Lipoprotein Binding Sites Research Articles

Glucocorticoids upregulate high-affinity, high-density lipoprotein binding sites in rat hepatocytes

Glucocorticoid hormones (GL) regulate high-density lipoprotein (HDL) plasma concentrations by increasing synthesis and secretion of HDL by the liver. However, little is known about the effect of GL on the uptake and processing of HDL by hepatocytes (HEP). To investigate this question, we studied the effects of dexamethasone (DEX) on the expression of high-affinity HDL-binding sites via the specific binding and internalization of iodine-labeled apolipoprotein E (apo E)-free HDL 3 in a culture of rat HEP. Specific binding and internalization of HDL 3 decreased by 60% in cells cultured in the absence of DEX for 48 hours. At concentrations of 10 −7 and 10 −5 mol/L, DEX prevented the decrease, maintaining specific binding and internalization versus the control level (at 24 hours). HDL-binding sites with a K d of 20 μg/mL were revealed on the surface of cultured HEP. HEP demonstrated a greater binding capacity in the presence of DEX at concentrations of 10 −7 and 10 −5 mol/L (125 v 45 ng/mg cell protein). The effect of the hormone has demonstrated to be dose-dependent at concentrations between 10 −9 and 10 −7 mol/L, leveling off at 10 −7. Higher concentrations did not induce a further increase in specific binding and internalization. Withdrawal of the hormone from culture medium was associated with a decrease in specific binding of the ligand by 60% in the following 24 hours. To investigate the effect of glucocorticoid deficiency on liver uptake of HDL in vivo, specific binding and internalization were studied in a culture of HEP isolated from adrenalectomized rats (AER) at 2 hours after seeding. The HEP demonstrated a 2.5-fold decrease in specific binding versus those from normal rats (NR). Correction of the glucocorticoid deficiency in AER by intramuscular injection of DEX at a dose of 5 μg per rat restored the specific binding of HDL 3 at 2 hours after seeding. Replacement of the hormone by incubation of HEP from AER with DEX increased specific binding and internalization of the ligand in a dose-dependent fashion until NR levels were attained. In both cases, the upregulating effect of DEX was stopped by 0.5 mmol/L cycloheximide. Lovastatin (100 nmol/L) blocked the DEX-dependent increase in cholesterol synthesis, but failed to prevent the DEX-induced increase in specific binding and internalization of 125I-HDL 3. It is concluded that the specific binding and internalization of 125I-HDL 3 by liver parenchymal cells are reversibly regulated by physiologic and pharmacologic concentrations of DEX. Its effect does not depend on availability of the pool of newly synthesized cholesterol, but it seems to be a result of the increase in the synthesis of HDL-binding protein.

Interaction of Lp(a) with Plasminogen Binding Sites on Cells

Lp(a) competes with plasminogen for binding to cells but it is not known whether this competition is due to the ability of Lp(a) to interact directly with plasminogen receptors. In the present study, we demonstrate that Lp(a) can interact directly with plasminogen binding sites on monocytoid U937 cells and endothelial cells. The interaction of Lp(a) with these sites was time dependent, specific, saturable, divalent ion independent and temperature sensitive, characteristics of plasminogen binding to these sites. The affinity of plasminogen and Lp(a) for these sites also was similar (Kd = 1-3 microM), but Lp(a) bound to fewer sites (approximately 10-fold less). Both gangliosides and cell surface proteins with carboxy-terminal lysyl residues, including enolase, a candidate plasminogen receptor, inhibited Lp(a) binding to U937 cells. Additionally, Lp(a) interacted with low affinity lipoprotein binding sites on these cells which also recognized LDL and HDL. The ability of Lp(a) to interact with sites on cells that recognize plasminogen may contribute to the pathogenetic consequences of high levels of circulating Lp(a).

Cellular binding site and membrane binding proteins for triglyceride-rich lipoproteins in human monocyte-macrophages and THP-1 monocytic cells.

