Mónica Cunningham

Lipid circulation in spiders. Transport of phospholipids, free acids and triacylglycerols as the major lipid classes by a high-density lipoprotein fraction isolated from plasma of Polybetes pythagoricus

Abstract A high-density lipoprotein (HDL) fraction was isolated from the hemolymphatic plasma of the spider Polybetes pythagoricus by density-gradient ultracentrifugation. Hydrated density (1.13 g/ml), electrophoretic mobility (SDS—PAGE) of apoproteins and lipid classes composition were determined. Lipids were identified by HP-TLC and auxiliary techniques; they were quantified by TLC-FID. The protein moiety is composed of two main apoproteins (250 and 76 kDa, respectively) and several polypeptides of low molecular weight. It resembles the apoliphorins of insects and some other arachnids. The lipid composition differs from most lipophorins. Phospholipids amount to more than 60% of total lipids, while diacylglycerols (2.4%) are supplanted by triacylglycerols (16.5%) as the main circulating energetic lipids.

Characterization of lipoprotein fractions with high content of hemocyanin in the hemolymphatic plasma of Polybetes pythagoricus

In order to evaluate the role of hemocyanin in the hemolymphatic transport of lipids, two hemocyanin-containing lipoprotein fractions, a very-high-density lipoprotein (VHDL) and a high-density lipoprotein (HDL) isolated from plasma of the spider Polybetes pythagoricus, were characterized. VHDL concentration was found to be twofold when compared to that of HDL, and both of them transport 70% of total circulating lipids. Their lipid moieties were mainly composed of phospholipids (65% and 55% in VHDL and HDL, respectively), triacylglycerols, and free fatty acids in a minor degree, and diacylglycerols, cholesterol, and hydrocarbons even in lesser amounts. Although hemocyanin was the predominant apoprotein, two other proteins were also present in minor amounts, especially in VHDL. The two lipoproteins were analyzed by gel filtration chromatography and separated into three subfractions. Two of them were composed of the heptamer (500 kDa) and the monomer (70 kDa) of hemocyanin, respectively, and different proportions of binding lipids. Free fatty acids (37% of total subfraction lipids) were the main lipid classes linked to the heptamer, whereas triacylglycerols (24%) and free fatty acids (22%) predominated among lipids associated with the hemocyanin monomer. The third subfraction contained the hexamer of hemocyanin (420 kDa), two minor polypeptides, and 65% of the VHDL total lipids. This subfraction is the principal means of transport of phosphatidylcholine and triacylglycerols in VHDL. It is here suggested that hemocyanin, as a single apoprotein or associated with other polypeptides, is responsible for the transport of the majority of plasma lipids in this invertebrate.

Embryo lipoproteins and yolk lipovitellin consumption during embryogenesis in Macrobrachium borellii (Crustacea: Palaemonidae)

The prawn Macrobrachium borellii has lecithotrophic eggs with highly-abbreviated development. The major yolk component is lipovitellin (LV), a lipoprotein with 30% lipids (by weight). LV consumption during embryogenesis was followed by ELISA and Western blot analysis using an anti-LV polyclonal antibody. No cross-reacting proteins were observed and LV-like lipoproteins were strongly recognized by the antibody in hemolymph (vitellogenin), yolk (LV) and embryos (LVe), as determined by Western Blot analysis. LV decreased significantly along development from 9.4 to 1.1 microg/mg egg. Consumption rate of LV was slow in early embryogenesis, followed by a rapid utilization in late embryonic stages. Significant LVe amounts were still present at hatching. LV apolipoproteins were selectively degraded during embryo development, being the highest molecular weight subunit the most affected. Comparison among in vitro, in vivo and theoretical proteolysis suggested that trypsin may be involved in LV degradation during late embryogenesis. Embryo lipoprotein (HDLe) synthesis was first detected at stage 6. HDLe shared the same density, MW and subunit composition as adult hemolymph HDL(1) and did not cross-react with LV-like lipoproteins. Though expressed at low concentration, it fulfilled embryo needs for lipid transport among organs.

First insight into the lipid uptake, storage and mobilization in arachnids: role of midgut diverticula and lipoproteins.

The importance of midgut diverticula (M-diverticula) and hemolymph lipoproteins in the lipid homeostasis of Polybetes phythagoricus was studied. Radioactivity distribution in tissues and hemolymph was analyzed either after feeding or injecting [1-(14)C]-palmitate. In both experiments, radioactivity was mostly taken up by M-diverticula that synthesized diacylglycerols, triacylglycerols and phospholipids in a ratio close to its lipid class composition. M-diverticula total lipids represent 8.08% (by wt), mostly triacylglycerols (74%) and phosphatidylcholine (13%). Major fatty acids were (in decreasing order of abundance) 18:1n-9, 18:2n-6, 16:0, 16:1n-7, 18:0, 18:3n-3. Spider hemocyanin-containing lipoprotein (VHDL) transported 83% of the circulating label at short incubation times. After 24h, VHDL and HDL-1 (comparable to insect lipophorin) were found to be involved in the lipid uptake and release from M-diverticula, HDL-2 playing a negligible role. Lipoproteins labelled lipid changed with time, phospholipids becoming the main circulating lipid after 24h. These results indicate that arachnid M-diverticula play a central role in lipid synthesis, storage and movilization, analogous to insect fat body or crustacean midgut gland. The relative contribution of HDL-1 and VHDL to lipid dynamics indicated that, unlike insects, spider VHDL significantly contributes to the lipid exchange between M-diverticula and hemolymph.

