Delipidation of insect lipoprotein,lipophorin, affects its binding to the lipophorin receptor,LpR: Implications for the role of LpR-mediated endocytosis

The insect lipophorin receptor (LpR), an LDL receptor (LDLR) homologue that is expressed during restricted periods of insect development, binds and endocytoses high-density lipophorin (HDLp). However, in contrast to LDL, HDLp is not lysosomally degraded, but recycled in a transferrin-like manner, leaving a function of receptor-mediated uptake of HDLp to be uncovered. Since a hallmark of circulatory HDLp is its ability to function as a reusable shuttle that selectively loads and unloads lipids at target tissues without being endocytosed or degraded, circulatory HDLp can exist in several forms with respect to lipid loading. To investigate whether lipid content of the lipoprotein affects binding and subsequent endocytosis by LpR, HDLp was partially delipidated in vitro by incubation with α-cyclodextrin, yielding a particle of buoyant density 1.17 g/mL (HDLp-1.17). Binding experiments demonstrated that LpR bound HDLp-1.17 with a substantially higher affinity than HDLp both in LpR-transfected Chinese hamster ovary (CHO) cells and isolated insect fat body tissue endogenously expressing LpR. Similar to HDLp, HDLp-1.17 was targeted to the endocytic recycling compartment after endocytosis in CHO(LpR) cells. The complex of HDLp-1.17 and LpR appeared to be resistant to endosomal pH, as was recently demonstrated for the LpR–HDLp complex, corroborating that HDLp-1.17 is recycled similar to HDLp. This conclusion was further supported by the observation of a significant decrease with time of HDLp-1.17-containing vesicles after endocytosis of HDLp-1.17 in LpR-expressing insect fat body tissue. Collectively, our results indicate that LpR favors the binding and subsequent endocytosis of HDLp-1.17 over HDLp, suggesting a physiological role for LpR in selective endocytosis of relatively lipid-unloaded HDLp particles, while lipid reloading during their intracellular itinerary might result in decreased affinity for LpR and thus allows recycling.     

Structure of apoproteins in insect lipophorin

The two polypeptide chains of cockroach and locust lipophorins were separated and their amino acid compositions were determined. Circular dichroic spectra of the lipophorins and apolipophorin from 190 to 250 nm showed a single trough at 218 nm and a peak at 194 nm. Infrared spectra of the lipophorins in D2O showed a strong peak at 1625 cm-1 and a weak shoulder at 1693 cm-1 corresponding to v (pi, 0) and nu (0, pi) of antiparallel pleated sheet. The resonance frequency splitting delta nu = nu (0, pi) -nu (pi, 0) was 68 cm-1, which was larger than that of ordinary globular proteins containing antiparallel pleated sheet. From circular dichroic and infrared spectra it was concluded that lipophorins contained polypeptides rich in antiparallel pleated sheet with longer unbroken extensions than the case for ordinary globular proteins. Partial proteolytic digestion study of lipophorins with trypsin, chymotrypsin, and subtilisin showed that the larger apolipophorin (AL1) was exposed to the surface of the particle and the smaller apolipophorin (AL2) lay protected from the attack of the enzymes. Crosslinked products between AL1 and AL2 were readily obtained when dimethylsuberimidate or dimethyladipimidate was added to the lipophorin solution, without giving lipophorin dimers, suggesting that the two chains were located within 11 A from each other. Such structural features of insect lipoprotein were compared with other insect lipophorins and the human serum low-density lipoprotein (LDL). Similarities between lipophorins and LDL were found in the molecular weight, amino acid compositions, and the secondary structure of major apoproteins.     

Small-angle x-ray scattering study of insect lipophorin

The structure of lipophorin in insect blood (hemolymph) was investigated by a small-angle x-ray scattering method over the temperature range 0-45 degrees C. The small-angle x-ray scattering profile of lipophorin exhibited a symmetrical sphere with heterogeneous internal electron density. Cockroach and locust lipophorins, which contain hydrocarbons, demonstrated centrosymmetrical distribution of electron density inside the particles. A previous study suggested that the hydrocarbon-rich region is located in the core of lipophorin particle (Katagiri, C., Kimura, J., and Murase, N. (1985) J. Biol. Chem. 260, 13490-13495). Distance distribution functions, P (r), calculated for a simulated three-layer model (electron-rich shell, middle layer, and electron-deficient core) with radial electron density distribution, show good agreement with those observed experimentally for cockroach and locust lipophorins. The dimensions and electron density obtained for the middle layer reveal that this layer is occupied mainly by diacylglycerol and apolipophorin II. Thus, the present study together with previous reports strongly suggest that insect lipophorin is composed of centrosymmetrical three layers; an outer shell with apolipophorin I and phospholipid, a middle layer with diacylglycerol and apolipophorin II, and a core with hydrocarbons.     

