Degradation of apolipoprotein B-100 of human plasma low density lipoproteins by tissue and plasma kallikreins

Human plasma low density lipoproteins (LDL) contain one major apoprotein of apparent Mr = 550,000 designated apolipoprotein B-100 (apo-B-100) and in some LDL preparations, minor components termed apo-B-74 (Mr = 410,000) and apo-B-26 (Mr = 145,000). The structural and metabolic relationships among these LDL apoproteins remain obscure. In the present study, we show that the mixing of proteolytic inhibitors with blood at the moment of collection prevents the appearance of apo-B-74 and -26 in plasma LDL indicating that these peptides are derived by proteolytic degradation of apo-B-100. In order to simulate the degradation in vitro, LDL were digested with plasmin, trypsin, chymotrypsin, thrombin, and tissue and plasma kallikreins and the degradation products analyzed by polyacrylamide gradient gel electrophoresis. While plasmin, trypsin, and chymotrypsin caused extensive degradation of apo-B-100, thrombin, and tissue and plasma kallikreins generated limited cleavage patterns. LDL digested with thrombin contained stoichiometric amounts of two peptides with apparent Mr = 385,000 and 170,000. Mixing experiments showed that the thrombin-derived peptides of apo-B-100 did not co-migrate with apo-B-74 and B-26 during electrophoresis indicating that these peptides were different. In contrast, LDL digested with kallikrein contained stoichiometric amounts of two peptides with apparent molecular weights identical to apo-B-74 and -26. Together, the above results indicate that apo-B-74 and -26 are degradation products of apo-B-100 and are not produced by the action of thrombin. Whether the expression of a kallikrein-like activity in vivo accounts for the specific degradation of LDL B-100 to yield LDL B-74 and -26 remains to be determined.     

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.     

Direct determination of human and rabbit apolipoprotein B selectively precipitated with butanol-isopropyl ether

A method is described for the rapid, selective, and quantitative precipitation of apolipoprotein B from isolated hypercholesterolemic rabbit and human very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL). Lipoprotein samples are heat-treated at 100 degrees C in 1% SDS. The denatured apoprotein solutions are then mixed briefly with two volumes of butanol-isopropyl ether 45:55 (v/v) to precipitate the apoB. The supernatant solutions, containing the non-apoB proteins and lipids, are removed and the apoB pellet is washed once with water. To determine apoB specific activity, the apoB pellet is resolubilized in 0.5 M NaOH by heating for 30 min at 120 degrees C. The hydrolyzed apoB protein is quantitated by fluorescence of a fluorescamine derivative. The precipitation of apoB is quantitative and selective: 99.5% of rabbit 125I-labeled LDL-apoB and 97.5% of human 125I-labeled LDL-apoB is precipitated and less than 5% of 125I-labeled HDL added to unlabeled VLDL, IDL, or LDL is precipitated. Triglyceride and cholesteryl ester contamination of the apoB pellet is less than 2% of their original radioactivities.     

Unique structural properties of apolipoprotein B in low-density lipoproteins produced by several human hepatoma-derived cell lines

Previous work has shown that low-density lipoproteins (LDL) secreted by hepatoma-derived cell lines have an unusual composition compared to plasma LDL; rather than cholesteryl ester, the hepatoma cell-secreted LDL have a triacylglycerol core. We have found that they also have an increased negative charge, as judged by agarose electrophoresis. Since apolipoprotein B is a glycoprotein containing carbohydrate chains terminated with negatively charged sialic acid residues, we examined whether increased glycosylation of the apolipoprotein B from three hepatoma cell lines (Hep G2, Hep 3B and Huh 7) might account for the differences in LDL charge. The weight percent carbohydrate for Hep G2, Hep 3B and Huh 7 LDL-protein (1.1 +/- 0.2; 1.7 +/- 0.8; 0.4 +/- 0.1) was found to be extremely low compared with the 2.8-9% range we found for plasma LDL-protein, while the amount of LDL-lipid associated carbohydrate from hepatoma LDL was similar to that we found in plasma LDL. Furthermore, desialation of hepatoma cell-secreted LDL with neuraminidase did not normalize the negative charge to that of neuraminidase-treated plasma LDL. Western blots of thrombin proteolytic fragments indicated that, in addition to the T1-T4 fragments seen in plasma apolipoprotein B, apolipoprotein B of hepatoma-derived LDL produced four to five new fragments (T5-T9), suggesting increased exposure of proteolytic sites. Western blotting of the new fragments with antibodies specific for known apolipoprotein B sequences suggests that many of the new cleavage sites cluster in or near the putative LDL receptor recognition site.     