Triglyceride- and cholesterol-rich foam cells derived from monocyte-macrophages are commonly associated with some forms of hypertriglyceridemia. In this report, direct binding studies at 4 degrees C demonstrate that human monocyte-macrophages (HMM) 1-6 days after isolation from blood and human THP-1 monocytic cells, before and up to 7 days after differentiation with phorbol ester, exhibit a high affinity (Kd 3-6 nM), saturable, specific, and apolipoprotein (apo) E-independent binding site for the uptake and degradation of certain triglyceride-rich lipoproteins (TGRLP). Ligand blotting analysis identified two membrane binding proteins (MBP) of apparent molecular weights of 200 and 235 kDa (MBP 200 and MBP 235) in both cell types that share the same ligand specificity as the cellular site and bind hypertriglyceridemic (HTG) VLDL, trypsinized VLDL devoid of apoE (tryp-VLDL), and dietary plasma chylomicrons from normal subjects but not LDL, acetyl LDL, or normal VLDL with high affinity. Neither lipoprotein lipase nor apoE are required for TGRLP binding to the cells or the isolated MBPs. The cellular binding site and the MBPs are expressed at similar levels at all stages of differentiation, unlike the LDL or the acetyl LDL receptor. TGRLP that bind to the MBPs induce rapid, saturable, cellular triglyceride accumulation in monocytes as well as macrophages; normal VLDL does not. In addition, the cellular high affinity binding site and MBP 200 and 235 are not affected by the media sterol content, unlike the LDL receptor. Taken together, these data indicate that human monocyte-macrophages exhibit a high affinity, saturable, specific, apoE- and lipoprotein lipase-independent binding site and membrane binding proteins for TGRLP that differ in expression, specificity, and molecular size from receptors of the LDL receptor gene family or the acetyl LDL receptor. The shared characteristics of the cellular binding site with MBP 200 and MBP 235 suggest that they are candidates for the receptor-mediated, apoE-independent uptake of HTG-VLDL and chylomicrons by monocytes and macrophages and therefore may be involved in foam cell formation.

Analysis of the lipoprotein binding site of rat liver membrane

Analysis of the lipoprotein binding site of rat liver membrane

Protein kinase C-dependent desensitization of HDL3-activated phospholipase C in human platelets.

In isolated human platelets, exposure of subfraction 3 high-density lipoprotein (HDL3) binding sites to high concentrations of HDL3 (1 mg/mL) causes rapid desensitization of HDL3 (50 micrograms/mL)-stimulated breakdown of phosphatidylcholine, as shown in approximately a 70% depression of the maximal 1,2-diacylglycerol release activity by phospholipase C. This desensitization is HDL3 dose dependent (IC50, 150 +/- 20 micrograms/mL, n = 6) and time dependent (t1/2, < 30 seconds). It requires the binding of HDL3, as pretreatment of HDL3 by tetranitromethane does not cause the desensitization of HDL3-induced phospholipase C activity. Permeabilization of human platelets with 10 micrograms/mL digitonin, used to permit access of charged inhibitors to the cytosol, does not interfere with the pattern of HDL3 (1 mg/mL)-induced desensitization of HDL3 (50 micrograms/mL)-stimulated phospholipase C. Inhibitors of protein kinase C (100 mumol/L H-7 and 10 mumol/L staurosporine) markedly inhibit desensitization of HDL3-induced phospholipase C activity, whereas cAMP-dependent protein kinase inhibitor (1 mumol/L), heparin (100 nmol/L), or concanavalin A (0.25 mg/mL) were ineffective. HDL3-induced desensitization is accompanied at least by the phosphorylation of the 94- and 110-kD proteins. Inhibition of HDL3-induced desensitization by 100 mumol/L H-7 or 10 mumol/L staurosporine is characterized by a marked reduction of the phosphorylation state of these proteins in permeabilized platelets. Whereas protein kinase C inhibitors fully inhibited the phosphorylation of the 94- and 110-kD proteins, inhibitors of protein kinase A were less effective. These data establish that phosphorylation by protein kinase C represent a step in the desensitization of HDL3 binding sites in human platelets.

