The complex of the insect LDL receptor homolog, lipophorin receptor, LpR, and its lipoprotein ligand does not dissociate under endosomal conditions

The insect low-density lipoprotein (LDL) receptor (LDLR) homolog, lipophorin receptor (LpR), mediates endocytic uptake of the single insect lipoprotein, high-density lipophorin (HDLp), which is structurally related to LDL. However, in contrast to the fate of LDL, which is endocytosed by LDLR, we previously demonstrated that after endocytosis, HDLp is sorted to the endocytic recycling compartment and recycled for re-secretion in a transferrin-like manner. This means that the integrity of the complex between HDLp and LpR is retained under endosomal conditions. Therefore, in this study, the ligand-binding and ligand-dissociation capacities of LpR were investigated by employing a new flow cytometric assay, using LDLR as a control. At pH 5.4, the LpR-HDLp complex remained stable, whereas that of LDLR and LDL dissociated. Hybrid HDLp-binding receptors, containing either the beta-propeller or both the beta-propeller and the hinge region of LDLR, appeared to be unable to release ligand at endosomal pH, revealing that the stability of the complex is imparted by the ligand-binding domain of LpR. The LpR-HDLp complex additionally appeared to be EDTA-resistant, excluding a low Ca(2+) concentration in the endosome as an alternative trigger for complex dissociation. From binding of HDLp to the above hybrid receptors, it was inferred that the stability upon EDTA treatment is confined to LDLR type A (LA) ligand-binding repeats 1-7. Additional (competition) binding experiments indicated that the binding site of LpR for HDLp most likely involves LA-2-7. It is therefore proposed that the remarkable stability of the LpR-HDLp complex is attributable to this binding site. Together, these data indicate that LpR and HDLp travel in complex to the endocytic recycling compartment, which constitutes a key determinant for ligand recycling by LpR.     

Receptor-mediated endocytosis and intracellular trafficking of lipoproteins and transferrin in insect cells

While the intracellular pathways of ligands after receptor-mediated endocytosis have been studied extensively in mammalian cells, in insect cells these pathways are largely unknown. We transfected Drosophila Schneider line 2 (S2) cells with the human low-density lipoprotein (LDL) receptor (LDLR) and transferrin (Tf) receptor (TfR), and used endocytosis of LDL and Tf as markers. After endocytosis in mammalian cells, LDL is degraded in lysosomes, whereas Tf is recycled. Fluorescence microscopy analysis revealed that LDL and Tf are internalized by S2 cells transfected with LDLR or TfR, respectively. In transfectants simultaneously expressing LDLR and TfR, both ligands colocalize in endosomes immediately after endocytic uptake, and their location remained unchanged after a chase. Similar results were obtained with Spodoptera frugiperda Sf9 cells that were transfected with TfR, suggesting that Tf is retained intracellularly by both cell lines. The insect lipoprotein, lipophorin, is recycled upon lipophorin receptor (LpR)-mediated endocytosis by mammalian cells, however, not after endocytosis by LpR-expressing S2 transfectants, suggesting that this recycling mechanism is cell-type specific. LpR is endogenously expressed by fat body tissue of Locusta migratoria for a limited period after an ecdysis. A chase following endocytosis of labeled lipophorin by isolated fat body tissue at this developmental stage resulted in a significant decrease of lipophorin-containing vesicles, indicative of recycling of the ligand.     

Circulatory lipid transport: lipoprotein assembly and function from an evolutionary perspective