Different apolipoprotein B breakdown patterns in models of oxidized low density lipoprotein.

Low density lipoprotein (LDL) oxidation is characterized by alterations in biological properties and structure of the lipoprotein particles, including breakdown and modification of apolipoprotein B (apoB). We compared apoB breakdown patterns in different models of minimally and extensively oxidized LDL using Western blotting techniques and several monoclonal and polyclonal antibodies. It was found that copper and endothelial cell-mediated oxidation produced a relatively similar apoB banding pattern with progressive fragmentation of apoB during LDL oxidation, whereas malondialdehyde (MDA)- and hydroxynonenal (HNE) -modified LDL produced an aggregated apoB. It is conceivable that apoB fragments present in copper and endothelial cell oxidized LDL lead to the exposure on the lipoprotein surface of different protein epitopes than in aggregated MDA-LDL and HNE-LDL. Although all models of extensively oxidized LDL led to increased lipid uptake in macrophages, mild degrees of oxidation interfered with LDL uptake in fibroblasts and extensively oxidized LDL impaired degradation of native LDL in fibroblasts. We suggest that in order to improve interpretation and comparison of results, data obtained with various models of oxidized LDL should be compared to the simpliest and most reproducible models of 3 h and 18 h copper-oxidized LDL (apoB breakdown) and MDA-LDL (apoB aggregation) since different models of oxidized LDL have significant differences in apoB breakdown and aggregation patterns which may affect immunological and biological properties of oxidized LDL.     

Receptor binding activity of lipid recombinants of apolipoprotein B-100 thrombolytic fragments

Apolipoprotein (apo) B-100, the protein constituent of low density lipoproteins (LDL), is the determinant responsible for LDL binding to the apoB,E(LDL) receptor on cells. The current study was designed to identify the region(s) of apoB-100 that interact with the apoB,E(LDL) receptor. Apolipoprotein B-100 was fragmented by thrombin digestion, and the isolated fragments (T2, T3, T4) were recombined with cholesterol-induced canine high density lipoproteins (HDLc). Before the recombination, the receptor binding activity of apoE of the HDLc was abolished by reductive methylation and extensive trypsin treatment. This treatment permitted almost complete replacement of the small residual apoE fragments by the large apoB fragments. Recombinant apoB particles were isolated by ultracentrifugation and tested for binding to receptors on cultured human fibroblasts. The recombinant particles had chemical and physical properties similar to those of native HDLc. Recombinants of both the whole thrombolytic digest and of isolated fragments displayed specific binding to the apoB,E (LDL) receptor. Anti-apoB,E(LDL) receptor antibodies abolished 90% of the binding, and there was almost no specific binding to receptor-negative fibroblasts or to cells in which the receptors had been down-regulated. The binding of apoB-100 recombinants to the receptor also demonstrated calcium dependency; in addition, the surface binding of the recombinants was released by polyanionic compounds. All these recombinants had binding affinities comparable to one another but less than that of native LDL. Although T2, T3 and T4 recombinants can all bind specifically to the apoB,E(LDL) receptor, it remains to be established whether their activity represents physiologically relevant binding. Nevertheless, the present findings illustrate the potential of the recombinant method using HDLc lipids to reconstitute biological activity.     

