Scavenger receptor class B type I as a mediator of cellular cholesterol efflux to lipoproteins and phospholipid acceptors.

We recently reported that the rate of efflux of cholesterol from cells to high density lipoprotein (HDL) was related to the expression level of scavenger receptor class B type I (SR-BI). Moreover, the expression of this receptor in atheromatous arteries raises the possibility that SR-BI mediates cholesterol efflux in the arterial wall (Ji, Y., Jian, B., Wang, N., Sun, Y., de la Llera Moya, M., Phillips, M. C., Rothblat, G. H., Swaney, J. B., and Tall, A. R. (1997) J. Biol. Chem. 272, 20982-20985). In this paper we describe studies that suggest that the presence of phospholipid on acceptor particles plays an important role in modulating interaction with the SR-BI. Specifically, enrichment of serum with phospholipid resulted in marked stimulation of cholesterol efflux from cells that had higher levels of SR-BI expression, like Fu5AH or Y1-BS1 cells, and little or no stimulation in cells with low SR-BI levels, such as Y-1 cells. Stimulation of efflux by phospholipid enrichment was also a function of SR-BI levels in Chinese hamster ovary cells transfected with the SR-BI gene. Efflux to protein-free vesicles prepared with 1-palmitoyl-2-oleoylphosphatidyl-choline also correlated with SR-BI levels, suggesting that phospholipid, as well as protein, influences the interaction that results in cholesterol efflux. By contrast, cholesterol efflux from a non-cell donor showed no stimulation consequent to phospholipid enrichment of the serum acceptor. These results may help to explain observations in the literature that document an increased risk of atherosclerosis in patients with depressed levels of HDL phospholipid even in the face of normal HDL cholesterol levels.

Scavenger receptor BI promotes high density lipoprotein-mediated cellular cholesterol efflux.

Scavenger receptor BI (SR-BI) binds high density lipoproteins (HDL) with high affinity and mediates the selective uptake of HDL cholesteryl ester. We examined the potential role of SR-BI in mediating cellular cholesterol efflux. In Chinese hamster ovary cells stably transfected with murine SR-BI, overexpression of SR-BI resulted in a 3-4-fold stimulation of initial cholesterol efflux rates. Efflux rates correlated with SR-BI expression in cells and HDL concentration in the medium. When incubated with synthetic cholesterol-free HDL, SR-BI-transfected cells showed approximately 3-fold increases in initial rates of efflux compared with control cells, indicating that SR-BI expression enhances net cholesterol efflux mediated by discoidal HDL. In six different cell types, including cultured macrophages, the rate of efflux of cholesterol mediated by HDL or serum was well correlated with cellular SR-BI expression level. In addition, in situ hybridization experiments revealed that SR-BI mRNA was expressed in the thickened intima of atheromatous aorta of apolipoprotein E knockout mice. Thus, SR-BI is an authentic HDL receptor mediating cellular cholesterol efflux. SR-BI may facilitate the initial steps of HDL-mediated cholesterol efflux in the arterial wall as well as later steps of reverse cholesterol transport involving uptake of HDL cholesterol in the liver.

Modification of the cholesterol efflux properties of human serum by enrichment with phospholipid.

To investigate the importance of phospholipid in promoting cholesterol efflux from cells, phospholipid multilamellar vesicles were incubated with normal human serum and the efflux ability of these lipid-modified sera was tested. When incubated under appropriate conditions, both dimyristoylphosphatidylcholine (DMPC) and bovine brain sphingomyelin (BBSM) were shown to combine with components of human serum to form new protein:lipid complexes and to markedly enhance the ability of serum to promote efflux of cholesterol from Fu5AH cells. In particular, the high density lipoprotein (HDL) particles were altered in their composition and electrophoretic properties and the alpha-migrating species, which were reactive with antibodies to apo-A-I, were converted to larger, pre-beta-migrating particles, similar in electrophoretic properties to pre beta(2)-HDL. DMPC, but not BBSM, also generated particles with mobility similar to pre beta(2)-HDL; These species were demonstrably different from the discoidal complexes formed by reaction of DMPC with purified apoA-I. However, no change in cholesterol efflux potential was observed when serum was mixed with phospholipids that failed to interact or when cells were incubated with phospholipid multilamellar vesicles alone. To further identify the components of serum that become altered in their efflux potential after reaction with phospholipid, isolated lipoprotein fractions were incubated with DMPC or BBSM and it was found that only interaction with HDL caused enhancement of cholesterol efflux. In summary, cholesterol removal from the Fu5AH cells by serum can be promoted by adding phospholipid under conditions where new HDL-like complexes can be formed between the phospholipid and serum components, most notably apolipoprotein A-I.

