Concentrations and compositions of plasma lipoprotein subfractions of Lpb5-Lpu1 homozygous and heterozygous swine with hypercholesterolemia

Pigs with two mutant epitopes, Lpb5 of apolipoprotein B (apoB) and Lpu1 of a yet undefined apolipoprotein, specified by a haplotype Lpb5-Lpu1 and fed a cholesterol-free low fat diet show hypercholesterolemia. The purpose of this study was to establish whether a direct relationship exists between the swine lipoprotein concentration/composition and the genotype for the Lpb5-Lpu1 haplotype; i.e., homozygote versus heterozygote. Lipoproteins of fasted plasma from hypercholesterolemic swine, homozygous (HmHC) and heterozygous (HtHC) for Lpb5-Lpu1, and from normolipidemic (NL) pigs of other Lpb-Lpu haplotypes were separated into five layers by density gradient ultracentrifugation. Layer 1 contained particles of d less than 1.019 g/ml and layer 5 particles of d greater than 1.073 g/ml. Layers 2, 3, and 4 represented subfractions of low density lipoproteins (LDL). The plasma total cholesterol (TC) of the HmHC group (300 +/- 84 mg/dl) was different (P less than 0.05) from the HtHC group (200 +/- 80 mg/dl) and in both HmHC and HtHC, TC was significantly higher (P less than 0.0005 and P less than 0.005, respectively) than that of the NL group (69 +/- 14 mg/dl). The elevation in plasma TC was due to the increased TC in layers 2 and 3: a 13- and 7-fold increase in HmHC and a 7- and 4-fold increase in HtHC in layers 2 and 3, respectively. Parallel increases in unesterified cholesterol were observed in these two layers. Marked increases in apoB were also observed in layers 2 and 3 of HmHC and intermediate increases in apoB in the same two layers of HtHC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of new apolipoprotein B epitopes and haplotypes and their distribution in swine populations

Results from comparative immunogenetic studies on inheritance and identification of four new apolipoprotein B (apoB) allotypes and three additional apoB haplotypes and their distribution in miniature and domestic swine are presented. Immunological surveys on the four new and 16 previously described Lpb allotypes and genetic analysis of their segregation in progenies, of miniature and domestic swine and their crosses, indicate that three new allotypes designated Lpb9, Lpb10 and Lpb101 are individual (mutant) apoB epitopes, each representing a discriminating marker for one of the new apoB haplotypes specified by three new apoB alleles designated Lpb⁹, Lpb¹⁰ and Lpb¹⁰¹. The fourth allotype, Lpb20, is one of the common epitopes forming the alternative epitope pair with Lpb10, and is a constituent of each of the eight previously described and two new apoB haplotypes. The new apoB alleles have so far been found only in miniature swine, with Lpb¹⁰ being the most frequent in the Göttingen, Vietnamese Potbelly and Japanese Miniature, Lpb⁹ was detected only in Minnesota Miniature and Lpb101 only in Vietnamese Potbelly. The common allotype, Lpb20, shares immunological similarities with human apoB indicating its ancestral origin, whereas none of the alloreagents detecting the three individual apoB variants, Lpb9, Lpb10 or Lpb101, showed cross-reactivity with human apoB, suggesting their exclusive swine origin and evolvement during speciation through mutations.     

Lovastatin therapy reduces low density lipoprotein apoB levels in subjects with combined hyperlipidemia by reducing the production of apoB-containing lipoproteins: implications for the pathophysiology of apoB production