Ovarian vitamin E accumulation: evidence for a role of lipoproteins.

Reactive oxygen species, such as superoxide, hydrogen peroxide, and lipid peroxides, impair luteal function. Vitamin E, a lipophilic antioxidant vitamin, provides a major avenue of protection by scavenging free radicals and terminating lipid peroxidation. We previously showed that ovarian vitamin E levels increase after functional regression (loss of progesterone production) of the corpus luteum in the pseudopregnant rat and the objective of the present studies was to determine the mechanism(s) that resulted in such increased levels of vitamin E. Luteal vitamin E levels were significantly elevated after function regression and remained elevated. Luteal cholesterol ester levels, in contrast, decreased in parallel with the decrease in plasma progesterone levels, whereas plasma vitamin E, cholesterol, and cholesterol ester levels did not change. Because vitamin E is transported in blood by chylomicrons and lipoproteins, ovarian vitamin E levels were determined after treatments known to modify ovarian lipoprotein receptor content and serum lipoproteins. Acute treatment with aminoglutethimide during the mid-luteal phase decreased serum progesterone levels and increased luteal vitamin E and cholesterol ester levels. Daily treatment with 4-amino-pyrazolo-(3,4-d)pyrimidine reduced serum vitamin E and cholesterol ester levels, diminished the accumulation of vitamin E associated with luteal regression, significantly reduced luteal cholesterol esters levels, and increased luteal high density lipoprotein-binding sites. Analysis of the distribution of vitamin E between a membrane/particulate pellet and a lipid droplet/granule cytosol before and after luteal regression revealed no changes. Vitamin E levels were divided 60:40 between a crude particulate/membrane fraction and a cytosol/lipid droplet fraction, although functional regression produced a 2.5-fold increase in total luteal vitamin E levels. In conclusion, the uptake of vitamin E by the corpus luteum appears to be mediated by lipoprotein receptors and the increase in vitamin E that follows functional regression, we suggest, may be due to a diminished consumption of vitamin E by oxidative radicals, most likely generated during steroidogenesis.

Analysis of the selective uptake of the cholesteryl ester of human intermediate density lipoproteins by HepG2 cells

We have recently shown by the analysis of the association to HepG2 cells of human intermediate density lipoproteins (IDL) either labeled in cholesteryl ester (CE) with [ 3H]cholesteryl ethers (CEt) or in proteins by iodine-125 that the CE of IDL are selectively taken up by these cells. Our results also revealed that the addition of a sufficient quantity of HDL3 abolishes the binding of IDL to the ‘Lipoprotein binding site’ (LBS) and also the CE-selective uptake. This suggested that the LBS mediates this uptake (Brissette and Falstrault (1992) Biochim. Biophys. Acta. 1165, 84–92). This study was undertaken to analyze further the mechanism of the selective uptake of the CE of IDL by HepG2 cells to determine if the LBS is directly or indirectly involved. We show that the different labeling had no effect on the binding affinity of IDL to HepG2 cells. To verify that the apolipoprotein moiety of HDL3 was responsible for the abolishment of the CE selective uptake, we have studied the effect of free apoA-I and apoA-II on the association of IDL. Our results demonstrate that apoA-I and apoA-II are approximately 10-times better than HDL3 or apoA-I liposomes in abolishing the selective uptake of the CE from IDL. We also show that this correlates with a more efficient reduction of the binding of 125I-IDL to HepG2 cells by free apoA-I compared to apoA-I associated with lipids. Thus free apoA-I interfere with the binding of IDL to the LBS and free apolipoproteins have a better capacity to saturate the LBS than lipoproteins. Also, we found no evidence for the transfer of CE from the labeled IDL to HDL3 or to apolipoproteins used to abolish the interaction of IDL to the LBS. Thus our results indicate that the LBS is directly responsible for the selective uptake of CE.

