Phospholipases A2 of rod outer segment-free bovine retinae are different from well-known phospholipases A2.

We have recently demonstrated the presence of phospholipase A2 (PLA2) activity in a rod outer segment-free retinal fraction which we called P200 and which contains neuronal cells, Müller cells and rod inner segments. We report here our results on the characterization of this P200-PLA2 activity. We show that P200 probably contains more than one type of PLA2, as indicated by the results obtained with different chromatographically eluted PLA2-active fractions which were treated with either Ca2+, EGTA, dithiothreitol (DTT) or p-bromophenacyl bromide (pBPB), or heated. Moreover, the results from PLA2 assays using different substrates, as well as those obtained after treatment of the homogenate with H2SO4, guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) and ATP, suggest that P200-PLA2 are different from well-known secretory PLA2, cytosolic PLA2 and Ca2+-independent PLA2. Control experiments using our 'back-and-forth'-thin layer chromatography (bf-TLC) technique allowed us to confirm that, in our assay conditions, the release of fatty acids was due to PLA2 enzymes. These results, which constitute the first characterization of PLA2 of the neural retina, thus suggest that it contains novel types of PLA2 enzyme, in contrast to well-known PLA2.

Bovine retinal pigment epithelium contains novel types of phospholipase A2.

We have recently demonstrated the presence of phospholipase A2 (PLA2) activity in cells from bovine retinal pigment epithelium (RPE) [Jacob et al. (1996) J. Biol. Chem. 271, 19209-19218]. We report here our results on the characterization of this RPE-PLA2 activity. We show that RPE probably contains two types of PLA2 enzyme, as indicated by the results obtained with different PLA2-active fractions eluted from cation-exchange columns and treated with Ca2+/EGTA, dithiothreitol, p-bromophenacyl bromide or heat. These results, in addition to those from PLA2 assays using different substrates, also suggest that RPE-PLA2 enzymes are different from the well-known secretory, cytoplasmic and Ca2+-independent forms. Sequential extraction of RPE with (1) isotonic, (2) hypertonic and (3) detergent-containing PBS argues for the presence of weakly membrane-associated enzymes. Control experiments using 'back and forth' TLC allowed us to discriminate between PLA2 and phospholipase C/diacylglycerol lipase activity and confirmed that, in our assay conditions, the release of fatty acids was indeed due to PLA2 enzymes. These results, together with those obtained by treating RPE homogenates with H2SO4, guanosine 5'-[gamma-thio]triphosphate, ATP and different protease inhibitors, permitted us to make the first characterization of these RPE-PLA2 enzymes. We conclude that RPE contains novel types of PLA2 that are different from the secretory, cytoplasmic and Ca2+-independent forms.

Cytosolic phospholipase A2, cyclo-oxygenases and arachidonate in human stomach tumours.

Human stomach tumours usually form more prostaglandins (PGs) than their associated normal mucosa/submucosa, but the mechanisms are not fully understood. The key enzymes are cytosolic phospholipase A2 (cPLA2, Mr 85,000) and the cyclo-oxygenases (COXs) which exist in constitutive (COX-1) and inducible forms (COX-2). In human stomach tumours and associated macroscopically normal tissues, we determined the fatty acid composition by gas chromatography, amounts of cPLA2, COX-1 and COX-2 by immunoblotting with specific antibodies and cPLA2 enzyme activity using a tritiated substrate. Although compared to normal mucosa there was less arachidonate in tumours (P < 0.05), the arachidonate/total fatty acid ratio was higher. Mean amounts of cPLA2 and COX-1 and cPLA2 activity were similar in tumours and normal mucosa. However, substantial amounts of COX-2 were found in the tumours but not in the mucosa, which may explain why many gastric tumours form increased amounts of PGs.

Evolution of mammalian apolipoprotein A-I and conservation of antigenicity: correlation with primary and secondary structure.