Role of HDL phospholipid in efflux of cell cholesterol to whole serum: studies with human apoA-I transgenic rats.

Sera of transgenic rats expressing human apoA-I were tested for their ability to stimulate efflux of radiolabeled cholesterol from Fu5AH rat hepatoma cells. Expression of human apoA-I resulted in a dose-dependent increase in HDL, as measured by both HDL-cholesterol and HDL-phospholipid, and produced a decrease in rat apoA-I. In rats expressing high concentrations of human apoA-I (TgR[hAI]high, human apoA-I > 250 mg/dl), the increase in HDL-phospholipid was not proportional to the increase in human apoA-I, as illustrated by a HDL-PL/total apoA-I ratio of 0.84 +/- 0.19 compared to a ratio of 1.28 +/- 0.29 for control rats and of 1.28 +/- 0.39 for rats expressing low levels of human apoA-I (TgR[hAI]low, human apoA-I < 250 mg/dl). Compared to sera from control animals, efflux of cell cholesterol was increased by 26% in the sera from TgR[hAI]low, and by 76% in the TgR[hAI]high. An examination of the relationships between efflux and HDL-related parameters demonstrated a hyperbolic relationship between efflux and either HDL-cholesterol or HDL-apoA-I. In contrast, there was a strong linear association (r2 = 0.84) between cholesterol efflux and HDL-phospholipid, indicating that this parameter is the component of HDL that best reflects the serum's efflux efficiency. The importance of phospholipids in modulating cholesterol efflux was further explored by measuring the effect of supplementation of serum with dimyristoylphosphatidylcholine (DMPC) vesicles, apoA-I, or both DMPC vesicles and apoA-I. Whereas addition of human apoA-I had no effect on efflux, supplementation with DMPC vesicles produced a substantial increase in efflux that was further stimulated by the combination of DMPC vesicles and apoA-I. These results demonstrate that a major component of HDL that modulates cell cholesterol efflux is phospholipid.

Effect of apolipoprotein C-I peptides on the apolipoprotein E content and receptor-binding properties of beta-migrating very low density lipoproteins.

To evaluate the role of apolipoprotein (apo) C-I in inhibiting lipoprotein binding to the low density lipoprotein receptor-related protein (LRP), a putative lipoprotein remnant receptor, apoC-peptide fragments were prepared by chemical synthesis or by cyanogen bromide cleavage of intact apoC-I. In ligand-blotting assays, peptides corresponding to residues 1-38, 10-57, 20-57, 30-57, and 40-57 proved ineffective, but intact apoC-I was very effective, at inhibiting the binding of apoE-enriched beta-migrating very low density lipoproteins (beta-VLDL) to the LRP. Studies of the displacement of 125I-labeled apoE from apoE-enriched beta-VLDL showed that the largest peptide (residues 10-57) was two-thirds as effective as intact apoC-I; the other peptides were highly ineffective (residues 40-57, 1-38) or only partly effective (residues 20-57, 30-57). Changes in the intrinsic tryptophan fluorescence and helix content indicated that the largest peptide was similar to apoC-I in lipid binding affinity, while the other peptide fragments showed little or no affinity for either unilamellar or multilamellar vesicles of dimyristoyl-phosphatidylcholine. These findings suggest that the ability of apoC-I fragments to displace apoE from beta-VLDL is largely, but perhaps not exclusively, a reflection of their ability to bind to membranous bilayers and that apoC-I blocking of the interaction between apoE-rich beta-VLDL and the LRP probably involves displacement of a critical amount of the apoE from the surface of this lipoprotein.

Structural and functional domains of apolipoprotein A-I within high density lipoproteins.