LRIG1 protein in human cells and tissues

We have recently cloned the human LRIG1 gene (formerly LIG1). LRIG1 is a predicted integral cell-surface protein showing similarities to Kekkon-1, the Drosophila melanogaster epidermal growth-factor-receptor antagonist. A specific peptide antibody, LRIG1-151, was raised in rabbits and used to study the LRIG1 protein. LRIG1 migrated in denaturing polyacrylamide gel electrophoresis under reducing conditions as two species with apparent molecular weights of 143 kDa and 134 kDa, and as two fragments corresponding to an N-terminal 111-kDa species and a C-terminal 32-kDa species. Under non-reducing conditions, both apparent monomers and apparent higher molecular weight complexes were evident. Immunoblotting analysis of cell-surface-biotinylated lysates and confocal microscopy revealed that LRIG1 was localized to the cell surface in ZR-75 cells expressing endogenous LRIG1 and in COS-7 cells expressing a synthetic LRIG1-GFP fusion protein. Immunohistochemical analysis of normal human tissues showed staining for LRIG1 in epithelia in various organs, scattered neurons, and muscles. Immunoblotting demonstrated LRIG1 protein in tissue lysates from normal human prostate, mammary epithelial cells, ileum, stomach, lung, and cerebral cortex. These results demonstrate that LRIG1 is an integral cell-surface membrane protein that is expressed by specific cells in various human tissues and that its 143-kDa form might be cleaved into 111-kDa and 32-kDa fragments.     

Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins

We have previously demonstrated that astrocytes synthesize and secrete apolipoprotein E in situ. In the present work, primary cultures of rat brain astrocytes were used to study apolipoprotein E synthesis, secretion, and metabolism in vitro. The astrocytes in culture contained immunoreactive apolipoprotein E in the area of the Golgi apparatus. Incubation of the astrocytes with [35S]methionine resulted in the secretion of labeled immunoprecipitable apolipoprotein E, which constituted 1-3% of the total secreted proteins. The apolipoprotein E secreted in culture and the apolipoprotein E in rat brain extracts differed from serum apolipoprotein E in two respects: both had a slightly higher apparent molecular weight (approx. 36,000) and more acidic isoforms than serum apolipoprotein E. Sialylation of the newly secreted apolipoprotein accounted for the difference in both the apparent molecular weight and isoelectric focusing pattern of newly secreted apolipoprotein E and plasma apolipoprotein E. The astrocytes possessed apolipoprotein B,E(LDL) receptors capable of binding and internalizing apolipoprotein E-containing lipoproteins. The uptake of lipoproteins by the cells led to a reduction in the number of cell surface receptors and to the intracellular accumulation of cholesteryl esters. Since apolipoprotein E is present within the brain, and since brain cells can express apolipoprotein B,E(LDL) receptors, apolipoprotein E-containing lipoproteins may function to redistribute lipid and regulate cholesterol homeostasis within the brain.     

De novo production of low density lipoproteins: fact or fancy

Many investigators, observing an apparent dilution in the plasma specific activity (SA) of apolipoprotein B-100 (apoB) in low density lipoprotein (LDL) as compared with that in very low density lipoprotein (VLDL) after injection of radiolabeled VLDL, have formulated kinetic hypotheses incorporating the concept of de novo production of LDL to explain their data in humans and other mammals. These hypotheses, with rare exception, do not account for the kinetic heterogeneity known to exist in the apoB of human VLDL on the basis of size and in the apoB of rabbit VLDL on the basis of size and presence of apolipoprotein E. When a logical analysis of such kinetic heterogeneity of apoB in plasma VLDL is performed, it becomes clear that the apparent dilution of the SA of apoB in LDL relative to that in VLDL can be explained without the requirement for de novo production of LDL. Although this alternative hypothesis, incorporating the concept of kinetic heterogeneity of apoB in VLDL, does not exclude the process of de novo production of LDL, which so many investigators have invoked to explain their data, it does raise a question as to the existence of such a process since an alternative hypothesis can explain such data just as well. Clearly, more experimental data on the kinetic heterogeneity of human and other mammalian VLDL are needed before a reasonable choice can be made between these two hypotheses.     