We have evaluated the immunoreactivity of 20 monoclonal antibodies (mAbs) directed against human apolipoprotein (apo)A-I with a panel of high density lipoproteins (HDL) from 13 mammalian species. The pattern of cross-reactivity showed that 20 mAbs had different specificity. While not all mAbs recognized apoA-I from all of the different species, the antigenicity of some sequences was well conserved. Thus, mAb A05 cross-reacted with all species except guinea pig and rat. In contrast, the mAb 4H1, which recognized residues 2-8, required a specific proline in position 3, as no immunoreactivity was found in the species missing this amino acid. Furthermore, the presence of a threonine residue in place of serine (in position 6) in the cynomolgus monkey was associated with a 20-fold loss of immunoreactivity in radioimmunometric assay with 4H1. As most of the epitopes were found in CNBr fragments 2 and 3, we sequenced these regions in four species (horse, goat, sheep, and cat) and analyzed the alignment of most known sequences to evaluate their consensus. Except for the rat and the chicken, considerable identity was observed. This permitted us to deduce the involvement of the residues in some antigenic epitopes. In the middle of apoA-I, a conservative mutation Asp103-->Glu was found sufficient to eliminate all reactivity of this epitope for A11 (residues 99-105 ... 12l6-132) in five species (rabbit, cow, goat, sheep, and rat). The residues essential to the expression of two other epitopes overlapping with A11 were also characterized. Edmundson-wheel representation of 18-residue repeated sequences of the different apoA-I species (for the eight amphipatic helices of residues 46-63, 68-85, 101-118, 123-140, 143-160, 167-184, 189-206, and 222-239) showed that secondary structure of apoA-I was more conserved than the antigenic epitopes. The N-terminal region, residues 1 to about 98, is rich in both strictly preserved sequences and epitope expression in most of the species surveyed. This evolutionary conservation of the N-terminal domain suggests an important yet unknown function.

High molecular weight phospholipase A2 and fatty acids in human colon tumours and associated normal tissue.

Human colon tumours usually form more prostaglandins (PGs) than associated normal tissues, but the mechanism(s) are not fully understood. We analysed fatty acid compositions, in particular arachidonate, and measured the amount and the activity of high molecular weight cytoplasmic phospholipase A2 (cPLA2) of these tissues. Total lipids extracted from homogenised surgical specimens were transesterified and fatty acids were analysed by gas chromatography. cPLA2 was separated by SDS-PAGE, Western-blotted, immunoblotted using a specific antibody to cPLA2 and semiquantified following enhanced chemiluminescence using a scanning densitometer. cPLA2 biological activity was also assayed using 1-stearoyl, 2-[1-14C]-arachidonyl, L-3-phosphatidylcholine. Compared with normal mucosa/submucosa, there was more total arachidonate in tumours (P < 0.01), and increased levels of cPLA2 occurred in 6 of 17 tumours. In conclusion, the higher amounts of tumour total arachidonate and the sometimes higher levels of cPLA2, might help to explain why some human colon tumours form increased amounts of PGs.

Presence of a light-independent phospholipase A2 in bovine retina but not in rod outer segments.

Rod outer segments (ROS) are responsible for the visual transduction process. Rhodopsin, which constitutes 85-90% of ROS proteins, absorbs light photons, changes its conformation, and then binds to a heterotrimeric G-protein called transducin. As a consequence, transducin dissociates into Talpha and Tbetagamma subunits. The presence in ROS of a phospholipase A2 (PLA2) stimulated by light and guanosine 5'-O-(3-thio)triphosphate was first demonstrated in 1987 (Jelsema, C. L.(1987) J. Biol. Chem. 262, 163-168). This led that author to conclude that ROS PLA2 could be involved in the phototransduction process, and raised the possibility of receptor-mediated activation of PLA2 via G-proteins in cell types other than rods. However, the biochemical characteristics and the role of this PLA2 have not been fully elucidated. We have tried to reproduce some of the results previously reported in order to further characterize this enzyme. We have found that, in our hands, there is neither light-dependent nor GTP-dependent PLA2 activity in intact purified ROS. We also failed to detect PLA1 activity in those ROS preparations. Nevertheless, we detected significant amounts of PLA2 activity in two subretinal fractions adjacent to ROS: RPE (enriched with retinal pigment epithelial cells) and P200 (presumably containing neuronal cells, Müller cells, and rod inner segments). The enzyme present both in RPE and P200 is light- and GTP-independent, Ca2+- and Mg2+-independent, and seems to be optimally active in the alkaline pH range. Our results suggest that there is, if any, vanishingly little PLA2 or PLA1 activity in intact purified ROS and that the activity levels previously reported in the literature could have been due to a contamination by either RPE or P200. This is supported by our observation that some contaminated ROS preparations were "PLA2 active."