Decrease in high density lipoprotein binding sites is associated with decrease in intracellular cholesterol efflux in dedifferentiated aortic smooth muscle cells

One of the key features of atherosclerosis formation and progression is ‘dedifferentiation’ of contractile arterial smooth muscle cells (SMC) in synthetic cells. In primary cultures and subcultures before 10 and after 200 passages, SMC exhibit contractile-like, synthetic and transformed phenotypes, respectively, providing a good model for studing dedifferentiation process in vitro: the rationale for comparing these phenotypes to SMC in vivo rests in similar changes in cytoenzymatic and cytoskeletal features. In vivo, dedifferentiated SMC are transformed into foam cells by accumulating lipids. Thus, the aim of this study was to determine wether cholesterol metabolism undergoes changes in dedifferentiated cells and the three cultured phenotypes were compared in regard to their cholesterol efflux mechanisms. Phenotypic changes were shown to be associated with decrease in intracellular cholesterol apoprotein mediated efflux and translocation but also with decrease in high affinity binding sites for native HDL. Thus, the dedifferentiation process triggers a need for increased supply of cholesterol for membrane synthesis and efflux down-regulation mechanisms are aimed at maximizing cholesterol availability to the cell. Plasma membrane cholesterol efflux, which seems to be apoprotein-independent, decrease slightly with cell dedifferentiation suggesting either modifications in the dedifferentiated cell membranes physical properties. Taken together, these different results showed that dedifferentiation of arterial SMC is associated with decrease in the different steps of the efflux process, which could constitute one of the early events in their foam cell transformation.

Analysis of the lipoprotein binding site of rat liver membranes.

Intermediate density lipoproteins (IDL) were shown to bind to high- and low-affinity binding sites on rat liver membranes. The low-affinity sites were named lipoprotein binding sites (LBS), since they bind all classes of lipoproteins. This study was undertaken to further characterize the interaction of 125I-labelled IDL with the LBS of rat liver membranes to determine the chemical nature of the LBS. We found that the binding of IDL to the LBS is insensitive to EDTA and sensitive to heparin and that it is present on plasma membranes. Also, membranes were pretreated with various enzymes that have an effect on the membrane constituents, and the activity of the LBS on these treated membranes was determined. Our results reveal that the LBS of rat liver membranes is insensitive to heparinase I, chondroitinase ABC, and phospholipase C, while it is partially sensitive to phospholipase A2 and sensitive to proteases and heat. Rat liver membrane proteins were solubilized with Triton X-100, reconstituted in liposomes, and analyzed for their ability to bind lipoproteins. 125I-labelled IDL were shown to bind to high- and low-affinity sites that are similar, in affinity and specificity, to the ones observed with intact rat liver membranes, indicating that a LBS activity is detectable on these liposomes. We found that the binding capacity of low-affinity sites in liposomes containing either no protein or containing proteins solubilized from Escherichia coli membranes is five times weaker than low-affinity sites in liposomes containing liver membrane proteins. Thus, a protein solubilized from rat liver membranes has LBS activity when reconstituted in liposomes. Taken altogether our results provide new information on the binding of IDL to the LBS and indicate that the LBS activity is in part mediated by a protein. Thus, the LBS appears to be a bona fide receptor.

Characteristics of high-density lipoprotein binding sites in cultured parenchymal, endothelial, and Kupffer's cells from rat liver

Liver endothelial cells posses surface high-affinity binding sites for HDL3, whose affinity is 4 times higher than that of the sites on hepatocytes and Kupffer's cells. The maximal number of high-affinity binding sites on endothelial cells is 437 ng/mg protein, which surpasses this parameter on the hepatocyte surface several times.

Glucocorticoid-dependent regulation of the expression of high-density lipoprotein binding sites in rat hepatocytes

It is shown that dexamethasone increases the number of HDL binding sites in cultured hepatocytes bothin vivo andin vitro. The glucocorticoid acts in a dose-dependent and reversible manner within a wide range of concentrations, including physiological and subphysiological doses.