Structural and RNAi characterization of the German cockroach lipophorin receptor,and the evolutionary relationships of lipoprotein receptors

Background  

Lipophorin receptors (LpRs) have been described in a number of insects, but functional studies have been reported only in locusts and mosquitoes. The aim of the present work was to characterize the LpR of the cockroach Blattella germanica, not only molecularly but also functionally using RNAi techniques, and to place LpRs in a phylogenetical context among lipoprotein receptors.     

Structural studies of lipophorin in insect blood by differential scanning calorimetry and 13C nuclear magnetic relaxation measurements. Location of hydrocarbons

The possible structure of lipophorin in insect blood (hemolymph) was investigated by differential scanning calorimetry (DSC) and 13C nuclear magnetic relaxation studies. The DSC heating curves of intact lipophorins showed endothermic peaks between -3 and 40 degrees C for lipophorins which contain hydrocarbons, whereas no such peaks were observed for lipophorins which do not contain this lipid. Hydrocarbon fractions isolated from the lipophorins showed endothermic peaks similar to those obtained from intact lipophorin in terms of the transition temperatures, the shapes, and the enthalpy changes. 13C spin lattice relaxation times of the (CH2)n resonance of hydrocarbons of intact lipophorin were measured as a function of temperature and revealed that the motions of hydrocarbon chains changed coincidentally with the onset and offset of phase transition. These data suggest the presence of a hydrocarbon-rich region within the lipophorin particles.     

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.     

Human apolipoprotein E N-terminal domain displacement of apolipophorin III from insect low density lipophorin creates a receptor-competent hybrid lipoprotein

The surface of Manduca sexta low density lipophorin (LDLp) particles was employed as a template to examine the relative lipid binding affinity of the 22 kDa receptor binding domain (residues 1–183) of human apolipoprotein E3 (apo E3). Isolated LDLp was incubated with exogenous apolipoprotein and, following re-isolation by density gradient ultracentrifugation, particle apolipoprotein content was determined. Incubation of recombinant human apo E3(1–183) with LDLp resulted in a saturable displacement of apolipophorin III (apo Lp-III) from the particle surface, creating a hybrid apo E3(1–183)-LDLp. Although subsequent incubation with excess exogenous apo Lp-III failed to reverse the process, human apolipoprotein A-I (apo A-I) effectively displaced apo E3(1–183) from the particle surface. We conclude that human apo E N-terminal domain possesses a higher intrinsic lipid binding affinity than apo Lp-III but has a lower affinity than human apo A-I. The apo E3(1–183)-LDLp hybrid was competent to bind to the low density lipoprotein receptor on cultured fibroblasts. The system described is useful for characterizing the relative lipid binding affinities of wild type and mutant exchangeable apolipoproteins and evaluation of their biological properties when associated with the surface of a spherical lipoprotein.     

Transport of hydrocarbons by the lipophorin of insect hemolymph

When [I-¹⁴C]acetate was injected into the American cockroach, the labeled acetate was incorporated preferentially into the hydrocarbon fraction and, subsequently, the labeled hydrocarbon was released into the hemolymph where it was associated with the lipophorin (formerly called diacylglycerol-carrying lipoprotein). The label was traced to the three hydrocarbons, n-pentacosane, 3-methylpentacosane and 6,9-heptacosadiene that had been shown previously to be associated with the lipophorin. The specific capacity of lipophorin to accept hydrocarbons from oenocytes, which are believed to be the site of hydrocarbon synthesis, was demonstrated in vitro, and the uptake of hydrocarbon by lipophorin was retarded by respiratory poisoning. When lipophorin containing ¹⁴C-labeled hydrocarbon was injected into the hemocoele of cockroach, the labeled hydrocarbon soon appeared at the cuticular surface where it was deposited specifically. The above observations and our previous data support the postulate that insect lipophorin serves as the true carrier molecule for the transport of hydrocarbons from the site of synthesis (oenocyte) to the site of deposition (cuticle), in addition to its function of transporting diacylglycerol and cholesterol from the fat body and intestine.     

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.     

Turnover of protein and diacylglycerol components of lipophorin in insect haemolymph

Lipophorin, radiolabelled in the protein or diacylglycerol moiety, was purified from adult locusts injected previously with [¹⁴C]protein hydrolysate or sodium[1-¹⁴C]palmitate. The radiolabelled lipophorin was injected into adult male locusts and haemolymph samples taken periodically to determine the rate of disappearance of radioactivity from the haemolymph. Lipophorin was also purified from locusts that had been injected four days previously with ([¹⁴C]protein)-lipophorin to demonstrate that the radioactivity observed in the haemolymph at this time is due to radiolabelled lipophorin. The results indicate that the half-life of the protein component of lipophorin in resting insects is about 5–6 days whereas that of the diacylglycerol component is only about 2–3 hr.The results are consistent with the hypothesis that lipophorin functions as a “reusable shuttle” to transport a variety of lipid classes between sites of absorption, storage and utilisation.     