Colocalization of cytosolic phospholipase A2, 5-lipoxygenase, and 5-lipoxygenase-activating protein at the nuclear membrane of A23187-stimulated human neutrophils.

The distribution of cytosolic phospholipase A2 (cPLA2), arachidonate 5-lipoxygenase, and 5-lipoxygenase-activating protein (5-LAP) was investigated in subcellular fractions of human neutrophils disrupted by three techniques. As determined by immunoblot analysis, the bulk of cPLA2 and 5-lipoxygenase was detected in cytosolic fractions of unstimulated neutrophils disrupted by sonication or cavitation. After cell stimulation with the calcium ionophore A23187, both proteins accumulated primarily in nuclei-containing fractions; this accumulation was accompanied by a loss of these enzymes from cytosolic fractions. Further resolution of nuclear fractions revealed that 5-lipoxygenase and cPLA2 were localized in a fraction that contained nuclear membranes. In comparison, 5-LAP was localized to the nuclear-membrane fraction of resting and activated neutrophils, as determined by immunoblotting and photoaffinity labeling. In agreement with the immunoblot data, A23187 stimulation markedly enhanced 5-lipoxygenase enzymatic activity in the nuclear-membrane fraction, which was accompanied by decreased cytosolic 5-lipoxygenase activity. Similarly, neutrophil activation caused increased phosphorylation of cPLA2, a process that is known to result in enhanced catalytic activity. Our data demonstrate that in activated human neutrophils, the key proteins involved in leukotriene synthesis colocalize at the nuclear membrane, in a catalytically active state.

A continuous fluorescence-based assay for the human high-molecular-weight cytosolic phospholipase A2.

A sensitive method for continuously monitoring the activity of the human cytosolic phospholipase A2 (cPLA2) is described. Recombinant cPLA2 efficiently hydrolyzes fatty acid esters of 7-hydroxycoumarin, producing the free fatty acid and the highly fluorescent 7-hydroxycoumarin. All of the observed 7-hydroxycoumarinyl ester hydrolase activity (7-HCEase) in a preparation of the purified recombinant cPLA2 was due to this enzyme since: (1) all of the ester hydrolase activity comigrated on nondenaturing polyacrylamide gel with a protein characterized as the cPLA2 by Western analysis; (2) the immunoreactive protein also possessed both phospholipase A2 and lysophospholipase activities; and (3) arachidonyl trifluoromethyl ketone, a potent inhibitor of the phospholipase A2 activity of cPLA2, also inhibited the 7-HCEase activity. A study of the 7-HCEase activity demonstrated that when 7-hydroxycoumarinyl gamma-linolenate was dispersed in a phospholipid matrix it was hydrolyzed by cPLA2 at a rate comparable to that of an arachidonyl-containing phospholipid substrate and with an identical reaction progress curve. In the presence of phospholipid vesicles, the cPLA2-catalyzed hydrolysis of hydrophobic 7-hydroxycoumarinyl esters was stimulated by submicromolar concentration of free calcium and showed a preference for polyunsaturated substrates. The cPLA2-catalyzed hydrolysis of the water-soluble substrate 7-hydroxycoumarinyl 6-heptenoate was catalyzed by cPLA2 in the absence of calcium and other lipids.