We prepared discoidal and spherical model high density lipoprotein (HDL) with apolipoprotein A-I and 1-palmitoyl-2-oleoyl phosphatidylcholine at various lipid:protein ratios and compared their reactivity with exo- and endopeptidases to that of human HDL2 and HDL3. Limited proteolysis with trypsin, Staphylococcus V8 protease, and elastase yielded a major stable peptide of 11,000-11,500 daltons under conditions which completely degraded lipid-free A-I. By Western blotting this protease-resistant fragment was shown to consist of the amino-terminal 90-100 residues of the A-I protein; the residues on the carboxyl side of this peptide are therefore protease-sensitive and appear to correlate with the putative "hinge" region, which is especially reactive with antibodies. The amino terminus was very resistant to digestion with a variety of aminopeptidases, whereas carboxypeptidases could remove up to 70 residues from the lipid-free A-I protein or 12-24 residues from A-I in various HDL. When these truncated forms of A-I, in combination with lipid, were used to examine binding interactions with rat hepatic plasma membranes, it was found that removal of up to 20-24 residues from the carboxyl terminus had no significant effect on binding, whereas removal of 70 residues completely eliminated specific binding to the membranes. Taken together, our data indicate that there is a protease-resistant domain constituted by the first 90 residues of A-I, which, in HDL, contain little of the class of amphipathic helix characteristic of the rest of the molecule and most likely form a structure dominated by protein-protein interactions. At the carboxyl end of the protein, there is a functional domain constituted by residues 149-219 that possesses the capacity to bind to proteins on hepatic membranes.

Properties of phosphatidylethanolamine-containing phospholipid-apolipoprotein complexes modified by lecithin-cholesterol acyltransferase.

The effect of the inclusion of phosphatidylethanolamine (PE), a phospholipid with unusual packing properties, on the substrate properties of protein-lipid complexes toward lecithin-cholesterol acyltransferase (LCAT) has been studied. Recombinant particles of apolipoprotein A-I with dimyristoylphosphatidylcholine (DMPC), dilauroylphosphatidylethanolamine (DLPE) and cholesterol were prepared at a molar ratio of 1:140:14 (A-I/DMPC/cholesterol) or 1:70:70:14 (A-I/DMPC/DLPE/cholesterol); the efficiency of cholesterol incorporation into complexes containing phosphatidylethanolamine was found to be very pH-dependent, with enhanced cholesterol incorporation at elevated pH values. By incubating the complexes with either purified human LCAT or the d greater than 1.21 g/ml fraction of rat serum as a source of LCAT activity, it was found that a high degree of cholesterol esterification could be achieved with either complex; however, the DLPE-containing complex possessed a much smaller Stokes' diameter than the DMPC-only particle despite compositional similarities between these complexes. With respect to particle diameter the DLPE-containing particles behaved more like complexes prepared with egg yolk lecithin than did complexes prepared with DMPC alone. When human LDL was added to the incubations to provide a source of additional cholesterol, the products were markedly different. Concomitant with an increased cholesteryl ester core was an increase in the protein stoichiometry in both types of particles, from 2 to 3 or 4 apo A-I per particle. The proportion of DLPE to DMPC in the products was reduced from 1:1 to 0.3:1, reflecting a preferential hydrolysis of PE by LCAT, and the Stokes' diameters of the DMPC-only and the DLPE-containing complexes were closely similar. We conclude that the presence of elevated proportions of certain phospholipid species may significantly alter both the physical properties of the particles and their substrate properties with regard to reactions with enzymes of lipid metabolism.

High density lipoprotein subpopulations from galactosamine-treated rats and their transformation by lecithin:cholesterol acyltransferase.