Low-density lipoprotein receptor binding determinants switch from apolipoprotein E to apolipoprotein B during conversion of hypertriglyceridemic very-low-density lipoprotein to low-density lipoproteins

Using thrombin and trypsin as probes, we determined: first, that low-density lipoprotein (LDL) receptor binding determinants switch from apolipoprotein (apo) E to apo-B within the very-low-density lipoprotein (VLDL) Sf 20-60 region of the metabolic cascade from VLDL1 (Sf 100-400) of hypertriglyceridemic (HTG) human subjects to LDL. Second, two different conformations of apo-E exist in HTG-VLDL Sf greater than 60, one accessible (greater than or equal to 1 mol/mol of particle) and one inaccessible (1-2 mol/mol) to both thrombin and the LDL receptor; normal VLDL (Sf greater than 60) have only the inaccessible conformation and therefore do not bind to the LDL receptor. Third, thrombin degrades apo-B into large fragments, three of which have electrophoretic mobilities similar to B-48, B-74, and B-26; this, however, has no effect on apo-B-mediated receptor binding. Fibroblast studies showed that thrombin could abolish receptor uptake of HTG-VLDL1 and HTG-VLDL2 (Sf 60-100), had little or no effect on HTG-VLDL3 (Sf 20-60), and no effect on uptake of intermediate-density lipoprotein (IDL) or LDL. Trypsin abolished the binding of HTG-VLDL1 and HTG-VLDL2, reduced that of HTG-VLDL3, but had little to no effect on IDL or LDL binding. Immunochemical techniques revealed that thrombin cleaved some apo-E into the E-22 and E-12 fragments; after trypsin treatment no apo-E was detected in any HTG-lipoprotein. Normal VLDL subclasses contained less apo-E than the corresponding HTG-VLDL subclasses and it was not cleaved by thrombin. Apo-B immunoreactivities of VLDL subclasses were not significantly changed after treatment with thrombin, although thrombin cleaved some of the B-100 of each VLDL subclass, and all apo-B in IDL and LDL, into 4-6 major large fragments. Trypsin converted all of the apo-B of each lipoprotein into smaller fragments (Mr less than 100,000). We conclude that apo-E of the thrombin-accessible conformation mediates uptake of HTG-VLDL1 and HTG-VLDL2 but that apo-B alone is sufficient to mediate receptor binding of IDL and LDL; the switch from apo-E to apo-B as the primary or sufficient binding determinant occurs within the VLDL3 (Sf 20-60) region of the metabolic cascade, where receptor binding first appears in VLDL subclasses from normal subjects.     

Apolipoprotein gene expression in the rabbit: abundance, size, and distribution of apolipoprotein mRNA species in different tissues

We have used human apolipoprotein cDNAs as hybridization probes to study the relative abundance and distribution of apolipoprotein mRNAs in rabbit tissues by RNA blotting analysis. The tissues surveyed included liver, intestine, lung, pancreas, spleen, stomach, skeletal muscle, testis, heart, kidney, adrenal, aorta, and brain. We found that liver is the sole or major site of synthesis of apoA-II, apoA-IV, apoB, apoC-I, apoC-II, apoC-III, and apoE, and the intestine is a major site of synthesis of apoA-I, apoA-IV, and apoB. Minor sites of apolipoprotein mRNA synthesis were as follows: apoA-I, liver and skeletal muscle; apoA-IV, spleen and lung; apoB, kidney; apoC-II and apoC-III, intestine. ApoE mRNA was detected in all tissues surveyed with the exception of skeletal muscle. Sites with moderate apoE mRNA (10% of the liver value) were lung, brain, spleen, stomach, and testis. All rabbit mRNAs had forms with sizes comparable to their human counterparts. In addition, hybridization of hepatic and intestinal RNA with human apoA-IV and apoB probes produced a second hybridization band of approximately 2.4 and 8 kb, respectively. Similarly, hybridization of rabbit intestinal RNA with human apoC-II produced a hybridization band of 1.8 kb. The 8 kb apoB mRNA form may correspond to the apoB-48 mRNA, whereas the apoA-IV- and apoC-II-related mRNA species have not been described previously. This study provides a comprehensive survey of the sites of apolipoprotein gene expression and shows numerous differences in both the abundance and the tissue distribution of several apolipoprotein mRNAs between rabbit and human tissues. These findings and the observation of potentially new apolipoprotein mRNA species are important for our understanding of the cis and trans acting factors that confer tissue specificity as well as factors that regulate the expression of apolipoprotein genes in different mammalian species.     