Arachidonyl trifluoromethyl ketone, a potent inhibitor of 85-kDa phospholipase A2, blocks production of arachidonate and 12-hydroxyeicosatetraenoic acid by calcium ionophore-challenged platelets.

Arachidonyl trifluoromethyl ketone (AACOCF3) is a potent and selective slow binding inhibitor of the 85-kDa cytosolic phospholipase A2 (cPLA2) (Street, I. P., Lin, H.-K., Laliberté, F., Ghomashchi, F., Wang, Z., Perrier, H., Tremblay, N. M., Huang, Z., Weech, P. K., and Gelb, M. H. (1993) Biochemistry 32, 5935-5940). AACOCF3 and a number of its structural analogues have been used to investigate the role of cPLA2 in the cellular generation of free arachidonic acid (AA) and in eicosanoid biosynthesis. AACOCF3 inhibited the release of AA from calcium ionophore-challenged U937 cells (IC50 = 8 microM, 2 x 10(6) cells ml-1) and from platelets (IC50 = 2 microM, 4 x 10(7) cells ml-1). Arachidonyl methyl ketone (AACOCH3) and AACH(OH)CF3, both of which are noninhibitory to the purified cPLA2, did not inhibit the production of AA in the ionophore-challenged cells. In addition to the release of AA, AACOCF3 also inhibited the production of 12-hydroxyeicosatetraenoic acid (12-HETE) and thromboxane B2, two of the major metabolites of AA produced by platelets. The inhibition of 12-HETE biosynthesis showed a dose dependence similar to that of AA release in ionophore-challenged platelets; however, when platelet 12-HETE production was stimulated with 10 microM AA to circumvent the PLA2-dependent step, AACOCF3 no longer inhibited the production of 12-HETE. In contrast, AACOCF3 blocked thromboxane B2 formation by both calcium ionophore- and AA-challenged platelets, indicating that the compound affects the cyclooxygenase pathway in addition to AA release. The crude cytosol and membrane fractions from platelets were assayed for calcium-dependent and calcium-independent PLA2 activities and for the susceptibility of each to inhibition by AACOCF3. At AACOCF3 concentrations as high as 10 mol %, only one of the observed PLA2 activities was inhibited by more than 25%. The AACOCF3-susceptible PLA2 (77% inhibition at 1.6 mol %) was found in the cytosolic platelet fraction and showed the functional characteristics of the cPLA2. These results suggest that the cPLA2 plays an important role in the generation of free AA for 12-HETE biosynthesis in platelets.

NMR structural studies of the tight complex between a trifluoromethyl ketone inhibitor and the 85-kDa human phospholipase A2.

Arachidonyl trifluoromethyl ketone (AACOCF3) is a slow- and tight-binding inhibitor of the human cytosolic phospholipase A2 (cPLA2) [Street et al. (1993) Biochemistry 32, 5935]. 19F and 13C NMR experiments have been carried out to elucidate the structure of the cPLA2.AACOCF3 complex. One mole of AACOCF3 per mole of enzyme is tightly bound in the active site while excess molar equivalents of the inhibitor associate loosely and nonspecifically with hydrophobic regions of the protein. Incubation of the cPLA2.AACOCF3 complex with a 10-fold molar excess of a structurally related inhibitor allows the slow dissociation of the enzyme-inhibitor complex to be followed with 19F NMR. These results establish that the bound inhibitor is in slow exchange with the free ligand and that inhibition of the cPLA2 by AACOCF3 is not due to irreversible modification of the protein. AACOCF3 labeled with 13C at the carbonyl position was used to determine the nature of the bound inhibitor species. A comparison of the 13C NMR chemical shift value obtained from labeled enzyme-inhibitor complex (delta c 101.0 ppm) with the chemical shift values obtained from model compounds suggests that the enzyme-bound inhibitor species is a charged hemiketal. These results are very similar to those obtained previously with alpha-chymotrypsin and a peptidyl trifluoromethyl ketone inhibitor [Liang, T.-C., & Abeles, R. H. (1987) Biochemistry 26, 7603] and, by analogy with the serine proteases, a structural model for the cPLA2.AACOCF3 complex is proposed.