It is known that an acute hepatotoxicity is produced in rats by intraperitoneal administration of galactosamine; a consequence of this treatment is a marked deficiency of lecithin:cholesterol acyltransferase (LCAT) activity in the plasma compartment. In this study high density lipoprotein (HDL) from galactosamine-treated rats was isolated, resolved into subpopulations, and characterized. In contrast to HDL from control rats, which elutes from gel filtration columns as a single peak and has a diameter of 13.1 nm, HDL from the galactosamine-treated animals was found to elute in five major zones with diameters of 7.8-35 nm. Characterization of these subpopulations has revealed that the larger fractions are enriched in apolipoprotein E, phospholipid, and cholesterol, but contain little cholesteryl ester, while the smallest two fractions contain mainly apolipoprotein A-I, are enriched in phospholipid, and have 50-60% of their cholesterol in the ester form. Incubation of HDL from treated rats with a source of LCAT activity plus low and very low density lipoproteins caused transformation of these subpopulations into a species which, by size and composition, was essentially identical to control rat HDL. In addition, when the subpopulations were individually incubated with purified human lecithin:cholesterol acyltransferase and bovine serum albumin, there was a similar convergence toward a moderate particle size approximating control rat HDL. Cross-linking studies showed that incubation with LCAT activity reduced the heterogeneity of the treated rat HDL. We conclude that the galactosamine treatment induces a complex mixture of HDL that bears strong similarities to the small, apoA-I rich and large, apoE-rich particles seen in LCAT deficiency or secreted by hepatic cells in culture. Furthermore, these species appear to coalesce in the presence of the d greater than 1.21 g/ml fraction of control serum to yield a fairly homogeneous population that resembles control rat HDL in size, composition, and apoprotein content.

Effect of phosphatidylethanolamine on the properties of phospholipid-apolipoprotein complexes.

Plasma high density lipoproteins (HDL) are synthesized in intestinal mucosal cells and hepatocytes and are secreted into the blood. Factors influencing the structure and function of these HDL, such as lipid and protein composition, are poorly understood. It appears, however, that intracellular, discoidal HDL are enriched, relative to plasma HDL, in phosphatidylethanolamine (PE), a phospholipid known to generate unusual, nonbilayer structures of putative physiological significance. Although incubation of dimyristoylphosphatidylcholine (DMPC) with apolipoprotein A-I at the gel-liquid crystalline phase transition temperature results in the spontaneous formation of lipid-protein complexes, the presence of proportionately small amounts of PE prevents the formation of such complexes, suggesting that PE profoundly alters the phase properties of the phospholipid bilayers. However, by using a detergent-mediated method for the formation of PE-rich model nascent HDL from phospholipids and apolipoprotein A-I, lipid-protein complexes containing as much as 75% DLPE could be formed, thus demonstrating that the presence of PE causes a kinetic, rather than a thermodynamic, barrier to spontaneous complex formation. The products contained a DLPE:DMPC molar ratio similar to that of the initial incubation mixture; however, as the mole percentage of DLPE increased, the products became less heterogeneous, the buoyant density of the products increased, and the Stokes diameter of the products decreased. Similar results were obtained when dimyristoylphosphatidylethanolamine (DMPE) and dipalmitoylphosphatidylethanolamine (DPPE) were employed in lieu of DLPE. Electron microscopy of complexes containing DLPE and DMPC at a 1:1 molar ratio showed that these particles possessed a discoidal, bilayer morphology similar to that seen with complexes containing only phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of heparin-induced lipolytic activity on the structure of rat high-density lipoprotein.

Following its secretion into the plasma compartment, the high-density lipoprotein (HDL) is presumed to be acted upon by both soluble enzymes, such as lecithin:cholesterol acyltransferase (LCAT), and membrane-associated enzymes, such as lipoprotein lipase and hepatic lipase. Rats were injected intravenously with heparin to release membrane-associated lipolytic activities into the circulation and the collected plasma was incubated overnight at 37 degrees C in the presence or absence of an LCAT inhibitor or an inhibitor of lipoprotein lipase (1 M NaCl). It was observed that lipoprotein lipase accounted for most of the triglyceride hydrolase activity in the heparin-treated plasma, and that the heparin-releasable activities caused an increase in HDL density but no measurable change in particle size when LCAT was inhibited. Heparin treatment caused about a 60% decrease in plasma triacylglycerol during the interval between injection of heparin and blood collection. Although this caused marked compositional changes in the d less than 1.063 g/ml lipoproteins, no changes were observed in the lipid composition or apoprotein distribution in the HDL. Subsequent incubation for 18 h at 37 degrees C produced marked increases in the apoE content of HDL from heparin-treated plasma even when LCAT was inhibited. Time-course studies showed that in the presence of an LCAT inhibitor there was considerable conversion of phosphatidylcholine to lysophosphatidylcholine in heparin-treated plasma, and that this activity was diminished by 1 M NaCl, but that no phospholipolysis was observed in control plasma. By contrast, both heparin-treated and control plasma possessed substantial triglyceride hydrolase activity. The concurrent action of lipases and LCAT was observed to reduce the maximum level of cholesterol esterification which could be achieved in the absence of lipase activity. It is concluded that changes in HDL particle size are mainly attributable to LCAT, but that lipase activities, which are either free in rat plasma or releasable by heparin, play a role in restructuring the phospholipid moiety and altering the protein composition of the HDL, especially with respect to apoE, a potential ligand to cellular receptors.