Subunit structure of thrombin-activated porcine factor VIII

Factor VIII (fVIII) is synthesized as a single chain having a domainal sequence A1-A2-B-A3-C1-C2. Analysis of the proteolyic cleavage of fVIII by thrombin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) identifies three fragments designated fVIIIA1, fVIIIA2, and fVIIIA3-C1-C2 with fragment(s) derived from the B domain being difficult to visualize. The appearance of these fragments is associated with the development of coagulant activity, but the activity is labile without further apparent proteolysis. In this study, porcine fVIII was reacted with thrombin until peak coagulant activity was obtained and then subjected to cation-exchange (Mono S) high-pressure liquid chromatography. Coagulant activity was recovered in a single peak that contained all three fragments and was stable for weeks at 20 degrees C in 0.65 M NaCl/0.01 M His-HCl/0.005 M CaCl2 at pH 6.0. Analytical ultracentrifugation of activated fVIII was done to test whether all three fragments were associated. The apparent molecular weight of activated fVIII from equilibrium sedimentation increased from 148,000 to 161,000 as the loading concentration was increased from 0.06 to 0.16 mg/mL. This agrees well with the summed apparent molecular weights of fVIIIA1, fVIIIA2, and fVIIIA3-C1-C2 calculated from SDS-PAGE analysis (148,000) or from the amino acid sequence of human fVIII (159,000). This establishes the major species in the preparation as a fVIIIA1/A2/A3-C1-C2 heterotrimer and additionally indicates either weak self-association of the trimer and/or incomplete association of the individual subunits to form the trimer. Velocity sedimentation of activated fViii revealed a single boundry (S020,w = 7.2 S).(ABSTRACT TRUNCATED AT 250 WORDS)     

A cross-species comparison of the apolipoprotein B domain that binds to the LDL receptor

Apolipoprotein (apo)-B-100 is the ligand that mediates the clearance of low density lipoprotein (LDL) from the circulation by the apoB,E (LDL) receptor pathway. Clearance is mediated by the interaction of a domain enriched in basic amino acid residues on apoB-100 with clusters of acidic residues on the apoB,E (LDL) receptor. A model has been proposed for the LDL receptor binding domain of apoB-100 based on the primary amino acid sequence (Knott, T. J., et al. 1986. Nature. 323: 734-738). Two clusters of basic residues (A: 3147-3157 and B: 3359-3367) are apposed on the surface of the LDL particle by a disulfide bridge between Cys 3167 and 3297. Support for this single domain model has been obtained from the mapping of epitopes for anti-apoB monoclonal antibodies that block the binding of apoB to the LDL receptor. Here we test this model by comparing the nucleotide (from 9623 to 10,442) and amino acid sequence (from 3139 to 3411) of apoB-100 in seven species (human, pig, rabbit, rat, Syrian hamster, mouse, and chicken). Overall, this region is highly conserved. Cluster B maintains a strong net positive charge and is homologous across species in both primary and secondary structure. However, the net positive charge of region A is not conserved across these species, but the region remains strongly hydrophilic. The secondary structure of the region between clusters A and B is preserved, but the disulfide bond is unique to the human sequence. This study suggests that the basic region B is primarily involved in the binding of apoB-100 to the apoB,E (LDL) receptor.     