Comparative binding of bile acids to serum lipoproteins and albumin

Characteristics of the binding of lithocholic acid (LC), chenodeoxycholic acid (CDC), and cholic acid to human plasma proteins were studied. Affinity of the different plasma protein fractions for the bile acids studied decreased with increased polarity of the steroid nucleus of the bile acid. Binding of LC, CDC, and cholic acid to the lipoprotein-free, albumin-rich plasma fraction was characterized by two classes of binding sites with respective KDs of 2, 5, and 51 microM, and of 39, 2,387, and 5,575 microM, while corresponding Bmax values were similar for the different bile acids, at around 6 and 100 nmol/mg protein. Bile acid binding to the different lipoprotein fractions was characterized by a single population of binding sites, with a KD ranging from 47 to 66 microM for LC, 695 to 1010 microM for CDC, and 2,511 to 2,562 microM for cholic acid. Bmax values, at 416-913 nmol/mg protein, were similar among the different bile acids studied. Both glycine- and taurine-conjugated, as well as unconjugated LC competitively inhibited [24-14C]LC binding to low density (LDL) and to high density lipoproteins (HDL) to the same extent, while the more polar LC-3-sulfate, CDC, and cholic acid were increasingly less potent in displacing LC binding from the respective lipoproteins. Furthermore, all bile acids studied shared the same lipoprotein binding site. The lipoprotein fluorescence at 330-334 nm, following excitation at 280 nm, was diminished after incubation with LC, suggesting that the bile acid masks the tryptophan residues of the protein moiety. Finally, the initial rate of uptake of 1 microM LC, in isolated hamster hepatocytes, at around 0.045 nmol.sec-1.mg cell wt-1, was not affected by the protein carrier. However, for the same concentration of LC, bound to either LDL or HDL, LC binding resulted in 75-77% of the total [24-14C]LC nonspecifically bound to the hepatocyte, compared to 65% when bound to albumin, and 45% in the absence of protein. The studies show that, under conditions when the serum bile acid concentration exceeds the capacity of the high affinity class of albumin binding sites for bile acids, lipoproteins have similar or greater affinity to bind bile acids than does albumin. The ability of lipoproteins to increase the nonspecific association of lithocholic acid with liver cells may also facilitate bile acid association with extrahepatic tissues. As lipoproteins, in contrast to albumin, are targeted to most cells, they may play a major role in the transport of potentially toxic bile acids to peripheral cells.

Cholesterol synthesis and lipoprotein reuptake during synaptic remodelling in hippocampus in adult rats

Apolipoprotein E is synthesized and secreted by astrocytes in the hippocampus following lesions of the entorhinal cortex. It was proposed that apolipoprotein E, by analogy to its role in cholesterol transport in circulation, could be involved in the salvage and reutilization of non-esterified cholesterol released during terminal breakdown. The salvaged cholesterol could then be transported to neurons by apolipoprotein E-complexes and taken up via the apolipoprotein E/apolipoprotein B (low-density lipoprotein) receptor. To test this hypothesis, we have examined low-density lipoprotein receptor binding in brain sections of rats undergoing hippocampal reinnervation. The number of neuronal cells labelled by fluorescent Dil-low-density lipoprotein as well as the density of [ 125I]low-density lipoprotein binding sites in the dentate gyrus were found to increase in parallel with the extent of cholinergic reinnervation occurring in the deafferented hippocampus. In contrast, hippocampal cholesterol synthesis fell by more than 60% at eight days post-lesion, but eventually returned to control levels at 30 days post-lesion. The transient loss of cholesterol synthesis coincided with a peak in hippocampal apolipoprotein E expression. A concomitant accumulation of sudanophilic lipids (cholesterol esters and phospholipids) was detected in the outer molecular layer of the dentate gyrus and in the hilar region. The present findings suggest that non-esterified cholesterol released during terminal breakdown is esterified, transported via the apolipoprotein E transport system to neurons undergoing reinnervation, and take-up through the low-density lipoprotein receptor pathway where it is presumably used as a precursor molecule for the synthesis of new synapses and terminals.

