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 studies on lipophorin, an insect lipoprotein

An insect high density lipoprotein, lipophorin, can be rapidly isolated from larval Manduca sexta (tobacco hornworm) hemolymph by single vertical spin density gradient ultracentrifugation. The two apolipoproteins (Mr = 245,000 and 78,000; designated apoLp-I and apoLp-II, respectively) were readily dissociated and separated in 6 M guanidine HCl by gel permeation chromatography. ApoLp-I and apoLp-II showed no immunological cross-reactivity on electrophoretic blots of sodium dodecyl sulfate-polyacrylamide gels. ApoLp-I and apoLp-II from lipophorin of adult M. sexta behaved identically to their larval counterparts. Amino acid compositions of larval apoLp-I and apoLp-II were similar except with respect to tryptophan and cysteine; apoLp-I contained 32 residues/mol of tryptophan (1.5 mol%) and 22 residues/mol (1.1 mol%) of cysteine; apoLp-II contained 2 residues/mol of tryptophan (0.2 mol%) and 14 residues/mol of cysteine (2.1 mol%). In double immunodiffusion tests, antiserum against apoLp-I or whole lipophorin strongly precipitated lipophorin, while antiserum against apoLp-II caused only minor precipitation. This indicates relatively greater exposure of apoLp-I to the aqueous environment.     

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.     

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.     

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.     

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.     

Characterization of Colorado potato beetle lipophorin: A hydrocarbon-rich diacylgycerol-poor lipophorin

• 1.1. The lipid composition of lipophorin from the Colorado potato beetle, Leptinotarsa decemlineata Say, was analyzed.
• 2.2. This insect lipophorin contains 44% lipid and is characterized by large amounts of hydrocarbons and small amounts of diacylglycerol.
• 3.3. This is the first observation of a diacylglycerol-poor insect lipophorin in haemolymph.
• 4.4. Since the main energy source for flight in the Colorado potato beetle is proline, the low diacylglycerol content in lipophorin must be related to its peculiar flight metabolism.
• 5.5. This lipophorin, however, can still take up appreciable amounts of diacylglycerol from the locust fat body. Hydrocarbon uptake by this lipophorin was also demonstrated.
• 6.6. The main function of this lipophorin therefore seems to be transport of hydrocarbons from oenocytes to the cuticle.

     

Trehalose, the insect blood sugar, inhibits loading of diacylglycerol by lipophorin from the fat body in locusts

Trehalose, the insect blood sugar, was found to inhibit diacylglycerol uptake by lipophorin from the fat body in vitro. Trehalose inhibited diacylglycerol uptake by about 40%-50% at various physiological concentrations. This suggests that trehalose may play a dual role in the hemolymph, i.e. serving as the insect's fuel and as a regulator in lipid transport.     

Blue lipophorin from Podisus maculiventris

A chromoprotein responsible for the blue coloration of the hemolymph in the spined soldier bug, Podisus maculiventris (Say), was isolated and identified as lipophorin. With the exception of its blue color the lipoprotein shares similar molecular characteristics with the hemolymph lipophorins of other Hemipterans and insects of several different orders. Its ability to carry a blue chromophore, biliverdin IX γ, adds a new feature to this multifunctional lipoprotein. © 1992 Wiley-Liss, Inc.     

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.     

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.     

Lipid thermotropic transitions in Triatoma infestans lipophorin

The structure and lipid thermotropic transitions of highly purified lipophorin of Triatoma infestans were examined by several techniques: steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), cis-parinaric acid (cis-PnA) and trans-parinaric acid (trans-PnA), light scattering fluorescence energy transfer between the lipophorin tryptophan residues and the bound chromophores, DPH, trans-parinaric acid cis-parinaric acid, gel electrophoresis, and gel filtration. Fluorescence polarization of PnAs and DPH revealed a reversible lipid thermotropic transition in intact lipophorin at about 20 degrees C and 18 degrees C, respectively. In lipophorin, lipid dispersion fluorescence polarization of DPH detected a lipid transition approximately at 20 degrees C, while trans-PnA showed a gel phase formation at a temperature below 30 degrees C. Similar experiments in which trans-PnA was incorporated into diacylglycerols and phospholipids extracted from the lipophorin revealed gel phase formation below 30 degrees C and 24 degrees C, respectively. Light scattering measurements showed that lipophorin particles aggregate irreversibly at 45 degrees C, increasing the molecular weight, as determined by gel filtration on Sephacryl S-300, from 740,000 to values larger than 1,500,000. The particle aggregation did not change the physical properties of the lipophorin studied by fluorescence polarization, indicating that the aggregation is apparently a non-denaturing process. Energy transfer between the lipophorin tryptophans and the bound chromophores cis-PnA, trans-PnA, and DPA revealed a different location of the fluorescent probes within the lipophorin. Temperature-dependence on the energy transfer efficiency for all probes confirmed a change in the ordering of the lipophorin lipids at 24 degrees C.     

昆虫受精囊的结构与功能

昆虫受精囊是在雌雄交配之后,作为雌性贮存、释放精子和使卵受精的器官,其结构、功能及活动调节对保持精子活性和卵子受精有重要的影响。该文对昆虫受精囊结构、功能及其活动调节的研究进展做一介绍,以期为相关研究提供有益的资料。     

Structure of Colorado potato beetle lipophorin: differential scanning calorimetric and small-angle X-ray scattering studies

The structure of lipophorin, isolated from hemolymph of the Colorado potato beetle, was investigated by differential scanning calorimetry (DSC) and small-angle X-ray scattering. The DSC heating curves of intact lipophorin showed endothermic peaks that were similar to peaks obtained with the hydrocarbon fraction isolated from this lipophorin. The observed peaks correlated with the transition of the hydrocarbons from an ordered into a more disordered state. Changes in structure of the lipophorin particles with increasing temperature were also observed by small-angle X-ray scattering studies. The structural organization of lipophorin was further elucidated by simulation analysis, using a three-layered symmetrical sphere as a model. These studies revealed that lipophorin from the Colorado potato beetle is a sphere with a maximum diameter of 175 A. The sphere is composed of three radially symmetrical layers of different electron densities. The outer layer (37.5-39.5 A in thickness) is composed of phospholipid, apolipophorin I, and part of apolipophorin II. The middle layer (5-10 A) contains diacylglycerol, the rest of apolipophorin II, and probably beta-carotene. The core of the particle (40-45 A) only contains hydrocarbons. This structure differs from another model, previously proposed for cockroach and locust lipophorins [Katagiri, C., Sato, M., & Tanaka N. (1987) J. Biol. Chem. 262, 15857-15861], in the small size of the middle layer. The volume of the middle layer correlated well with the low diacylglycerol content of this lipophorin.     

Larval Musca domestica lipophorin biosynthesis

• 1.1. Larval Musca domestica lipophorin biosynthesis was studied in vitro.
• 2.2. The newly synthesized lipophorin has a density a little lower than the circulating lipophorin after 1 hr of incubation. After 3 hr of incubation the fat body cells transfer lipids to the lipophorin that attains the density of circulating lipophorin.
• 3.3. The lipophorin synthesized in vitro is identical to circulating lipophorin in density and in electrophoretical behavior.
• 4.4. However these two molecules must have differences since the circulating lipophorin transfers lipids to fat body cells while the synthesized in vitro does not.
• 5.5. The biosynthesis of Musca lipophorin shows differences with the Manduca sexta lipophorin biosynthesis.

     

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.