Structural characteristics of tenecin 3, an insect antifungal protein

Tenecin 3, an antifungal protein, previously isolated from the insect Tenebrio molitor, inhibits growth of the fungus Candida albicans. However, the antifungal mechanism and functions of tenecin 3 remain unknown. As an initial step to study the mechanism and functions, physical and structural properties of tenecin 3 were examined by circular dichroism (CD) analysis and 2D nuclear overhauser effect spectroscopy. These analyses suggest that tenecin 3 has a propensity of random structure with very loose turn-like elements. The CD results also indicate that this random structural propensity is not significantly affected by temperature, pH, and by the presence of organic solvents or sodium dodecyl sulfate (SDS) micelles. However, the hydrodynamic studies suggest that tenecin 3 is not in extended form in spite of its random structural feature.     

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.     

Treatment of low density lipophorin with lipoprotein lipase: diacylglycerol content has no effect on dissociation of apolipophorin III from low-density lipophorin.

The mechanism of the conversion of low-density lipophorin (LDLp) to high-density lipophorin (HDLp) in long-distance flight insects was investigated using a lipoprotein lipase from a bacterium, Alcaligenes sp. Diacylglycerol of LDLp was steadily hydrolyzed in vitro by the lipase, resulting in a 90% loss of diacylglycerol from LDLp during incubation. The "lipase-treated LDLp" thus obtained still contained associated apolipophorin-III (apoLp-III). These data suggest that the dissociation of apoLp-III is independent of the depletion of diacylglycerol from LDLp, and that the decrease in particle diameter caused by the depletion of diacylglycerol does not force the dissociation of apoLp-III from the lipophorin particle. Some physico-chemical properties of the lipase-treated LDLp were measured.     

Genetic studies of an apoA-I lipoprotein variant

Summary Chromosomal Q polymorphism was studied in 116 Turkmen, aboriginals of the Kara-Kum desert of Central Asia. Propylquinacrine mustard was used as fluorochrome. Of the 116 subjects aged 16–20 years, 109 (94.0%) were found to have Q-polymorphic variants, while seven (6.0%) showed complete absence of Q bands with fluorescence levels 4 and 5. There was a total of 351 polymorphic Q bands, 0–7 per individual, with a mean of 3.0 in the population. No differences between sexes were observed in the frequency of Q bands. The observed homo- and heteromorph frequencies proved to be in complete agreement with those predicted by the law of Hardy-Weinberg. Chromosome 3 with pericentric inversion of the Q-heterochromatin band was found in two (1.7%) of the 116 subjects.The following questions were examined: (1) the possible selective value of chromosomal Q-heterochromatin material in the adaptation of human populations to extreme environmental factors, in particular to the desert climate; (2) intracial heterogeneity in Europoids of Eurasia; (3) the taxonomic value of Q polymorphism in ethnic anthropology.     

Isolation and fluorescence studies on a lipophorin from the weevil diaprepes abbreviatus

Lipophorin was isolated from larvae of a root weevil, Diaprepes abbreviatus (Coleoptera: Curculionidae), using density gradient ultracentrifugation. D. abbreviatus lipophorin contained two apoproteins, apolipophorin-I (M_r = 226,000) and apolipophorin-II (M_r = 72,100) and had a density of 1.08. Relative to other larval lipophorins, D. abbreviatus lipophorin contained little cysteine (determined as cysteic acid) and methionine. Fluorescence spectroscopy of intrinsic tyrosine and tryptophan residues excited at 290 nm revealed a single broad emission peak at 330 nm. Upon denaturing and delipidating lipophorin in guanidine HCl, this peak resolved into two peaks with maxima at 305 and 350 nm. Excitation spectra suggested that the two peaks were due to tyrosine and tryptophan, respectively. Fluorescence quenching agents, iodide and acrylamide, were used to determine accessibility of tyrosine and tryptophan residues to the aqueous environment. Iodide, a polar quenching agent, did not quench fluorescent emission from native lipophorin; quenching by iodide increased to moderate levels when lipophorin was denatured in guanidine HCl. Acrylamide quenched the fluorescence of native lipophorin moderately and very efficiently quenched fluorescence of denatured lipophorin. No difference was observed between fluorescence quenching of denatured vs. denatured and delipidated lipophorin by either iodide or acrylamide.     