Use of bacterial expression cloning to localize the epitopes for a series of monoclonal antibodies against apolipoprotein B100.

Bacterial expression of apolipoprotein (apo) B cDNA constructs has been used to map a series of monoclonal antibodies (mAbs) to apoB by immunoblotting. In some cases assignments have been confirmed and refined by (i) semipurification of expressed protein, CNBr digestion, and assignment of the immunoreactive fragments; (ii) controlled digestion of the cDNA with the exonuclease Bal31 and bacterial expression of the truncated proteins that result; or (iii) expression of specific segments of cDNA amplified by the polymerase chain reaction. Forty mAbs were mapped to a minimum of 17 separate determinants on apoB. Tryptic fragments have been used to confirm the epitope assignments. In addition, this approach in conjunction with immunoassay, enables some deductions to be made about the trypsin-accessible regions in low density lipoprotein (LDL). The cleavage pattern obtained predicts retention of structure in the cysteine-rich domain of the amino terminus and also in the LDL receptor binding region. Trypsinized LDL was shown to bind to the LDL receptor by an authentic process, using monoclonal antibodies as competing ligands. In conjunction with the previous paper (Milne, R. W., Theolis, R., Maurice, R., Pease, R. J., Weech, P. K., Rassart, E., Fruchart, J.-C., Scott, J., and Marcel, Y. L. (1989) J. Biol. Chem. 265, 19754-19760) the mapped mAbs have been used to define the receptor-binding domain of apoB100 in LDL.     

Radioimmunoassay of rat apolipoprotein B peptides in lipoproteins and tissues

Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) separates rat apolipoprotein B (apoB) into one lower and two higher molecular weight components. Of the latter, peptide I (PI) corresponds to human B-100, while the slightly faster-migrating peptide II (PII) lacks a human counterpart; the smaller species peptide III (PIII) corresponds to human B-48. We describe here a competitive radioimmunoassay which separately measures the amounts of total (i.e., PI + PII + PIII) and larger (i.e., PI + PII) rat apoB peptides, with the amounts of PIII obtained by difference. Standard rat PIII and combined PI + PII (PI,II) were isolated by high-pressure gel filtration liquid chromatography in the presence of SDS, and the PI,II was used as an immunogen to raise rabbit antisera which were capable of binding all three forms of rat apoB. However, Scatchard analysis showed this binding to represent two distinct types of antibodies: one high-affinity class which bound only PI,II and a second class which bound all apoB peptides with equal but lower affinity. Thus, since 125I-labeled PIII was displaced equally effectively by PI,II and PIII, but 125I-labeled PI,II was displaced only by PI,II, the unabsorbed antiserum could be used to measure either total apoB or PI,II alone, depending on the choice of labeled ligand. The validity of the assay for apoB peptides in very-low-density and low-density lipoproteins and in liver microsomes was verified by comparison with peptide determinations by SDS-PAGE.     

The enhanced cellular uptake of very-low-density lipoprotein enriched in apolipoprotein E.

We have recently reported an increased clearance of plasma very-low-density lipoprotein (VLDL) after intravenous injection of apolipoprotein (apo) E in Watanabe heritable hyperlipidemic (WHHL) rabbits. In the present study, we have investigated the cellular uptake of VLDL enriched in apo E (VLDL-E) which had been incubated with purified rabbit apo E. VLDL-E was taken up approx. 2-fold more than VLDL in human skin fibroblast, human monocyte-derived macrophage and Hep G2 cell and its degradation was least in macrophage. To characterize the binding of VLDL-E, we performed a binding assay using hepatic endosome isolated from estradiol-treated rats and we observed both increased EDTA-sensitive and -resistant binding of VLDL-E on endosome. Ligand blotting of hepatic endosome demonstrated two major bands of LDL receptor (130 and 260 kDa protein) and a minor band of LDL receptor-related protein (580 kDa protein) with a ligand of VLDL-E. These results suggested that VLDL-E was endocytosed in liver through a similar pathway among three cell types, and enrichment of apo E in VLDL enhanced the uptake of VLDL not only via an EDTA-sensitive binding site (classical LDL receptor) but also via other binding sites including an EDTA-resistant binding site and an LDL receptor-related protein.     