Analysis of the binding and association of human intermediate density lipoproteins to HepG2 cells

The binding of human intermediate density lipoproteins (IDL) to HepG2 cells was studied. We found that human 125I-IDL interact with a binding site of high-affinity ( K d 0.74 μg/ml, B max 0.049 μg/mg cell protein) and a binding site of lower affinity ( K d 86.8 μg/ml; B max 0.53 μg/mg cell protein). The high-affinity binding sites show characteristics of LDL-recepto since they interact with IDL and low-density lipoprotcins (LDL) and are calcium dependent. The low-affinity binding sites are calcium-independent and interact with IDL, LDL, high density lipoproteins-3 (HDL 3), apolipoprotein (apo) E-liposomes, apoCs-liposomes, apoA-I-liposomes but not with liposomes containing albumin or erythrocyte membrane proteins. Therefore, HepG2 cells have on their surface a binding site that resembles or is identical to the lipoprotein binding site (LBS) that we found on rat liver membranes (Brissette and Noël (1986) J. Biol. Chem. 261, 6847-6852). Internalization, degradation and cholesterol ester selective uptake were determined in the presence or in the absence of a sufficient amount of human HDL 3 to abolish the interaction of IDL to the LBS in order to obtain information on the function of this site. Our results suggest that the LBS participates in the internalization of IDL but not in their degradation and that it is responsible for the selective uptake of cholesterol esters of IDL.

Platelets in patients with homozygous familial hypercholesterolemia are sensitive to Ca(2+)-mobilizing activity of low density lipoproteins.

While some data suggest that Ca(2+)-mobilizing effects of low density lipoprotein (LDL) in platelets are mediated by a specific membrane receptor, the data about the nature of this lipoprotein-binding site are contradictory. This work was performed in order to assess possible involvement of apolipoprotein (apo) B,E receptor, present in most cell types. To answer the question we compared effects of LDL in normal platelets and those obtained from patients with homozygous familial hypercholesterolemia (HFH), characterized by absence of functional apo B,E receptors. We have found that in accordance with previous results LDL induced instant reversible elevation of free cytoplasmic calcium concentration ([Ca2+]i) in fura-2-loaded platelets. The effect was observed both in healthy and HFH groups. Neither half-maximal effective concentrations nor maximal effects of LDL differed significantly between two groups. Ca(2+)-mobilizing effects of lipoproteins were potentiated about 4-fold by epinephrine and completely blocked by prostaglandin E1 both in platelets of healthy and HFH subjects. The similarity of lipoprotein effects in control and HFH platelets is evidence that apo B,E receptor does not mediate the Ca(2+)-mobilizing activity of LDL in this cell type.

Lipid utilization by human lymphocytes is correlated with high-density-lipoprotein binding site activity.