Structural study of the asparagine-linked oligosaccharides of lipophorin in locusts

The complete structure of oligosaccharides from locust lipophorin was studied. The asparagine-linked oligosaccharides were first liberated from the protein moiety of lipophorin by digestion with almond glycopeptidase (N-oligosaccharide glycopeptidase, EC 3.5.1.52). Two major oligosaccharides (E and F), separated by subsequent thin-layer chromatography, were analyzed by methylation analysis and 1H-NMR. Based on the experimental data, the whole structure of oligosaccharide E was identified as Man alpha 1----2Man alpha 1----6(Man alpha 1----2Man alpha 1----3) Man alpha 1----6(Man alpha 1----2Man alpha 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc. The data also revealed that oligosaccharide F is identical with oligosaccharide E in the structure, except for one glucose residue that is linked to the nonreducing terminal Man alpha 1----2 residue.     

Fluorescence studies on the lipoprotein complex of the fatty acid synthetase from the insect Ceratitis capitata

The fatty acid synthetase complex from the insect Ceratitis capitata forms a stable lipoprotein complex. The intrinsic fluorescence of the complex was studied by observing the emission spectra with different excitation wavelengths, both in the native complex and after temperature with sodium cholate and sodium dodecyl sulfate. The excitation spectrum of the native form also was recorded. The fluorescence behavior of the native enzyme showed two families of tryptophan residues. Cholate influenced the fluorescence, suggesting that phospholipids are the conformational support at this level. The two families of fluorescing tryptophan residues were similarly accessible to quenching by acrylamide. Thermal changes in the fluorescence characteristics were observed; warming caused a decrease in the quantum yield as well as a red shift in the emission maximum. The high fluorescence remaining after the thermal transition suggested that the lipid-protein interaction was affected but maintained shielding of the fluorophore by the lipids. Fluorescent probe molecules 1,6-diphenyl-hexa-1,3,5-triene (DPH) and dansylphosphatidylethanolamine (DPE) also were used. DPH uptake was temperature dependent, with a middle point consistent with the thermal conformation transition, indicating that internal lipids are nonrandomly distributed within the complex. DPE uptake did not reach the saturation of the complex, suggesting that its solubilization sites would be located on the lipoprotein surface.     

Structural and functional heterogeneity in an insect muscle.

Singing muscles of the katydid, Neoconocephalus robustus (Insecta, Tettigoniidae) are neurogenic, yet perform at contraction-relaxation frequencies as high as 212 Hz (Josephson and Halverson, '71). The mechanical and electrical responses of different bands of one of these muscles (the dorsal longitudinal muscle, DLM) has been examined with respect to ultrastructural features of each part which may be related to muscle performance. The DLM is composed of three bands and is innervated by four motoneurones. The cell bodies of three of these motoneurones occur ipsilaterally in the prothroracic ganglion; the cell body of the other motoneurone is contralateral in the mesothoracic ganglion. Three of the motoneurones (as yet unidentified fast axons) initiate extraordinarily fast twitches (rise time equal 7.3 msec, half duration equals 14.3 msec, 25 C), the fourth (an unidentified slower axon) evokes twitches which are considerably slower (rise time equals 18.9 msec, half duration equals 5.10 msec). Whereas the ventral and medial bands of the muscle are innervated only by fast axons (some fibers of the medial band are doubly innervated), the dorsal band is innervated by both a fast axon and the slower axon. A few fibers of the dorsal band are doubly innervated. The structure of fibers from the ventral and medial bands is very similar, with short sarcomeres (4.0 and 4.3 mum, respectively) and thin strap-like myofibrils delineated by well-developed sarcoplasmic reticulum (SR). Twenty-four percent of the volume of ventral band fibers is SR and the diffusion distance from SR to the center of the adjacent myofibril averages 0.083 mum. Twenty percent of the medial band fiber volume is SR, with a diffusion distance of 0.118 mum. Ventral and medial band fibers contain about 40% mitochondria, and 33% myofibrils. The dorsal band fibers have longer sarcomeres (9.5 mum), and only 10% of the fiber volume is SR. The muscle fibrils of the dorsal band are larger and consequently the diffusion distance is greater (0.227 mum) than in the ventral and medial bands. Mitochondria comprise 23% of the volume of dorsal band fibers. Most dorsal band mitochondria are aggregated into distinct clumps. Although some dorsal band fibers are innervated by a fast axon and some by the slower axon, the dorsal band fibers are structurally homogeneous, suggesting that neurotrophic effects are not important in maintaining the structure of dorsal band fibers. The mechanical-electrical performance and ultrastructure of the ventral and medial bands suggest their roll as fast, metabolically active but weak muscles, used in singing; the dorsal band as a slower but stronger muscle, perhaps involved in postural movements of the wing during singing.