We investigated the metabolism of very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL) apolipoprotein B (apoB) in seven patients with combined hyperlipidemia (CHL), using 125I-labeled VLDL and 131I-labeled LDL and compartmental modeling, before and during lovastatin treatment. Lovastatin therapy significantly reduced plasma levels of LDL cholesterol (142 vs 93 mg/dl, P less than 0.0005) and apoB (1328 vs 797 micrograms/ml, P less than 0.001). Before treatment, CHL patients had high production rates (PR) of LDL apoB. Three-fourths of this LDL apoB flux was derived from sources other than circulating VLDL and was, therefore, defined as "cold" LDL apoB flux. Compared to baseline, treatment with lovastatin was associated with a significant reduction in the total rate of entry of apoB-containing lipoproteins into plasma in all seven CHL subjects (40.7 vs. 25.7 mg/kg.day, P less than 0.003). This reduction was associated with a fall in total LDL apoB PR and in "cold" LDL apoB PR in six out of seven CHL subjects. VLDL apoB PR fell in five out of seven CHL subjects. Treatment with lovastatin did not significantly alter VLDL apoB conversion to LDL apoB or LDL apoB fractional catabolic rate (FCR) in CHL patients. In three patients with familial hypercholesterolemia who were studied for comparison, lovastatin treatment increased LDL apoB FCR but did not consistently alter LDL apoB PR. We conclude that lovastatin lowers LDL cholesterol and apoB concentrations in CHL patients by reducing the rate of entry of apoB-containing lipoproteins into plasma, either as VLDL or as directly secreted LDL.     

Immunogenetic polymorphism of lipoproteins in swine. 1. Four additional serum beta-lipoprotein allotypes (Lpp2, Lpp4, Lpp5 and Lpp15) in the Lpp system.

Four additional swine serum lipoprotein allotypes are described. Specific anti-allotype reagents were obtained from alloimmune precipitating sera produced in lipoprotein-defined-type recipients immunized with normal sera and subsequently with lipoprotein fractions. Identification studies indicate that the four serologically defined low-density lipoprotein (LDL) variants, designated Lpp2, Lpp4, Lpp5 and Lpp15, are members of a previously described Lpp system. The individual specificities, Lpp2, Lpp4 and Lpp5, are determined by three co-dominant autosomal genes, Lpp2, Lpp4 and Lpp5, respectively, whereas the common specificity, Lpp15, is controlled by a complex of genetic information of the Lpp2 and Lpp4 genes, and by the two previously described alleles, Lpp1 and Lpp3; Lpp15 occurs on the same molecule with respective individual specificity. The Lpp5 and Lpp15 antigens behave as a pair of alternative allotypic specificities. The double immunodiffusion test in agar was employed to demonstrate independent phenotypic expression of each allelic gene in the Lpp heterozygous animals, for the analysis of the immune sera, and for lipoprotein testing of 3305 sera. Marked differences in gene frequencies were found between the swine breeds tested. As a result of characteristic frequencies, only nine of 15 possible Lpp genotypes were found in the breeding herds tested; the remaining six genotypes were obtained from testcross matings.     

Immunochemical evidence that human apoB differs when expressed in rodent versus human cells

LDL from human apolipoprotein B-100 (apoB-100) transgenic (HuBTg+/+) mice contains more triglyceride than LDL from normolipidemic subjects. To obtain novel monoclonal antibody (MAb) probes of apoB conformation, we generated hybridomas from HuBTg+/+ that had been immunized with LDL isolated from human plasma. One apoE-specific and four anti-apoB-100-specific hybridomas were identified. Two MAbs, 2E1 and 3D11, recognized an epitope in the amino-terminal 689 residues of apoB in native apoB-containing lipoproteins (LpBs) from human plasma or from the supernatant of human hepatoma HepG2 cells, but did not react with LpB from HuBTg+/+ mice or LpB secreted by human apoB-100-transfected rat McArdle 7777 hepatoma cells. 2E1 reacted weakly and 3D11 reacted strongly with apoB from HuBTg+/+ mice after SDS-PAGE. The lack of expression of the 2E1 and 3D11 epitopes on native LpB from HuBTg+/+ mice did not solely reflect the abnormal lipid composition of murine LpB. Both epitopes were detected in all human plasma samples tested and in all human plasma LpB classes. Therefore, human apoB expressed by rodent hepatocytes or hepatoma cells appears to adopt a different conformation or undergoes different posttranslational modification than apoB expressed in human hepatocytes or hepatoma cells.     