The nature and physiological importance of high-density lipoprotein (HDL) binding sites on unstimulated (resting) and mitogen-activated (blast) human peripheral blood lymphocytes were investigated. Specific HDL binding on resting and blast T-lymphocytes was saturable at 50 micrograms of 125I-HDL/ml and of high affinity, with Kd values of 8.1 x 10(-8) M and 6.5 x 10(-8) M, respectively, and Bmax. values of 79 ng and 180 ng/mg of cell protein respectively at 4 degrees C. Binding of HDL double-labelled with fluorescent dioctadecylindocarbocyanine (Dil) and isotope (125I) as well as of single fluorescence- or isotope-labelled HDL was inhibited competitively by HDL apoproteins. Studies of the cholesterol flux between the cells and HDL showed that HDL, low-density lipoprotein (LDL) or BSA at a concentration of 100 micrograms/ml in the tissue culture medium did not result in a significant difference in exogenous [3H]cholesterol efflux from the cell membrane at 37 degrees C. Proliferating T-blasts incorporated more cholesterol from HDL or LDL than did resting lymphocytes. When the cells were pulsed with 125I-HDL and chased in fresh lipid-free medium, up to 80% of the radioactivity released was not precipitable with trichloroacetic acid. This percentage decreased in a competitive manner when unlabelled HDL was present in the chase incubation medium. Finally, cultivation of lymphocytes with conditioned medium from macrophages increased Dil-HDL binding/uptake, while it was decreased by mevinolin-induced inhibition of hydroxymethylglutaryl-coA reductase. In conclusion, human lymphocytes possess a HDL binding site (receptor) responsible for lipid binding/uptake and concomitant internalization and degradation of apoproteins from HDL, but not for reverse cell membrane cholesterol transport. The activity of the binding site is up-regulated during cell proliferation and down-regulated during cell growth suppression.

Characteristics of the binding of human and bovine high-density lipoproteins by bloodstream forms of the African trypanosome, Trypanosoma brucei brucei

Bloodstream forms of Trypanosoma brucei brucei are unable to synthesize cholesterol but appear to bind and take up plasma low-density lipoproteins (LDL) from their host. Whether cholesterol homeostasis of this unicellular parasite also requires interactions with host high-density lipoprotein (HDL) particles is unknown. Equilibrium binding of radioiodinated apolipoprotein E-depleted human HDL 3 ( d = 1.125−1.21 g/ ml) and bovine HDL ( d = 1.063−1.21 g/ ml) by T.b.brucei was rapid (< 30 min) at 4°C and was characterized by a saturable, specific component. There were five times the number of high-affinity binding sites for human HDL 3 as for bovine HDL (64,000 vs. 11,500 per trypanosome) and their binding affinity was greater with an equilibrium dissociation constant ( K d ) of 157 nM compared to 315 nM for bovine HDL). Binding of rat and rabbit HDL 3 was similar to bovine HDL. By contrast, equilibrium binding of human LDL was slower (~ 6 h) and the number of high-affinity binding sites ( K d = 23 nM) was much lower for this ligand (660 per trypanosome). Total binding of HDL 3 was independent of divalent cations and was only slightly inhibited by heparin, but when the trypanosomes were preincubated with trypsin or pronase the binding was markedly reduced. After 30 min at 37°C, binding of bovine HDL and human HDL 3 was 10–20% higher than at 4°C; after 45 min trypanolysis occurred with human HDL 3 but not with bovine HDL. Chemical modification of HDL 3 by treatment with cyclohexanedione, by acetylation or by reductive alkylation had little effect on its ability to compete with [ 125I]labelled HDL 3 for binding by the parasite. Nitrosylation of HDL 3 with tetranitromethane increased its binding ability, suggesting that trypanosomes might possess scavenger receptors, and native HDL 3 was less effective than nitrosylated HDL 3 in displacing bound [ 125I]labelled nitrosylated HDL 3. These findings suggest that, in addition to a receptor for LDL, T.b.brucei has other lipoprotein binding sites which separately recognize HDL from permissive host species such as bovine, trypanolytic HDL such as human HDL 3, and more negatively charged HDL particles such as nitrosylated HDL 3.

The human asialoglycoprotein receptor is a possible binding site for low-density lipoproteins and chylomicron remnants.