A rapid method for the synthesis of protein-lipid complexes using adsorption chromatography.

A novel and rapid method for the detergent-mediated synthesis of protein-lipid complexes has been developed and has several advantages over detergent dialysis methods. This new method involves co-incubation of human apolipoprotein A-I (apoA-I), the major protein component of high density lipoproteins (HDL), and dipalmitoylphosphatidylcholine for 1 hr in the presence of cholate, after which removal of greater than 99.7% of the detergent is achieved by a 2-hr batch adsorptive chromatography procedure. Complexes prepared by this method had a density of 1.10 g/ml, similar to plasma HDL. Chemical cross-linking of these products demonstrated that there was complete conversion of apoA-I to a protein-lipid complex that contained two molecules of apoA-I. One major band was resolved by gradient gel electrophoresis in the region of the gel expected for newly synthesized HDL. Results are described which show the application of this method to the study of lipid variation on the structure of model HDL, including the alteration of lipid-protein molar ratios and the addition of cholesterol.

Structural modifications of rat serum high density lipoprotein by pancreatic phospholipase A2.

An important and unusual aspect of the high density lipoprotein (HDL) in the rat is its tendency to undergo marked alterations in structure in response to physiological perturbations. In this study, the role of the surface lipids for maintenance of HDL integrity were investigated. Hydrolysis by pancreatic phospholipase A2 of the phospholipids of rat HDL in the presence of the d greater than 1.21 g/ml fraction of rat serum results in an increase in the particle diameter and an uptake of apo-E and apo A-IV from the lipoprotein-free fraction; augmentation of the albumin concentration in the incubation mixture intensified the observed changes, probably due to enhancement of the compositional changes brought about by phospholipase treatment. Phospholipase A2, treatment of the d less than 1.21 g/ml fraction of rat serum produces only minor changes in the properties of the isolated HDL. These data suggest that changes in apoprotein content reflect an uptake of A-IV and E by the rat HDL, rather than a net loss of apo A-I. Likewise, titration of the action of pancreatic phospholipase A2 on HDL apoprotein composition showed that initially a modest increase in apo A-IV content occurred, but with more extensive phospholipolysis there was a considerably greater increase in the apo-E content of the particle. The data suggest that hydrolysis of phospholipids such as occurs physiologically through the action of lecithin:cholesterol acyl transferase and hepatic lipase may alter the HDL structure independently from changes effected in the neutral lipid core.

Proteolysis in vitro of low and high density lipoproteins in human plasma by Cerastes cerastes (Egyptian sand viper) venom.

Envenomation by snake venoms would be expected to result in proteolysis of plasma proteins as well as of cellular constituents. Incubation of human serum with crude venom from Cerastes cerastes showed that the plasma lipoproteins were a target of this venom. Fractionation of the crude venom by gel filtration revealed that high density lipoprotein (HDL) was susceptible almost exclusively to the highest mol. wt fraction of venom and that proteolysis was due to a metalloprotease. Although HDL was degraded only by this metalloprotease, the low density lipoprotein (LDL) was proteolyzed by both metalloproteases and serine proteases present in several fractions of the venom. Despite extensive degradation, LDL remained intact, as judged by gradient gel electrophoresis. The selectivity of venom fractions may prove useful in the study of lipoprotein structure.