Circulatory transport of neutral lipids (fat) in animals relies on members of the large lipid transfer protein (LLTP) superfamily, including mammalian apolipoprotein B (apoB) and insect apolipophorin II/I (apoLp-II/I). Latter proteins, which constitute the structural basis for the assembly of various lipoproteins, acquire lipids through microsomal triglyceride transfer protein (MTP)—another LLTP family member—and bind them by means of amphipathic structures. Comparative research reveals that LLTPs have evolved from the earliest animals and additionally highlights the structural and functional adaptations in these lipid carriers. For instance, in contrast to mammalian apoB, the insect apoB homologue, apoLp-II/I, is post-translationally cleaved by a furin, resulting in their appearance of two non-exchangeable apolipoproteins in the insect low-density lipoprotein (LDL) homologue, high-density lipophorin (HDLp). An important difference between mammalian and insect lipoproteins relates to the mechanism of lipid delivery. Whereas in mammals, endocytic uptake of lipoprotein particles, mediated via members of the LDL receptor (LDLR) family, results in their degradation in lysosomes, the insect HDLp was shown to act as a reusable lipid shuttle which is capable of reloading lipid. Although the recent identification of a lipophorin receptor (LpR), a homologue of LDLR, reveals that endocytic uptake of HDLp may constitute an additional mechanism of lipid delivery, the endocytosed lipoprotein appears to be recycled in a transferrin-like manner. Binding studies indicate that the HDLp–LpR complex, in contrast to the LDL–LDLR complex, is resistant to dissociation at endosomal pH as well as by treatment with EDTA mimicking the drop in Ca²⁺ concentration in the endosome. This remarkable stability of the ligand–receptor complex may provide a crucial key to the recycling mechanism. Based on the binding and dissociation capacities of mutant and hybrid receptors, the specific binding interaction of the ligand-binding domain of the receptor with HDLp was characterized. These structural similarities and functional adaptations of the lipid transport systems operative in mammals and insects are discussed from an evolutionary perspective.     

An insect homolog of the vertebrate very low density lipoprotein receptor mediates endocytosis of lipophorins

A novel member of the low density lipoprotein (LDL) receptor family was identified, which is expressed in locust oocytes, fat body, brain, and midgut. This receptor appeared to be a homolog of the mammalian very low density lipoprotein receptor as it contains eight cysteine-rich repeats in its putative ligand-binding domain. When transiently expressed in COS-7 or stably expressed in LDL receptor-deficient CHO cells, the receptor mediates endocytic uptake of high density lipophorin (HDLp), an abundant lipoprotein in the circulatory compartment of insects. Moreover, in the latter cell line, we demonstrated that an excess of unlabeled HDLp competed with fluorescent labeled HDLp for uptake whereas an excess of human LDL did not affect uptake. Expression of the receptor mRNA in fat body cells is down-regulated during adult development, which is consistent with the previously reported down-regulation of receptor-mediated endocytosis of lipophorins in fat body tissue (Dantuma, N. P., M.A.P. Pijnenburg, J. H. B. Diederen, and D. J. Van der Horst. 1997. J. Lipid Res. 38: 254-265). The expression of this receptor in various tissues that internalize circulating lipophorins and its capability to mediate endocytosis of HDLp indicate that this novel member of the LDL receptor family may function as an endocytic lipophorin receptor in vivo.     

Lipophorin receptor-mediated lipoprotein endocytosis in insect fat body cells

High-density lipophorin (HDLp) in the circulation of insects is able to selectively deliver lipids to target tissues in a nonendocytic manner. In Locusta migratoria, a member of the LDL receptor family has been identified and shown to mediate endocytosis of HDLp in mammalian cells transfected with the cDNA of this receptor. This insect lipophorin receptor (iLR) is temporally expressed in fat body tissue of young adult as well as larval locusts, as shown by Western blot analysis. Fluorescence microscopy revealed that fat body cells internalize fluorescently labeled HDLp and human receptor-associated protein only when iLR is expressed. Expression of iLR is down-regulated on Day 4 after an ecdysis. Consequently, HDLp is no longer internalized. By starving adult locusts immediately after ecdysis, we were able to prolong iLR expression. In addition, expression of the receptor was induced by starving adults after down-regulation of iLR. These results suggest that iLR mediates endocytosis of HDLp in fat body cells, and that expression of iLR is regulated by the demand of fat body tissue for lipids.     

Insect lipophorin conversions. Compositional analysis of high- and low-density lipophorin of Acherontia atropos and Locusta migratoria.

The formation of low-density lipophorin (LDLp) in insect hemolymph, resulting from association of high-density lipophorin (HDLp) with both lipid and apolipophorin III, is considered to provide a reutilizable lipid shuttle for flight muscle energy supply. The changes in lipid and apolipoprotein composition of LDLp, isolated after flight activity, compared to that of HDLp in the hemolymph at rest, were studied in two evolutionary divergent insects, the hawkmoth Acherontia atropos and the migratory locust, Locusta migratoria. Using FPLC on Superose 6 prep grade as a novel technique to separate the apolipophorins of HDLp and LDLp, the ratio of apolipoprotein I, II, and III in HDLp of both species was demonstrated to be 1:1:1, whereas flight activity resulted in a ratio of 1:1:10 in LDLp. Injection of adipokinetic hormone into resting moths showed that, depending on the dose, the number of apolipophorin III molecules in LDLp can exceed that recovered after the physiological condition of flight. Analysis of the lipophorin lipids demonstrated that in addition to the considerable increase in diacylglycerol in the LDLp particle, which is consistent with the role LDLp in energy supply, particularly the hydrocarbons were increased compared to HDLp, rendering the mechanism of LDLp formation from HDLp even more complex.     

