Role of intramolecular disulfide bond formation in the assembly and secretion of apolipoprotein B-100-containing lipoproteins.

Apolipoprotein B-100 (apoB) is essential for the hepatic assembly and secretion of triglyceride-rich very low density lipoprotein (VLDL). The mechanism of VLDL assembly was explored by perturbing apoB folding in HepG2 cells with the thiol reducing agent dithiothreitol (DTT). Although apoB contains eight known disulfide bonds, seven of which are positioned in the amino-terminal 21% of the protein, its assembly and secretion was only partially blocked in cells treated with 2 mM DTT, a condition that fully blocks the secretion of other disulfide-bonded proteins. Nonreducing gel electrophoresis of an apoB-derived proteolytic peptide revealed that apoB escapes the secretory block normally caused by DTT because its amino-terminal disulfide bonds undergo maturation to a DTT-resistant form after completing synthesis of only the first approximately 20-25% of the protein. If, however, DTT was used under conditions that prevented the initial formation of amino-terminal disulfide bonds, lipoprotein secretion was blocked. Reduced forms of apoB were extremely labile and, unlike other disulfide-bonded proteins, incapable of achieving secretion competence posttranslationally. These results indicate that disulfide bond formation within the amino-terminus of apoB is essential for the proper folding and assembly of its downstream lipophilic sequences. The onset of DTT resistance while still a nascent polypeptide chain is consistent with a model in which the amino-terminal domain of apoB undergoes an independent folding and maturation process, the completion of which may represent an initiation phase of triglyceride-rich lipoprotein assembly.

Dietary fatty acid modification of HDL phospholipid molecular species alters lecithin: cholesterol acyltransferase reactivity in cynomolgus monkeys.

In the following report, cynomolgous monkeys, fed atherogenic diets containing either saturated, monounsaturated, polyunsaturated (n-6 Poly) or fish oil (n-3 Poly) fat as 35% of total calories, provide a model for the study of dietary fat effects on plasma lipoproteins and atherosclerosis. We have previously described the ability of polyunsaturated fat diets to lower plasma described the ability of polyunsaturated fat diets to lower plasma high density lipoprotein (HDL) cholesterol levels and alter HDL subpopulation distribution in the primate model. These experiments investigate possible mechanisms responsible for such modifications. Animals fed polyunsaturated fat had significantly lower plasma concentrations of HDL cholesterol, total plasma cholesterol, and apolipoprotein A-I. Such changes were reflected in the distribution of protein among HDL subfractions, with the most remarkable modification in subclass distribution being the preponderance of small HDL particles in the n-3 Poly-fed animals. Striking alterations were also observed in the distribution of phosphatidylcholine (PC) molecular species (diet effect P < 0.0001 for all major molecular species). Phosphatidylcholine isolated from lipoproteins were used to make recombinant HDL (rHDL) particles. The reaction rate of purified lecithin:cholesterol acyltransferase (LCAT) with particles made from n-3 Poly-derived PC was 50% of that determined using rHDL formed with PC from other dietary groups (P < 0.0001). When the distribution of LCAT-derived rHDL cholesteryl esters was analyzed, LCAT demonstrated little selectivity for certain PC molecular species except in n-3 Poly-derived rHDL where 18:2-containing PC was selectively utilized. These data demonstrate that differences in dietary fat intake can significantly alter HDL PC concentration and molecular species distribution. We suggest that diet-induced alterations in HDL PC molecular species modify the type of cholesteryl esters produced during the LCAT reaction thereby affecting the plasma cholesteryl ester pool. We also propose that dietary n-3 Poly affects cholesteryl ester metabolism in part via LCAT by lowering PC (LCAT substrate) availability, altering the rate of the LCAT reaction, and decreasing HDL cholesterol concentrations; however, n-6 Poly dietary fat effects on HDL concentration appear to be through some mechanism other than LCAT.

