreated last instar larvae.
Assuntos
Apolipoproteínas/farmacologia , Insetos/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Apolipoproteínas/química , Apolipoproteínas/genética , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , DNA Complementar/metabolismo , Dimiristoilfosfatidilcolina/química , Escherichia coli/metabolismo , Insetos/imunologia , Insetos/ultraestrutura , Lipopolissacarídeos/farmacologia , Lipoproteínas LDL/química , Dados de Sequência Molecular , Proteínas Recombinantes/farmacologiaRESUMO
The chemical composition of a poly(benzoxazine) thermoset polymer (a copolymer of bisphenol-A benzoxazine and tert.-butylphenol benzoxazine) has been studied by pyrolysis-gas chromatography (Py-GC). Major pyrolysates have been identified and the possible degradation pathways have been investigated. A specific pyrolysate was identified for quantitative analysis after carefully proving the linear relationship between the pyrolysate signal intensity and monomer concentration over a wide range of compositions. A method to determine the concentration of the monomer that potentially acts as a cross-linking unit has been developed. In this study, Py-GC was shown to be an excellent analytical technique for the qualitative and quantitative analysis of thermoset polymers.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Oxazinas/análise , Polímeros/químicaRESUMO
A few years ago, it was shown that intrahemocoelic injection of the insect apolipoprotein apolipophorin III (apoLp-III) stimulates an immune response in larvae of the greater wax moth, Galleria mellonella. Since the mode of action of this activation process is unknown, we followed apoLp-III's pathway in the early phase of the immune-stimulating process, using biotin as a probe. Biotinylated apoLp-III was injected and localized using avidin-coupled horseradish peroxidase. The labeled protein was fully functional; the added amount of biotin per apoLp-III molecule used in this study only slightly decreased its ability to associate with phospholipase C-treated human low-density lipoprotein, as well as the immune-stimulating capability of apoLp-III.Gel electrophoresis with subsequent staining of biotin moieties and lipids revealed that apoLp-III undergoes lipid association in vivo within the first few minutes after injection. After two hours, no biotinylated apoLp-III was detectable in cell-free hemolymph. At this time, a subpopulation of hemocytes showed a distinct peroxidase staining. Control injections of biotinylated bovine serum albumin did not lead to similar results, giving evidence for the specificity of the phenomena observed. The results indicate that lipid association of apoLp-III occurs prior to endocytosis by immune-competent hemocytes, which is followed by the induction of a humoral immune response.
RESUMO
Injection of heat-killed bacteria into larvae of the greater wax moth Galleria mellonella is followed by changes in lipoprotein composition in the hemolymph. Density gradient centrifugation experiments revealed that within the first four hours after injection, a part of larval lipoprotein, high-density lipophorin (HDLp), was converted into a lipoprotein of lower density. SDS-polyacrylamide gel electrophoresis analysis of the gradient fractions and sequencing of protein fragments, established that the exchangeable apolipoprotein apolipophorin III (apoLp-III), a potent immune-activator, was associated with this newly formed lipophorin. To investigate further the influence of lipophorin-associated apoLp-III on immune-related reactions, we performed in vitro studies with isolated hemocytes from G. mellonella and lipophorins from the sphinx moth Manduca sexta, as a natural source of high amounts of low-density lipophorin (LDLp) and HDLp. The hemocytes were activated to form superoxide radicals upon incubation with LDLp, but not with HDLp. Fluorescence-labeled LDLp was specifically taken up by granular cells. This process was inhibited by adding an excess of unlabeled LDLp, but not by HDLp. We hypothesize that larval lipophorin formed in vivo is an endogenous signal for immune activation, specifically mediated by the binding of lipid-associated apoLp-III to hemocyte membrane receptors.