Wax moth,Galleria mellonella fat body receptor for high-density lipophorin (HDLp)

To identify and characterize the HDLp (high-density lipophorin) receptor from Galleria mellonella (LpRGm), we used techniques of ligand blotting. This method was, to our knowledge, first used to characterize the lipophorin receptor (LpR) in insects. LpRGm had an approximate molecular weight of 97 kDa under non-reducing conditions and bound the HDLp specifically. The time-course of lipophorin binding to their receptor protein was rapid. The binding of lipophorins to their receptors was saturable with a Kd of 34.33+/-4.67 microg/ml. Although Ca2+ was essentially required in the binding of HDLp to their receptors, interestingly increasing concentration of Ca2+ has shown to have a slight inhibitory effect. EDTA was used here as Ca2+ chelating reagent, because Mg2+ in the binding buffer did not affect the binding of HDLp to their receptors, and inhibited the binding of HDLp and LpRGm absolutely. Suramin (polysulfated polycyclic hydrocarbon), known to inhibit the binding of lipoproteins to their receptors, effectively abolished the binding of HDLp to their receptors. LpRGm showed the stage specific binding activity especially in day 1-3 last instar larval, prepupal, and day 1-3 adult stages.     

Alternative lipid mobilization: The insect shuttle system

Lipid mobilization in long-distance flying insects has revealed a novel concept for lipid transport in the circulatory system during exercise. Similar to energy generation for sustained locomotion in mammals, the work accomplished by non-stop flight activity is powered by oxidation of free fatty acids (FFA) derived from endogenous reserves of triacylglycerol. The transport form of the lipid, however, is diacylglycerol (DAG), which is delivered to the flight muscles associated with lipoproteins. In the insect system, the multifunctional lipoprotein, high-density lipophorin (HDLp) is loaded with DAG while additionally, multiple copies of the exchangeable apolipoprotein, apoLp-III, associate with the expanding particle. As a result, lipid-enriched low-density lipophorin (LDLp) is formed. At the flight muscles, LDLp-carried DAG is hydrolyzed and FFA are imported into the muscle cells for energy generation. The depletion of DAG from LDLp results in the recovery of both HDLp and apoLp-III, which are reutilized for another cycle of DAG transport. A receptor for HDLp, identified as a novel member of the vertebrate low-density lipoprotein (LDL) receptor family, does not seem to be involved in the lipophorin shuttle mechanism operative during flight activity. In addition, endocytosis of HDLp mediated by the insect receptor does not seem to follow the classical mammalian LDL pathway.Many structural elements of the lipid mobilization system in insects are similar to those in mammals. Domain structures of apoLp-I and apoLp-II, the non-exchangeable apolipoprotein components of HDLp, are related to apoB100. ApoLp-III is a bundle of five amphipathic -helices that binds to a lipid surface very similar to the four-helix bundle of the N-terminal domain of human apoE. Despite these similarities, the functioning of the insect lipoprotein in energy transport during flight activity is intriguingly different, since the TAG-rich mammalian lipoproteins play no role as a carrier of mobilized lipids during exercise and besides, these lipoproteins are not functioning as a reusable shuttle for lipid transport. On the other hand, the deviant behavior of similar molecules in a different biological system may provide a useful alternative model for studying the molecular basis of processes related to human disorders and disease.     