Metabolic pathways of apolipoprotein B in heterozygous familial hypercholesterolemia: studies with a [3H]leucine tracer.

The kinetics of apolipoprotein B (apoB) were measured in seven studies in heterozygous, familial hypercholesterolemic subjects (FH) and in five studies in normal subjects, using in vivo tracer kinetic methodology with a [3H]leucine tracer. Very low density (VLDL) and low density lipoproteins (LDL) were isolated ultracentrifugally and LDL was fractionated into high and low molecular weight subspecies. ApoB was isolated, its specific radioactivity was measured, and the kinetic data were analyzed by compartmental modeling using the SAAM computer program. The pathways of apoB metabolism differ in FH and normal subjects in two major respects. Normals secrete greater than 90% of apoB as VLDL, while one-third of apoB is secreted as intermediate density lipoprotein IDL/LDL in FH. Normals lose 40-50% of apoB from plasma as VLDL/IDL, while FH subjects lose none, metabolizing all of apoB to LDL. In FH, there is also the known prolongation of LDL residence time. The leucine tracer, biosynthetically incorporated into plasma apoB, permits distinguishing the separate pathways by which the metabolism of apoB is channeled. ApoB synthesis and secretion require 1.3 h. ApoB is secreted by three routes: 1) as large VLDL where it is metabolized by a delipidation chain; 2) as a rapidly metabolized VLDL fraction converted to LDL; and 3) as IDL or LDL. ApoB is metabolized along two pathways. The delipidation chain processes large VLDL to small VLDL, IDL, and LDL. The IDL pathway channels nascent, rapidly metabolized VLDL and IDL particles into LDL. It thus provides a fast pathway for the entrance of apoB tracer into LDL, while the delipidation pathway is a slower route for channeling apoB through VLDL into LDL. LDL apoB is derived in almost equal amounts from both pathways, which feed predominantly into large LDL. Small LDL is a product of large LDL, and the major loss of LDL-apoB is from small LDL. Two features of apoB metabolism in FH, the major secretory pathway through IDL and the absence of a catabolic loss of apoB from VLDL/IDL, greatly facilitate measuring the metabolic channeling of apoB into LDL.     

Cytotoxicity of lipid-free apolipoprotein B

To investigate the effect of apolipoprotein B (apoB) on cell viability, we used lipid-free apoB as a model for denatured apoB. Lipid-free apoB had cytotoxicity to J774 macrophages, CHO cells and HepG2 cells, whereas apoB bound to low density lipoprotein (LDL) and lipid-free apolipoprotein A-I had no effect on cell viability. Lipid-free apoB induced apoptosis in J774 macrophages assessed by caspase-3 activation and annexin V binding. LDL receptor, heparan sulfate proteoglycans, and class A scavenger receptor were involved in the binding/uptake of lipid-free apoB, but lipid-free apoB binding/uptake by the cells did not correlate with cytotoxicity. Lipid-free apoB disrupted the lipid bilayer of large unilamellar vesicles containing calcein. We evaluated the interaction between apoB and cellular membrane by monitoring the change in intracellular Ca²⁺ concentration using Fura-2, and found that lipid-free apoB rapidly disrupted the cellular membrane in the absence or presence of the inhibitors for cellular binding/uptake mediated by the receptors. Therefore, it is suggested that lipid-free apoB induces cell death by disturbance of the plasma membrane. In addition to other lipid component in modified LDL, apoB itself has an ability to induce apoptosis and plays a crucial role in the development of atherosclerotic lesions.