Assuntos
Proteínas de Transporte/análise , Hemolinfa/química , Hemolinfa/imunologia , Lipoproteínas/análise , Mariposas/imunologia , Proteínas de Saccharomyces cerevisiae , Animais , Apolipoproteínas/análise , Apolipoproteínas/química , Apolipoproteínas/farmacocinética , Bactérias/imunologia , Carbocianinas , Proteínas de Transporte/química , Proteínas de Transporte/farmacocinética , Fracionamento Celular , Endocitose , Corantes Fluorescentes , Glicoproteínas/análise , Glicoproteínas/imunologia , Homeostase/imunologia , Proteínas de Insetos , Lipoproteínas/química , Lipoproteínas/farmacocinética , Proteínas de Membrana , Dados de Sequência Molecular , Análise de Sequência de Proteína , Serina Endopeptidases/análise , Serina Endopeptidases/imunologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein consisting of five amphipathic alpha-helices. The protein is able to open reversibly on associating with hydrophobic surfaces and plays a role both in lipid transport and induction of immune responses. Point mutations were introduced at positions 66 (N-->D) and/or 68 (K-->E) between helices 2 and 3, a region possibly serving as a hinge for the opening of the molecule when associating with lipids. The lipid-binding properties of the mutant proteins were analyzed and compared with their immune inducing activities. Structural properties of the proteins were studied by far UV circular dichroism spectroscopy and their abilities to form discoidal complexes of dimyristoyl phosphatidylcholine (DMPC) vesicles were investigated. In comparison to wild-type apoLp-III, apoLp-III(N66D/K68E), and apoLp-III(K68E) displayed significantly decreased lipid-binding abilities and immune stimulating activities, while these effects were less noticeable with apoLp-III(N66D). The secondary structure of the double mutant apoLp-III(N66D/K68E) was similar to that of wild-type apoLp-III. A noticeable reduction of alpha-helical content could be observed for the single mutants apoLp-III(N66D) and apoLp-III(K68E), which was accompanied by an increase in percentage amount of beta-turns. The stability of the secondary structure determined by heat denaturation was not affected by mutagenesis. Furthermore, the ability of all proteins to form discoidal complexes of equal size and shape in the presence of dimyristoyl phosphatidylcholine indicated that the mutagenesis did not affect the molecular architecture in the lipid-associated conformation. The relationship between reduced lipid association and reduced immune stimulating activity supports the hypothesis that apoLp-III-induced immune activation is triggered by the conformational change of the protein.
Assuntos
Apolipoproteínas/química , Apolipoproteínas/metabolismo , Dimiristoilfosfatidilcolina/metabolismo , Mariposas/fisiologia , Substituição de Aminoácidos , Animais , Apolipoproteínas/ultraestrutura , Sítios de Ligação , Proteínas de Transporte/metabolismo , Dimiristoilfosfatidilcolina/química , Larva , Ligantes , Lipoproteínas LDL/metabolismo , Microscopia Eletrônica , Modelos Moleculares , Mariposas/imunologia , Mutagênese Sítio-Dirigida , Mutação Puntual , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fosfolipases Tipo C/metabolismoRESUMO
Apolipophorin III (apoLp-III) from the greater wax moth Galleria mellonella is an exchangeable insect apolipoprotein that consists of five amphipathic alpha-helices, sharing high sequence identity with apoLp-III from the sphinx moth Manduca sexta whose structure is available. To define the minimal requirement for apoLp-III structural stability and function, a C-terminal truncated apoLp-III encompassing residues 1-91 of this 163 amino acid protein was designed. Far-UV circular dichroism spectroscopy revealed apoLp-III(1-91) has 50% alpha-helix secondary structure content in buffer (wild-type apoLp-III 86%), increasing to essentially 100% upon interactions with dimyristoylphosphatidylcholine (DMPC). Guanidine hydrochloride denaturation studies revealed similar stability properties for wild-type apoLp-III and apoLp-III(1-91). Resistance to denaturation for both proteins increased substantially upon association with phospholipid. In the absence of lipid, wild-type apoLp-III was monomeric whereas apoLp-III(1-91) partly formed dimers and trimers. Discoidal apoLp-III(1-91)-DMPC complexes were smaller in diameter (13.5 nm) compared to wild-type apoLp-III (17.7 nm), and more molecules of apoLp-III(1-91) associated with the complexes. Lipid interaction revealed that apoLp-III(1-91) binds to modified spherical lipoprotein surfaces and efficiently transforms phospholipid vesicles into discoidal complexes. Thus, the first three helices of G. mellonella apoLp-III contain the basic features required for maintenance of the structural integrity of the entire protein.