Binding of locust high-density lipophorin to fat body proteins monitored by an enzyme-linked immunosorbant assay

Binding of locust high-density lipophorin (HDLp) to fat body proteins coated on immunoassay plates was studied using the ELISA method and ligand blotting techniques. HDLp binding proved to be correlated with the amount of fat body protein coated. From the concentration-dependent total HDLp binding an equilibrium dissociation constant could be calculated (Kd = 1.6 x 10(-8) M). Heparin inhibits the HDLp binding, indicating that positively charged groups are involved in the HDLp-fat body interaction. These groups were shown to be arginyl residues, as the arginine-specific treatment of HDLp by 1,2-cyclohexanedione resulted in a approximately 50% decrease in the binding ability of HDLp. HDLp binding is also affected by the pH. A decrease from pH 7.5 to pH 6.5 increases the binding affinity by approximately 250%. A monoclonal antibody specific for apolipophorin-II (apoLp-II) hampered the HDLp binding significantly, whereas a monoclonal anti-apoLp-I had no effect. Locust fat body HDLp binding proteins are highly specific for locust HDLp.     

Insect adipokinetic hormones: release and integration of flight energy metabolism

Insect flight involves mobilization, transport and utilization of endogenous energy reserves at extremely high rates. Peptide adipokinetic hormones (AKHs), synthesized and stored in neuroendocrine cells, integrate flight energy metabolism. The complex multifactorial control mechanism for AKH release in the locust includes both stimulatory and inhibitory factors. The AKHs are synthesized continuously, resulting in an accumulation of AKH-containing secretory granules. Additionally, secretory material is stored in large intracisternal granules. Although only a limited part of these large reserves appears to be readily releasable, this strategy allows the adipokinetic cells to comply with large variations in secretory demands; changes in secretory activity do not affect the rate of hormone biosynthesis. AKH-induced lipid release from fat body target cells has revealed a novel concept for lipid transport during exercise. Similar to sustained locomotion of mammals, insect flight activity is powered by oxidation of free fatty acids derived from endogenous reserves of triacylglycerol. However, the transport form of the lipid in the circulatory system is diacylglycerol (DAG) that is delivered to the flight muscles associated with lipoproteins. While DAG is loaded onto the multifunctional insect lipoprotein, high-density lipophorin (HDLp) and multiple copies of the exchangeable apolipoprotein III (apoLp-III) associate reversibly with the expanding particle. The resulting low-density lipophorin (LDLp) specifically shuttles DAG to the working muscles. Following DAG hydrolysis by a lipophorin lipase, apoLp-III dissociates from the particle, regenerating HDLp that is re-utilized for lipid uptake at the fat body cells, thus functioning as an efficient lipid shuttle mechanism. Many structural elements of the lipoprotein system of insects appear to be similar to their counterparts in mammals; however, the functioning of the insect lipoprotein in energy transport during flight activity is intriguingly different.     

Lipophorin levels in the yellow fever mosquito,Aedes aegypti,and the effect of feeding

High density lipophorin (HDLp) is the major lipid transport vehicle in insect hemolymph. Using an indirect ELISA, levels of HDLp were measured in the yellow fever mosquito, Aedes aegypti. The level of lipophorin, when normalized to the total weight of the insect, was similar in the different developmental stages. Starvation (access to water only) of adult females did not affect the level of HDLp nor its density when compared to sugar-fed females. On the other hand, blood feeding (of normally sugar-fed females) resulted in a three-fold increase of the HDLp level at 40 h after feeding. This increase was accompanied by a slight but significant increase in the density of HDLp at 24 h after feeding. Ingestion of a lipid-free protein meal or a lipid-supplemented protein meal induced changes in HDLp level and density that were comparable to those induced by ingestion of a blood meal. Ingestion of a blood meal, following starvation (access to water only) from the moment of adult emergence, did not induce an increase in HDLp level. The results presented indicate that, in contrast to other insect species, A. aegypti responds to an increased need for lipid transport in the hemolymph by increasing the amount of HDLp. Arch. Insect Biochem. Physiol. 34:301–312, 1997. © 1997 Wiley-Liss, Inc.     

Interaction of lipophorin with the plasma membrane of locust flight muscles

Binding of high-density lipophorin (HDLp) to a plasma membrane preparation of locust flight muscle tissue was studied using a radiolabelled ligand binding assay and ligand blotting techniques. Analysis at 33 degrees C of the concentration-dependent total binding of tritium-labelled HDLp ([3H]HDLp) to the membrane preparation revealed the presence of a single specific binding site with an equilibrium dissociation constant of Kd = 9 (+/- 2) X 10(-7) M and a maximal binding capacity of 84 (+/- 10) ng X (micrograms protein)-1. Unlabelled HDLp as well as unlabelled low-density lipophorin (LDLp) competed with [3H]HDLp for binding to the identified binding site. In addition, ligand blotting demonstrated that both HDLp and LDLp bind specifically to a 30-kDa protein in the plasma membrane preparation, suggesting the involvement of this protein in the binding of lipophorins to the isolated membranes. A possible relationship between the identified binding of lipophorins and the observed co-purification of lipophorin lipase activity with the plasma membranes is discussed.     