Enzymatically induced alterations in the structure of rat serum lipoproteins.

Incubation of freshly isolated rat serum induces a large number of changes in the properties of the serum lipoproteins, especially the high density lipoproteins (HDL). The particle diameter of the HDL increases from about 10.4 nm to 12.3 nm and the protein content appears to increase by about 60,000 Daltons. Reactions catalyzed by lecithin:cholesterol acyltransferase (LCAT) lead to a marked decrease in cholesterol and phospholipid content, and an even greater increase in cholesteryl ester content. Especially noteworthy are the marked increases in apoE and apoA-IV which are found associated with HDL as a result of this process. Data indicate that the affinity of apoE and apoA-IV for the HDL particles may be influenced by the proportion of surface to core lipid and by the presence of products of the LCAT reaction. Changes in the apoprotein content of very low density lipoproteins are also observed, with A-I and A-IV appearing in this density interval. All of the above changes can be prevented by the inclusion of 5,5'dithiobis(2-nitrobenzoate) or p-chloromercuriphenylsulfonate during the incubation, or by heat treatment of serum at 56 degrees C for 30 min; these treatments are known to inhibit LCAT activity. It is concluded that LCAT action is the major cause of the various changes in HDL structure that are observed and that alterations in apoprotein content occur to correct the resultant imbalance between core lipid and coverage of this core by amphiphilic components. Increased apoE association with cholesteryl ester-rich HDL may provide an efficient means for receptor-mediated removal of cholesterol from the circulation.

Membrane cholesterol uptake by recombinant lipoproteins.

Recombinant lipoproteins, prepared with apo A-I isolated from human high density lipoprotein (HDL) and various phospholipids (PLs), were compared with respect to their ability to remove cholesterol (Chol) from labelled erythrocyte ghost membranes. It was found that uptake of Chol was essentially complete following an 8 h incubation at 37 degrees C. Quantitation of the amount of cholesterol taken up showed that recombinants prepared from bovine brain sphingomyelin (BBSM) or dipalmitoyl phosphatidylcholine (DPPC) acquired the highest proportion of Chol (80-140 mol/mol protein), whereas shorter chain phospholipids like dimyristoyl phosphatidylcholine (DMPC) acquired little or no membrane Chol. Chemical analysis of the incubation products indicated that this latter result was due to loss of PL, presumably to the membrane, with consequent disruption of the recombinant particle. Results with DPPC:A-I recombinants of differing PL/protein ratios and sizes showed that Chol uptake was fairly constant at 0.70 mol Chol/mol PL. It is concluded that discoidal, phospholipid-rich recombinant lipoproteins can effectively take up substantial amounts of Chol from physiological membranes, provided that the PLs utilized form micellar complexes which are capable of retaining their structural integrity during the incubation with the membranes.

Isolation and lipid-binding properties of rat apolipoprotein A-IV.

Apolipoprotein A-IV was isolated from the d less than 1.21 g/ml fraction of rat serum by gel filtration followed by heparin-Sepharose affinity chromatography; this method also facilitated the preparation of apolipoprotein A-I and apolipoprotein E. The apolipoprotein A-IV preparation was characterized by SDS-gel electrophoresis, isoelectric focusing, amino acid analysis and immunodiffusion. The lipid-binding properties of this protein were studied. Apolipoprotein A-IV associated with dimyristoylphosphatidylcholine (DMPC) to form recombinants which contained two molecules of apolipoprotein A-IV and had a lipid/protein molar ratio of 110. The density of the DMPC/apolipoprotein A-IV particles was determined to be 1.08 g/ml and the particles were visualized by electron microscopy as discs which were 5.8 nm thick and 18.0 nm in diameter. The stability of the DMPC/apolipoprotein A-IV recombinants, as determined by resistance to denaturation, was comparable to the stability of DMPC/apolipoprotein A-I complexes. However, by competition studies it was found that apolipoprotein A-I competed for the binding to DMPC more effectively than did apolipoprotein A-IV. It is concluded that, while rat apolipoprotein A-IV resembles other apolipoproteins in its lipid-binding characteristics, it may be displaced from lipid complexes by apolipoprotein A-I.