Human group II phospholipase A2 expressed in Trichoplusia ni larvae--isolation and kinetic properties of the enzyme.

Human secreted synovial fluid/platelet-type group II phospholipase A2 (sPLA2) was expressed in Trichoplusia ni (cabbage looper) larvae and cultured Sf9 insect cells by infection with a recombinant baculovirus. Active sPLA2, with correct N-terminal proteolytic processing, was not secreted by Sf9 cells in culture. The enzyme was isolated from their homogenate without any need for refolding or renaturation of the protein. The enzyme was extracted from the 5000g pellet with 1 M KBr and isolated by chromatography on a cation exchange column followed by reverse-phase chromatography on a Butyl Aquapore column. The yield of active enzyme (25 micrograms/g insect) was comparable to yields obtained in CHO cells or Escherichia coli by other investigators. The recombinant enzyme had the correct N-terminal sequence, expected molecular weight, and reacted with antisera raised against peptides inferred from the cDNA sequence of the natural enzyme. Monoclonal antibodies were raised against the recombinant sPLA2 and they permitted the isolation of the natural enzyme from human serum by immunoaffinity. The recombinant sPLA2 showed a preference for substrate vesicles with a net negative charge. The baculovirus expression system provided active sPLA2 that can be produced economically in insects, purified simply, had well-defined kinetic properties, and should be useful in studies of inflammatory disorders.

Slow- and tight-binding inhibitors of the 85-kDa human phospholipase A2.

A trifluoromethyl ketone analogue of arachidonic acid in which the COOH group is replaced with COCF3 (AACOCF3) was prepared and found to be a tight- and slow-binding inhibitor of the 85-kDa cytosolic human phospholipase A2 (cPLA2). Enzyme inhibition was observed when AACOCF3 was tested in assays using either phospholipid vesicles or phospholipid/Triton X-100 mixed micelles. The fact that the inhibition developed over several minutes in both assays establishes that AACOCF3 inhibits by direct binding to the enzyme rather than by decreasing the fraction of enzyme bound to the substrate interface. From the measured values of the inhibitor association and dissociation rate constants, an upper limit of the equilibrium dissociation constant for the Ca(2+).AACOCF3.PLA2 complex of 5 x 10(-5) mole fraction was obtained. Thus, detectable inhibition of cPLA2 by AACOCF3 occurs when this compound is present in the assay at a level of one inhibitor per several thousand substrates. Arachidonic acid analogues in which the COOH group is replaced by COCH3, CH(OH)CF3, CHO, or CONH2 did not detectably inhibit the cPLA2. The arachidonyl ketones AACOCF2CF3 and AACOCF2Cl were found by 19F NMR to be less hydrated than AACOCF3 in phospholipid/Triton X-100 mixed micelles, and compared to AACOCF3 these compounds are also weaker inhibitors of cPLA2. In keeping with the fact that cPLA2 displays substrate specificity for arachidonyl-containing phospholipids, the arachidic acid analogue C19H39COCF3 is a considerably less potent inhibitor compared to AACOCF3.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytosolic phospholipase A2 from U937 cells: size of the functional enzyme by radiation inactivation.

We have studied the cytosolic phospholipase A2 (cPLA2) of human U937 cells by radiation inactivation in order to characterize the functional form of the native enzyme by a method that was independent of the discrepancies observed by SDS-PAGE and cDNA cloning. The Radiation Inactivation Size of cPLA2 was reproducible and gave a value of 76,800-80,100 daltons. We eluted the active enzyme from polyacrylamide-gradient gel electrophoresis at a molecular weight of 77,000, confirming the irradiation result. We conclude that cPLA2 is active as the monomeric enzyme and is composed of a single major functional domain that is sensitive to irradiation.