Characterization of termite lipophorin and its involvement in hydrocarbon transport

The transport of lipids constitutes a vital function in insects and requires the plasma lipoprotein lipophorin. In all insects examined to date, cuticular hydrocarbons are also transported through the hemolymph by lipophorin, and in social insects they play important roles not only in water proofing the cuticle but also in nestmate recognition. High-density lipophorin (HDLp), isolated from Reticulitermes flavipes plasma by KBr gradient ultracentrifugation, contains 66.2% protein and 33.8% lipids; hydrocarbons constitute its major neutral lipid (20.4% of total lipids). Anti-lipophorin serum was generated in rabbit and its specific association with lipophorin, and not with any other plasma proteins, was verified with Western blotting. Immunoprecipitation also confirmed that this antibody specifically recognizes lipophorin, because all hemolymph hydrocarbons of the termites R. flavipes and R. lucifugus and the cockroach Supella longipalpa, which associate only with lipophorin, were recovered in the immunoprecipitated protein. Cross-reactivity of the antiserum with lipophorin from related species was investigated by double immunodiffusion with 10 termite species in the genera Reticulitermes, Coptotermes, Zootermopsis, and Kalotermes, and with five cockroach species. Involvement of lipophorin in hydrocarbon transport was shown by injecting HDLp antiserum into Zootermopsis nevadensis and then monitoring the de novo biosynthesis of hydrocarbons and their transport to the cuticular surface; the antiserum significantly disrupted hydrocarbon transport. ELISA revealed a gradual increase in the lipophorin titer in successively larger R. flavipes workers, and differences among castes in lipophorin titers were highest between nymphs and first instar larvae.     

Lipid transfer particle mediates the delivery of diacylglycerol from lipophorin to fat body in larval Manduca sexta

This work analyzed the process of lipid storage in fat body of larval Manduca sexta, focusing on the role of lipid transfer particle (LTP). Incubation of fat bodies with [(3)H]diacylglycerol-labeled lipophorin resulted in a significant accumulation of diacylglycerol (DAG) and triacylglycerol (TAG) in the tissue. Transfer of DAG to fat body and its storage as TAG was significantly inhibited (60%) by preincubating the tissue with anti-LTP antibody. Lipid transfer was restored to control values by adding LTP to fat body. Incubation of fat body with dual-labeled DAG lipophorin or its treatment with ammonium chloride showed that neither a membrane-bound lipoprotein lipase nor lipophorin endocytosis is a relevant pathway to transfer or to storage lipids into fat body, respectively. Treatment of fat body with suramin caused a 50% inhibition in [(3)H]DAG transfer from lipophorin. Treatment of [(3)H]DAG-labeled fat body with lipase significantly reduced the amount of [(3)H]DAG associated with the tissue, suggesting that the lipid is still on the external surface of the membrane. Whether this lipid represents irreversibly adsorbed lipophorin or a DAG lipase-sensitive pool is unknown. Nevertheless, these results indicate that the main pathway for DAG transfer from lipophorin to fat body is via LTP and receptor-mediated processes.     

Insect vitellogenin/lipophorin receptors: Molecular structures, role in oogenesis, and regulatory mechanisms