Interaction between hepatic microsomal membrane lipids and apolipoprotein A-I.

Incubation of apoprotein A-I (apo-A-I), the major protein component of human high density lipoprotein, with rat liver microsomal membranes under conditions of elevated pH and ionic strength leads to the production of a soluble protein:lipid complex (A-I/MM complex). The A-I/MM complex, as purified by density gradient centrifugation and agarose column chromatography, possesses a lipid composition similar to the hepatic microsomal membrane and a protein/lipid ratio similar to that of plasma high density lipoproteins, but markedly different from that of recombinant particles prepared with synthetic lipids. The A-I/MM complex constitutes a more physiological recombinant particle than can be formed using synthetic lipids and may be a suitable model for the newly assembled intracellular high density lipoproteins. Incubation of the erythrocyte plasma membranes with apo-A-I under the same conditions as used with microsomal membranes fails to generate any lipid:apoprotein complexes. This membrane specificity for forming soluble lipoprotein complexes suggests that the microsomal membranes possess a unique feature, possibly their lipid composition, which render them particularly suitable to serve as lipid donors to the apoproteins which are undergoing assembly within the endoplasmic reticulum/Golgi organelles.

Hybrid association between human apolipoproteins A-I and A-II in aqueous solution and in phospholipid recombinants.

The formation of hybrid association products between apolipoprotein A-I and apolipoprotein A-II from human high-density lipoprotein was investigated in solutions of these apolipoprotein and in recombinant particles with dimyristoylphosphatidylcholine (DMPC). It was found that these two proteins interact in solution to form hybrid association products, but not to a marked degree. When these two proteins were incubated together with DMPC, it was likewise found that there was little tendency to reside on the same particle, as judged from the absence of hybrid oligomers by chemical cross-linking. By a modified immunoelectrophoretic method it was found that only about 15% of the A-II and 10% of the A-I were precipitated by the heterologous antiserum; from this it is concluded that 80-90% of these proteins do not form hybrid recombinants with the other protein. These results suggest that in the delipidated state, as well as in discoidal recombinants, there do not exist strong protein-protein interactions between A-I and A-II. This implies that even in the high-density lipoprotein, where both proteins coexist in the same particle, the A-II does not stabilize the molecular structure through interactions with A-I, and its role in this molecule remains obscure.

Selective proteolytic digestion as a method for the modification of human HDL3 structure.

Trypsin digestion of human high density lipoprotein (d 1.125-1.21 g/ml) on which the lysine residues have been masked with the reversible blocking group, 2,3,4,5-tetrahydrophthallic anhydride (THPA), was found to result in the fragmentation of the apoA-I component, but not the apoA-II component of this lipoprotein particle. Approximately 50-80% of the apoA-I polypeptide was found in a lipid-free fraction, while the residual apoA-I material plus the apoA-II moiety constituted a core particle that contained most of the original lipid. Immunological analysis indicated that such fragmentation did not affect the immunoreactivity of apoA-II, but that all immunoreactivity of apoA-I was lost within the first 30 min of trypsinization. By column chromatography and electron microscopy this core particle appeared identical in size with the untrypsinized THPA-modified HDL(3) material. Size analysis of the core particle peptides suggests that not all of the A-I molecules present on the HDL(3) are trypsinized to the same extent, which indicates possible nonequivalence of these peptide chains. Analysis of the amino acid composition revealed a somewhat greater proportion of hydrophobic residues in the lipid-bound fraction than in the lipid-free fraction. Analysis of tryptophan showed that almost all of this highly hydrophobic residue was found in the lipid-bound fraction; this suggests that lipid binding occurs preferentially in the more hydrophobic domains of the A-I molecule. Incubation of the core particle with intact apoA-I, obtained from either human or bovine HDL, showed that these proteins could be incorporated to regenerate an HDL(3) of selectively altered protein composition, compared to the original lipoprotein. It is concluded that some latitude is allowable in the surface/volume relationship in lipoproteins before reorganization of the particle is required; this might, for example, provide a mechanism whereby the HDL could serve in a storage role for the C apolipoproteins in plasma.-Swaney, J. B. Selective proteolytic digestion as a method for the modification of human HDL(3) structure.