Larval Lipophorin Research Articles

Lipophorin of lower density is formed during immune responses in the lepidopteran insect Galleria mellonella.

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.

Carotenoid transport by the larval lipophorin of the southwestern corn borer, Diatraea grandiosella

Carotenoid transport by the larval lipophorin of the southwestern corn borer, Diatraea grandiosella

A comparison of adult and larval Manduca sexta apolipophorin-III

Apolipophorin-III (apoLp-III) is known to be present in the hemolymph of larval and adult Manduca sexta, but is associated with lipophorin only in the adult. We tested two hypotheses which might account for the lack of apoLp-III in larval lipophorin: (1) there might be a difference in processing of the prepro precursor between adult and larval fat body; (2) there might be larval and adult specific transcription of the apoLp-III gene. ApoLp-III isolated from adult or larval hemolymph has exactly the same amino acid composition and therefore there is not a significant amount of the pro precursor present in hemolymph in either case. We did confirm the presence of the pro precursor in the fat body and showed the site of cleavage of the prepro precursor is between ser (−6) and ala (−5). Restriction mapping, cDNA sequencing and primer extension analysis all indicated that both larval and adult apoLp-III have the same mRNA transcripts and utilize the same coding sequences of a single apoLp-III gene. The data show that neither hypothesis can explain the lack of apoLp-III in larval lipophorin and that other explanations, perhaps involving the mechanism of lipophorin assembly in the fat body, will have to be sought. It was shown that the apoLp-III gene is expressed in a cyclic manner during larval development with maximum expression occurring during the feeding period and that adult fat body contains 2- to 10-fold higher levels of the apoLp-III mRNA.

Lipophorin in the larval and adult stages ofMusca domestica

The major Musca domestica hemolymph lipoprotein, lipophorin, was purified from larval and from adult animals. The housefly lipophorin is composed of two apoproteins, apolipophorin I (Mr ∽ 253,000) and apolipophorin II (Mr ∽ 85,000). The lipophorin contains about 3.9% carbohydrates and reacts positively with concanavalin A. The density of larval lipophorin is equal to 1.152 g/ml and of adult lipophorin to 1.106 g/ml. The amount of lipophorin per animal increases during the larval stage, is constant during pupal stage, and suffers a great reduction at the pharate adult stage. The amount of lipophorin remains stable during the whole first gonotrophic cycle of the housefly. Lipophorin is not detected in the eggs of this insect.

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.

Major hemolymph proteins from larvae of the black swallowtail butterfly, Papilio polyxenes

AbstractThree major hemolymph proteins of Papilio polyxenes larvae were isolated and characterized. Density gradient ultracentrifugation of hemolymph resulted in flotation of the major lipoprotein, lipophorin. P. polyxenes larval lipophorin is composed of two apoproteins, apolipophorin‐I and apolipophorin‐II, plus a mixture of lipids, to give a density of 1.13 g/ml. Immunoblotting experiments using antisera directed against Manduca sexta apolipophorin‐I and apolipophorin‐II, respectively, revealed cross‐reactivity of apoLp‐I with Manduca sexta apoLp‐I, and apoLp‐II with M. sexta apoLp‐II.Gel permeation chromatography of the subnatant obtained following density gradient ultracentrifugation revealed the presence of a major protein peak which was shown to contain three major serum proteins, two of which were isolated and characterized. One of these proteins was purified by lectin affinity chromatography. Both proteins have native molecular weights in the range of 450,000 and appear to be hexamers of a single subunit type. Major serum protein‐1 is nonglycosylated and has a subunit molecular weight of 75,000. Major serum protein‐2 is glycosylated and has a subunit molecular weight of 74,000. Amino acid analysis of this protein revealed a tyrosine plus phenylalanine content of 20 mole percent, characteristic of the arylphorin class of insect storage proteins. Using antibodies against M. sexta larval hemolymph proteins, both the P. polyxenes major serum proteins were shown to be immunologically related to serum proteins of other lepidopteran species.

Insect apolipophorin III. Purification and properties.

The hemolymph of adult Manduca sexta (tobacco hornworm) contains a 17,000-dalton protein that can associate reversibly with the insect lipoprotein lipophorin. The protein is abundant in the hemolymph of the adult, but is found in larval hemolymph in only small amounts, and does not associate with larval lipophorin. On the basis of its association with adult lipophorin, we have designated the protein apolipophorin III. Apolipophorin III was dissociated from adult lipophorin by guanidinium chloride treatment and isolated by gel permeation and ion exchange chromatography. The unassociated apolipophorin III was also purified from lipophorin-free hemolymph by gel permeation, ion exchange, and lectin chromatography. Both preparations have identical isoelectric points and amino acid composition as well as the following properties. Apolipophorin III is a non-glycosylated polypeptide lacking cysteine and tryptophan. The 17,000-dalton polypeptide dimerizes in solution to a protein of Mr = 34,000.