Localization of the major sites of rabbit apolipoprotein D gene transcription by in situ hybridization.

We have identified the sites of transcription of the apolipoprotein D (apoD) gene in the rabbit by in situ hybridization. We show here for the first time that 1) apoD mRNA production does not correlate with the sites of steroid hormone production in adrenal glands nor male genital tissues; and 2) the expression of the apoD gene is clearly higher in white than in gray matter throughout the central nervous system (CNS). Specifically, apoD mRNA was abundant near blood vessels and was expressed mostly in fibroblast-like cells, in particular in the testis, the efferent ducts, the ductus epididymis, the lung, and the subarachnoid space of the CNS. Other positive cell types were endothelial cells of adrenal sinusoidal capillaries and glial cells of the CNS. We detected apoD mRNA in both the adrenal cortex and medulla. White but not gray matter showed high levels of apoD mRNA throughout both the rabbit CNS and in human brain. The red pulp of spleen showed a strong hybridization. In prepubertal rabbits apoD mRNA levels were moderate in both testis and epididymis. Epididymal but not testicular expression increased with the onset of puberty and epididymal levels always exceeded those of the testes in animals showing spermatogenesis. Thus, the variation in levels of apoD mRNA among organs in vivo, that we and others have previously reported, can be explained by transcription being not only characteristic of cell type, with a few common cell types producing in each organ, but transcription also varied among cells of the same lineage.

Apolipoprotein D transcription occurs specifically in nonproliferating quiescent and senescent fibroblast cultures.

We studied apolipoprotein D (apoD) mRNA in primary cultures of human diploid fibroblasts (HDF). In early-passage HDF no apoD mRNA was detected in replicating cells in sparse culture, but the gene was expressed in quiescent cells in confluent and in serum-starved cultures. In contrast, late-passage HDF expressed apoD mRNA in sparse culture, but the level increased after attainment of confluence. Thus fibroblasts, the common cell-type expressing apoD mRNA in vivo, express this characteristic following growth-arrest. The same pattern of activation was found in another fibroblast cell line deficient in apoB/E (LDL) receptors, excluding a role for cellular cholesterol delivery by the LDL-receptor pathway controlling apoD expression.

Apolipoprotein D--an atypical apolipoprotein.

The structure of ApoD and its sites of synthesis have been discovered. These characteristics differ from those of the other apolipoproteins. The role of ApoD in the plasma lipoprotein system remains to be discovered, but the recent, rapid increase in our knowledge of this protein suggests that it plays an important role in the homeostasis or housekeeping of probably all organs. One of its functions is likely to be the transport of a hydrophobic ligand (a lipid) in a one-to-one molar ratio with itself. This transport is likely to occur unidirectionally between neighboring cells in an organ, and between perivascular cells and the blood circulation. The chemical structure of the natural ligand, or ligands, of ApoD in normal cells in vivo or in culture is not known, but ApoD has been shown to bind some steroids and bilirubin. Remarkable upregulation of synthesis of ApoD has been observed during regeneration of injured peripheral nerves. Perhaps the physiologic role of ApoD will prove to be more interesting and of equal importance in biology to the roles of the other apolipoproteins in cardiovascular disease.

Molecular characterization and differential mRNA tissue distribution of rabbit apolipoprotein D.

We report for the first time the quantification of relative apolipoprotein D (apoD) mRNA concentrations in a wide selection of organs and a detailed characterization of the rabbit protein. ApoD cDNA clones were isolated from a rabbit testis cDNA library by screening with a human apoD cDNA-derived RNA probe. The 912 nucleotide sequence of rabbit apoD cDNA contains a unique reading frame coding for a protein sharing 80% homology with human apoD. The two sequences have two potential asparagine-linked glycosylation sites at the same positions, almost superimposable hydrophobicity plot, and the antigenic proteins show similar charge polymorphism, Mr, and lipoprotein distribution. This high degree of similarity shows that the rabbit system can be used as a model for apoD studies. Moreover, the two consensus sequences of the hydrophobic ligand carrier (alpha 2-microglobulin) family present in human apoD are also found in the rabbit protein and these sequences coincide with the most conserved regions. The distribution of apoD mRNA among rabbit organs was determined by Northern blot and quantitative dot blot analysis. The highest levels of mRNA were found in spleen, adrenal glands, lungs, brain, testis, and kidneys. Moderate or low concentrations were detected in all the other organs tested including liver and small intestine. Thus, our results show that the apoD gene is expressed mainly in peripheral organs, with levels as high as 59-fold that of the liver, unlike other apolipoproteins. We suggest that apoD exerts its main function locally in peripheral organs.