Regulation of arachidonic acid metabolism in resident and BCG-activated alveolar macrophages: role of lyso(bis)phosphatidic acid.

To dissect mechanisms of arachidonic acid (20:4) metabolism in pulmonary alveolar macrophages (PAM), two distinct cell populations were investigated, resident and BCG-activated rabbit alveolar macrophages. After purified resident PAM were labeled overnight with [3H]20:4, radioactivity was localized primarily within lyso(bis)phosphatidic acid (L(bis)PA) (13.1% +/- 1.7), phosphatidylethanolamine (PE) (22.8% +/- 0.8), and phosphatidylcholine (PC) (26.7% +/- 1.7), with lesser amounts recovered in phosphatidylserine plus phosphatidylinositol (PS/PI) (9.2 +/- 0.8%). By contrast, analysis of the phospholipid classes from prelabeled BCG-activated PAM revealed that the amount of [3H]20:4 contained in L(bis)PA was profoundly decreased (4.7% +/- 0.4), p less than 0.003), whereas [3H]20:4 contained within other BCG phospholipids remained unchanged. Moreover, L(bis)PA, which composed 18.6% +/- 1.2 of the total phospholipid phosphorus of resident PAM, was reduced to 4.1% +/- 0.1 in BCG-activated macrophages (p less than 0.01). Phospholipase A2 from snake venom or from pancreas failed to release 20:4 from L(bis)PA, and lipase (phospholipase A1) from Rhizopus delmar liberated no more than one-third of this arachidonate. These results suggest that much of the arachidonate is not mobilized by classical phospholipases A1 and A2. When [3H]20:4-labeled PAM were stimulated with 1 microM 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a loss of [3H]20:4 was observed from L(bis)PA, PE, PC, and PS/PI, with a concomitant increase in the synthesis of Hete and leukotriene C4. BCG-activated PAM exposed to either TPA or 3.8 microM calcium ionophore A23187 liberated [3H]20:4 solely from PE and PC, with diminished 20:4 oxidative metabolism. Analysis of the specific radioactivities of phospholipids obtained from resident PAM prelabeled with [3H]20:4 or [32P]i demonstrated that the specific activity of [32P]L(bis)PA was negligible, whereas that of [3H]20:4 was quite high. In addition, L(bis)PA deacylation induced by TPA in resident PAM was always accompanied by a corresponding loss of [3H]20:4 from phosphatidylinositol (PI), suggesting that metabolism of this novel phospholipid proceeded by a deacylation-reacylation reaction rather than by de novo synthesis. BCG-activated PAM, which exhibited depressed eicosanoid formation, consistently failed to deacylate [3H]20:4 from L(bis)PA or PI. These studies demonstrate that, unlike 20:4 derived from PE and PC by BCG-activated PAM, L(bis)PA may indeed provide a novel source of 20:4 that is tightly coupled to the lipoxygenase pathway.

Phosphorylation and degradation of exogenous phosphatidylinositol incorporated into Friend erythroleukemic cells.

Phosphatidyl[2-3H]inositol (PtdIns) obtained from rat skeletal muscle and yeast was introduced into Friend erythroleukemic cells by use of the PtdInstransfer protein or by spontaneous route. The mammalian PtdIns incorporated by the transfer protein appeared metabolically inert while the spontaneously incorporated PtdIns was both phosphorylated to PtdIns-4-phosphate (i.e. 30% of the total PtdIns incorporated) and converted into lyso-PtdIns (i.e. 20% of the total PtdIns incorporated); formation of PtdIns, 4,5-bisphosphate was minimal. The extensive metabolism of the spontaneously incorporated PtdIns strongly suggests that this PtdIns does not rapidly equilibrate with the endogenous PtdIns pools. A similar spontaneous incorporation of yeast PtdIns was accompanied by a negligible degree of phosphorylation and hydrolysis. Evidence is provided that this difference in metabolism reflects the absence of arachidonate in the yeast PtdIns.