CHARACTERIZATION OF LIPOPROTEINS ISOLATED FROM THE HEMOLYMPH OF THE SPIDER LATRODECTUS MIRABILIS (ARANEAE, THERIDIIDAE)

Abstract Two high density lipoprotein fractions (HDL1 and HDL2) were isolated from the hemolymphatic plasma of the spider Latrodectus mirabilis (Holmberg 1876). For each, the hydrated density, the electrophoresis mobility of the apoproteins, and the lipid classes composition were determined. The HDL1 fraction carried 80% of the total plasma lipids, which were predominantly composed of phospholipids, free fatty acids, and triacylglycerols. The apoprotein composition of this fraction showed two main bands of 90 and 103 kDa. The HDL2 fraction was composed primarily of phospholipids, free fatty acids and cholesterol. This fraction contained hemocyanin as the principal apoprotein. When the HDL2 fraction was separated into three subfractions, all of them contained hemocyanin, with the main subfraction containing the hexameric form of the respiratory pigment. With regard to triacylglycerol transport, lipid and apoprotein compositions and hemocyanin role in the lipid transport, these lipoproteins (HDL1, HDL2) show similarities and differences when compared to the two spider species already studied.

Lipid binding capacity of spider hemocyanin

The spider hemocyanin capacity to bind different lipid classes was evaluated by measuring some binding kinetic parameters. A very high lipoprotein (VHDL) which contains hemocyanin, was isolated from Polybetes pythagoricus hemolymph plasma and delipidated. Hemocyanin was bound separately to labelled palmitic acid, phosphatidylcholine, cholesterol, and triolein resulting in several artificial lipoprotein structures. It was possible to corroborate in vitro the lipid-hemocyanin interactions which had been previously observed and, consequently, the apolipoprotein role played by the respiratory pigment of spiders. Lipoproteins were analysed by gel filtration chromatography, and three subfractions with different hemocyanin structures were obtained. The four lipid classes were only bound to the hexameric structure (420 Kda), possibly to low polarity sites. Upon radioactivity measurements of the protein-associated lipids, maximal binding ratios (Mr), dissociation constants (Kd), and the maximal binding effectiveness at low lipid concentrations (Eo) were calculated. Lipid/protein ratios were increased proportionally to each available lipid concentration, following a hyperbolic binding model. Values of saturation, affinity, and maximal binding efficiency to hemocyanin were found to be different for each lipid class assayed. The highest lipid/protein ratio (41.5) was obtained with the free fatty acid and the lowest (7.2) with triolein. Phosphatidylcholine and cholesterol showed the highest relative affinities for hemocyanin (Kd = 63 x 10(-5) M and 74 x 10(-5) M, respectively). Phosphatidylcholine at low concentrations, similar to the physiological ones, presented the highest Eo value. Maximal lipid/protein ratios reached in vitro, were greater than those in P. pythagoricus VHDL, pointing out that hemocyanin could play the apolipoprotein role even under physiological conditions with a very high plasma lipid concentration. J. Exp. Zool. 284:368-373, 1999.

Energy sources from the eggs of the wolf spider Schizocosa malitiosa: isolation and characterization of lipovitellins.

In oviparous species, proteins and lipids found in the vitellus form the lipoproteins called lipovitellins that are the major source of energy for the development, growth, and survival of the embryo. The energy resources provided by the lipovitellins have not yet been investigated in the Order Araneae. Using the wolf spider Schizocosa malitiosa (Lycosidae) as an experimental model, we identified and characterized the lipovitellins present in the cytosol, focusing on the energetic contribution of those lipoprotein particles in the vitellus. Two lipovitellins (LV) named SmLV1 and SmLV2 were isolated. SmLV1 is a high-density lipoprotein with 67% lipid and 3.6% carbohydrate, and SmLV2 is a very high-density lipoprotein with 9% lipid and 8.8% carbohydrate. Through electrophoresis in native conditions we observed that SmLV1 has a molecular mass of 559 kDa composed of three apolipoproteins of 116, 87, and 42 kDa, respectively. SmLV2 comprised several proteins composed of different proportions of the same subunits (135, 126, 109, and 70 kDa). The principal lipids of these lipovitellins are sphingomyelin + lysophosphatidylcholine, esterified sterols, and phosphatidylcholine. Lipovitellin-free cytosol contains abundant phospatidylcholine and triacylglyceride related to the yolk nuclei (the vitellus organizing center). The principal fatty acids of SmLV1 and SmLV2 are 18:2 n-6, 18:1 n-9, and 16:0. Spectrophotometry detected no pigments in either the lipovitellins or the cytosol. The egg caloric content was 92 cal/g, at proportions of 59.8% protein, 20.1% carbohydrate, and 19.9% lipid. SmLV1 and SmLV2 provided 19.5% and 17.1% of the calories, respectively. Both lipovitellins contribute mainly with proteins (15.8-18%), with the input of carbohydrates and lipids being lower than 1.3%.