Distribution and concentration of cholesteryl ester transfer protein in plasma of normolipemic subjects.

A MAb (TP-2) directed against human cholesteryl ester transfer protein (CETP) has been applied to the development of a competitive solid-phase RIA. Experiments with immobilized CETP have shown that upon incubation with plasma or HDL in the presence of Tween (0.05%) apo A-I (but not apo A-II) binds to CETP while TP-2 binding to CETP is concomitantly decreased. With high detergent concentration (0.5% Triton), the interference is eliminated and a specific RIA in which all plasma CETP fractions have the same affinity can be obtained. Plasma levels of CETP, apo A-I, lipids, and lipoproteins were measured in 50 normolipemic, healthy subjects of both sexes. CETP levels varied nearly fourfold with a mean value of 1.7 micrograms/ml. CETP was positively correlated only with apo A-I (r = 0.38) and HDL-triglyceride (r = 0.39). In 29 other normolipemic subjects, where several apolipoproteins were also measured, significant correlations of CETP with apo A-I (0.41), apo E (0.43), and HDL-cholesterol (0.41) were observed, but there was no significant relationship between CETP and either apo A-II, B, or D. In other experiments CETP was shown to be present mostly in HDL3 and VHDL, to display exclusively an alpha 2-electrophoretic migration, and to occur within discrete particles ranging in size from 129 to 154 kD. In conclusion, the association of CETP with apo A-I-containing lipoproteins probably explains the correlation between CETP and apo A-I levels. The relationship between CETP and apo E suggests either a common metabolism or a specific cooperative role in cholesterol ester transport for these proteins.

The use of monoclonal antibodies to localize the low density lipoprotein receptor-binding domain of apolipoprotein B.

Human apolipoprotein (apo) B-100 is composed of 4536 amino acids. It is thought that the binding of apoB to the low density lipoprotein (LDL) receptor involves an interaction between basic amino acids of the ligand and acidic residues of the receptor. Three alternative models have been proposed to describe this interaction: 1) a single region of apoB is involved in receptor binding; 2) groups of basic amino acids from throughout the apoB primary structure act in concert in apoB receptor binding; and 3) apoB contains multiple independent binding regions. We have found that monoclonal antibodies (Mabs) specific for a region that spans a thrombin cleavage site at apoB residue 3249 (T2/T3 junction) totally blocked LDL binding to the LDL receptor. Mabs specific for epitopes outside this region had either no or partial ability to block LDL binding. In order to define the region of apoB directly involved in the interaction with the LDL receptor we have tested 22 different Mabs for their ability to bind to LDL already fixed to the receptor. A Mab specific for an epitope situated between residues 2835 and 2922 could bind to its epitope on LDL fixed to its receptor whereas a second epitope between residues 2980 and 3084 is inaccessible on receptor-bound LDL. A series of epitopes near residue 3500 of apoB is totally inaccessible, and another situated between residues 4027 and 4081 is poorly accessible on receptor-bound LDL. In contrast, an epitope that is situated between residues 4154 and 4189 is fully exposed. Mabs specific for epitopes upstream and downstream of the region 3000-4000 can bind to receptor-bound LDL with a stoichiometry close to unity. Our results strongly suggest that the unique region of apoB directly involved in the LDL-receptor interaction is that of the T2/T3 junction.