Binding and internalization of chylomicron remnants from rat mesenteric lymph by HepG2 cells was inhibited by both excess remnants and low-density lipoprotein (LDL) to the same extent. Ligand blots revealed binding of remnants and LDL to the LDL receptor. Measures regulating LDL receptor activity greatly influenced the binding of remnants: ethinyloestradiol, the hydroxymethylglutaryl-CoA reductase inhibitor pravastatin and the absence of LDL all increased binding, whereas high cell density or the presence of LDL decreased binding. Also, asialofetuin, asialomucin, the neoglycoprotein galactosyl-albumin and an antibody against the asialoglycoprotein receptor all decreased substantially the binding of remnants. At high cell density, binding internalization and degradation of chylomicron remnants was inhibited by up to 70-80%, yet binding of LDL was inhibited by no more than 20-30%. In cross-competition studies, the binding of 125I-asialofetuin was efficiently competed for by asialofetuin itself or by the antibody, and also by LDL and remnants, yet remnants displayed an approx. 100-fold higher affinity than LDL. Likewise, remnants of human triacylglycerol-rich lipoproteins and asialofetuin interfered with each others' binding to HepG2 cells or human liver membranes. It is concluded that the LDL receptor mediates the internalization of chylomicron remnants into hepatocytes depending on its activity, according to demand for cholesterol. Additionally, the asialoglycoprotein receptor may contribute to the endocytosis of LDL, but predominantly of chylomicron remnants.

Characterization of high-density lipoprotein binding to guinea pig hepatic membranes: Effects of dietary fat quality and cholesterol feeding

The effects of dietary fat quality and cholesterol intake on expression of guinea pig hepatic membrane high-density lipoprotein (HDL) binding sites were studied. Animals were fed semisynthetic diets containing 7.5% (wt/wt) of either corn oil (CO), olive oil (OL), or lard. The cholesterol diet was prepared by incorporating 0.25% recrystallized cholesterol into standard guinea pig chow. Plasma cholesterol levels of guinea pigs on the CO diet were significantly lower ( P < .02) than animals on the OL or lard diets. HDL cholesterol levels did not differ between the polyunsaturated, monounsaturated, and saturated dietary fat groups. Guinea pigs on the high cholesterol diet had increased total and HDL cholesterol levels compared with animals on the chow diet ( P < .01). Initial studies demonstrated that HDL binding to hepatic membranes was temperature-dependent. A threefold increase in binding was observed when assays were performed at 37°C, as compared with 4°C, for all membrane preparations. Dietary fat quality and dietary cholesterol intake significantly altered HDL binding to hepatic membranes with increased HDL binding to membranes of animals fed polyunsaturated fat and the high cholesterol diet. At 37°C, HDL binding to hepatic membranes of CO-fed animals was 26% and 46% higher than for membranes of OL- and lard-fed guinea pigs, respectively. A high cholesterol intake increased HDL binding by 24% at both 4°C and 37°C. Scatchard analysis demonstrated that while membrane affinity for HDL (K d) was not affected by diet, changes did occur in the total number of HDL binding sites. B max values were significantly higher ( P < .05) for membranes from cholesterol- and CO-fed guinea pigs. These data demonstrate that in the guinea pig, HDL binding to hepatic membranes is regulated by both dietary fat quality and dietary cholesterol.

A specific binding site for lipoproteins containing apolipoprotein A-I on human hepatoma cell line HepG2.

Interactions of lipoproteins containing apolipoprotein (apo) A-I with or without apoA-II with human hepatoma cell line HepG2 were studied to investigate the ligand specificity for high density lipoprotein receptor on human hepatic cells and their metabolism. The two types of lipoproteins were isolated by immunoaffinity chromatography, in which apoE-containing lipoproteins were removed. Specific binding kinetics at 0 degrees C were observed for the apoA-I-containing lipoproteins with or without apoA-II (Kd = 18 or 20 micrograms protein/ml, Bmax = 110 or 120 ng/mg cell protein, respectively). The binding of these lipoproteins to HepG2 cells was competitively inhibited by excess unlabeled apoA-I-containing lipoproteins or apoA-I-phospholipid complexes, but not by apoA-II.phospholipid complexes. Interactions of these lipoproteins with HepG2 cells at 37 degrees C were further examined. These results suggested that HepG2 cells have a specific binding site for apoA-I-containing lipoproteins, and that apoA-I might be a crucial component in the binding of these lipoproteins to human hepatic cells.