Isolation and characterization of two vitellins from eggs of the spider Polybetes pythagoricus (Araneae: Sparassidae).

Despite vitellins being essential yolk proteins, their presence in spiders remains almost unknown. Two vitellins from the spider Polybetes pythagoricus, named LV1 and LV2, were isolated and their size, shape, lipids, fatty acids, proteins and carbohydrates moieties were determined. LV1 has a density similar to that of HDL with 49.3% lipids, and LV2 has a density similar to that of VHDL with 9.7% lipids. The major neutral lipid present in both vitellins was found to be esterified cholesterol, 16% for LV1 and 24% for LV2. The major fatty acid was 18:1n-9 in LV1 and LV2. Results from native PAGE showed a lipoprotein of 550 kDa for LV1 and three lipoproteins of 571, 400 and 257 kDa for LV2. SDS-PAGE evidenced two major apolipoproteins of 64 and 25 kDa in LV1. The three lipoproteins of LV2 were electroeluted and analyzed by SDS-PAGE, showing different proportions of the same apolipoproteins (181, 67 and 60 kDa). LVs were analyzed by spectrophotometry, immunochemical and electron microscopy, showing that the respiratory pigment hemocyanin was not present as apolipoprotein. This fact evidenced that these LVs were not related to hemolymphatic lipoproteins.

In vitro lipid transfer between lipoproteins and midgut-diverticula in the spider Polybetes pythagoricus.

It has been already reported that most hemolymphatic lipids in the spider Polybetes pythagoricus are transported by HDL1 and VHDL lipoproteins. We studied in vitro the lipid transfer among midgut-diverticula (M-diverticula), and either hemolymph or purified lipoproteins as well as between hemolymphatic lipoproteins. M-diverticula and hemolymph were labeled by in vivo (14)C-palmitic acid injection. In vitro incubations were performed between M-diverticula and either hemolymph or isolated lipoproteins. Hemolymph lipid uptake was associated to HDL1 (67%) and VHDL (32%). Release from hemolymph towards M-diverticula showed the opposite trend, VHDL 75% and HDL1 45%. Isolated lipoproteins showed a similar behavior to that observed with whole hemolymph. Lipid transfer between lipoproteins showed that HDL1 transfer more (14)C-lipids to VHDL than vice versa. Only 38% FFA and 18% TAG were transferred from M-diverticula to lipoproteins, while on the contrary 75% and 73% of these lipids, respectively, were taken up from hemolymph. A similar trend was observed regarding lipoprotein phospholipids. This study supports the hypothesis that HDL1 and hemocyanin-containing VHDL are involved in the uptake and release of FFA, phospholipids and triacylglycerols in the spider P. pythagoricus. The data support a directional flow of lipids from HDL1 and VHDL suggesting a mode of lipid transport between lipoproteins and M-diverticula.

Analysis of lipid and fatty acid composition of three species of scorpions with relation to different organs

Within arthropods most of the information related to the type of mobilization and storage of lipids is found in insects and crustaceans. Literature is scarce with relation to scorpions. This order is a remarkably important model of the biochemistry, since it is characterized as an animal with very primitive traits which have varied minimally through time. In the present study we characterize and compare lipids and fatty acids present in three species of scorpion: Timogenes elegans, Timogenes dorbignyi, and Brachistosternus ferrugineus, focusing the study on the main organs/tissues involved in the dynamics of lipids. As found in the fat body of insects, hepatopancreas of crustaceans and midgut diverticula of spiders, the hepatopancreas of the three species studied here turned out to be the organ of lipid storage (great quantity of triacylglycerides). With relation to the hemolymph and muscles, a great quantity of phospholipids was observed, which is possibly involved in membrane formation. It is important to highlight that unlike what happens in insects, in scorpions the main circulating energetic lipid is the triacylglyceride. This lipid is found in greater proportion in the hepatopancreas of females, surely for reproduction. The fatty acid of the different organs/tissues analyzed remained constant in the three species studied with certain characteristic patterns, thus observing saturated and unsaturated most abundant fatty acids of C16 and C18. Finally, it could be observed that in T. elegans, T. dorbignyi and B. ferrugineus scorpions, there is a lack of 20:4 that generates a special condition within fatty acids of arthropods.