Insect vitellogenin and lipophorin receptors (VgRs/LpRs) belong to the low-density lipoprotein receptor (LDLR) gene superfamily and play a critical role in oocyte development by mediating endocytosis of the major yolk protein precursors Vg and Lp, respectively. Precursor Vg and Lp are synthesized, in the majority of insects, extraovarially in the fat body and are internalized by competent oocytes through membrane-bound receptors (i.e., VgRs and LpRs, respectively). Structural analysis reveals that insect VgRs/LpRs and all other LDLR family receptors share a group of five structural domains: clusters of cysteine-rich repeats constituting the ligand-binding domain (LBD), epidermal growth factor (EGF)-precursor homology domain that mediates the acid-dependent dissociation of ligands, an O-linked sugar domain of unknown function, a transmembrane domain anchoring the receptor in the plasma membrane, and a cytoplasmic domain that mediates the clustering of the receptor into the coated pits. The sequence analysis indicates that insect VgRs harbor two LBDs with five repeats in the first and eight repeats in the second domain as compared to LpRs which have a single 8-repeat LBD. Moreover, the cytoplasmic domain of all insect VgRs contains a LI internalization signal instead of the NPXY motif found in LpRs and in the majority of other LDLR family receptors. The exception is that of Solenopsis invicta VgR, which also contains an NPXY motif in addition to LI signal. Cockroach VgRs still harbor another motif, NPTF, which is also believed to be a functional internalization signal. The expression studies clearly demonstrate that insect VgRs are ovary-bound receptors of the LDLR family as compared to LpRs, which are transcribed in a wide range of tissues including ovary, fat body, midgut, brain, testis, Malpighian tubules, and muscles. VgR/LpR mRNA and the protein were detected in the germarium, suggesting that the genes involved in receptor-endocytotic machinery are specifically expressed long before they are functionally required.     

Role of lipophorin in lipid transport to the insect egg

Lipid accounts for 40% of the dry weight of a mature Manduca sexta egg. Less than 1% of the total egg lipid is derived from de novo synthesis by the follicles. The remaining egg lipid originates in the fat body and is transported to the ovary by lipoproteins. Vitellogenin, the major egg yolk lipoprotein, accounts for 5% of the total egg lipid. The remaining 95% lipid is attributable to the hemolymph lipophorins, adult high density lipophorin (HDLp-A) and low density lipophorin (LDLp). When HDLp-A that is dual labeled with 3H in the diacylglycerol fraction and 35S in the protein moiety is incubated with follicles in vitro, the ratio of 3H:35S in the incubation medium does not vary and is similar to the ratio of the labels that are associated with the follicles. In an accompanying paper (Kawooya, J. K., Osir, E. O., and Law, J. H. (1988) J. Biol. Chem. 263, 8740-8747), we show that HDLp-A is sequestered by the follicles without subsequent hydrolysis of its apoproteins. These results, together with those presented in this paper, support our conclusion that HDLp-A is not recycled back into the hemolymph after it is internalized by the follicles and, therefore, does not function as a reusable lipid shuttle between the fat body and the ovary. When follicles are incubated with dual labeled LDLp, the diacylglycerol component of the particle is internalized by the follicles without concomitant endocytosis of its associated apoproteins. This LDLp particle is the major vehicle by which lipid is delivered to the ovary.     

Characterization of lipophorin binding to the fat body of Rhodnius prolixus

In insects, lipids are transported by a hemolymphatic lipoprotein, lipophorin. The binding of lipophorin to the fat body of the hematophagous insect Rhodnius prolixus was characterized in a fat body membrane preparation, obtained from adult females. For the binding assay, purified lipophorin was radiolabelled in the protein moiety (125I-HDLp), and it was shown that iodination did not affect the affinity of the membrane preparation for lipophorin. Under incubation conditions used, lipophorin binding to membranes achieved equilibrium after 40-60 min, but this time was longer when a low concentration of lipophorin was present in the medium. The capacity of the fat body membrane preparation to bind lipophorin was abolished when membranes were pre-treated with trypsin, and it was also affected by heat. When 125I-HDLp was incubated with increasing concentrations of membrane protein, corresponding increases in binding were observed. Lipophorin binding was sensitive to pH, and it was maximal between pH 6.0 and 7.0. The specific binding of lipophorin to the fat body membrane preparation was a saturable process, with a Kd of 2.1 +/- 0.4 x 10(-7)M and a maximal binding capacity of 289 +/- 88 ng lipophorin/microgram of membrane protein. Binding to the fat body membranes did not depend on calcium, but it was affected by ionic strength, being totally inhibited at high salt concentrations. Suramin also interfered with lipophorin binding and it was abolished in the presence of 2 mM suramin, but at concentrations of 0.05 and 0.1 mM it seemed to increase binding activity slightly. Fat body membrane preparation from Rhodnius prolixus was able to bind lipophorin from Manduca sexta larvae.