Immunogenetic polymorphism of lipoproteins in swine. 1. Four additional serum β-lipoprotein allotypes (Lpp2, Lpp4, Lpp5 and Lpp15) in the Lpp system1

Four additional swine serum lipoprotein allotypes are described. Specific anti-allotype reagents were obtained from alloimmune precipitating sera produced in lipoprotein-defined-type recipients immunized with normal sera and subsequently with lipoprotein fractions. Identification studies indicate that the four serologically defined low-density lipoprotein (LDL) variants, designated Lpp2, Lpp4, Lpp5 and Lpp15, are members of a previously described Lpp system. The individual specificities, Lpp2, Lpp4 and Lpp5, are determined by three co-dominant autosomal genes, Lpp², Lpp⁴ and Lpp⁵, respectively, whereas the common specificity, Lpp 15, is controlled by a complex of genetic information of the Lpp- and Lpp* genes, and by the two previously described alleles, Lpp¹ and Lpp³; Lpp15 occurs on the same molecule with respective individual specificity. The Lpp5 and Lpp15 antigens behave as a pair of alternative allotypic specificities. The double immunodiffusion test in agar was employed to demonstrate independent phenotypic expression of each allelic gene in the Lpp heterozygous animals, for the analysis of the immune sera, and for lipoprotein testing of 3305 sera. Marked differences in gene frequencies were found between the swine breeds tested. As a result of characteristic frequencies, only nine of 15 possible Lpp genotypes were found in the breeding herds tested; the remaining six genotypes were obtained from testcross matings.     

Interaction of LDL, Lp[a], and reduced Lp[a] with monoclonal antibodies against apoB

Five monoclonal antibodies (2A, 9A, 6B, L3, L7) produced in mice against human apolipoprotein B were investigated by competitive and inhibitive electroimmunoassay (EIA) for their reactivity with low density lipoprotein (LDL), lipoprotein[a] (Lp[a]), and reduced Lp[a]. All of the antibodies reacted with apoB of the different lipoproteins indicated by very similar slopes of the binding curves. None of them gave a positive reaction with apolipoprotein[a]. The amount of apoB required for 50% inhibition of antibody binding varied for the different antibodies and lipoproteins. Antibody 9A showed almost the same affinity for LDL, Lp[a], and reduced Lp[a]. Antibodies 2A and 6B bound about twofold better to LDL and reduced Lp[a] than to untreated Lp[a]. Antibodies L3 and L7 needed nearly threefold higher amounts of Lp[a]-apoB for 50% inhibition of antibody binding than of apoB of LDL and reduced Lp[a]. The amount of apoB required for 50% inhibition of antibody binding was somewhat higher in inhibitive assay than in competitive assay. We suggest that apo[a] covers certain epitopes of apoB in native Lp[a] leading to a reduced reaction with the monoclonal antibodies. However, it could also be that the binding of the [a]antigen to apoB via disulfide bridges causes profound conformational changes of the apoB region exposed to the surface.     

Low density lipoprotein heterogeneity in spontaneously hypercholesterolemic pigs

We previously described a strain of spontaneously hypercholesterolemic pigs carrying an apo-B allele termed Lpb ⁵. Lpb ⁵ pigs are heterogeneous with respect to the severity of their hypercholesterolemia. We have termed Lpb ⁵ pigs with severe hypercholesterolemia Lpb 5.1 pigs, and those with moderate hypercholesterolemia Lpb 5.2, Lpb 5.1 animals have a dramatic increase in buoyant LDL relative to dense LDL, with a buoyant-to-dense LDL ratio of 2.2. In contrast, Lpb 5.2 and control pigs have buoyant-to-dense LDL ratios of 0.7 and 0.5 respectively. This ratio appears to be a stable characteristic of the Lpb 5.1 phenotype because sexually mature boars have a dramatic decrease in total plasma cholesterol concentration with no decrease in their ratio of buoyant-to-dense LDL. We have previously demonstrated a fourteen-fold overproduction of buoyant LDL in the Lpb 5.1 pigs, with very little conversion of dense LDL to buoyant LDL. In the current work, very low density lipoprotein (VLDL) turnover experiments were conducted to determine whether VLDL conversion to buoyant LDL was increased in the Lpb 5.1 pigs. VLDL conversion to buoyant LDL could not account for the increased production of buoyant LDL in Lpb 5.1 pigs. Thus, we cannot account for the increased production of buoyant LDL in the Lpb 5.1 pigs from any measurable plasma lipoprotein source. We have therefore termed this production of buoyant LDL in the Lpb 5.1 pigs direct buoyant LDL production.     

Evolution of low density lipoprotein structure probed with monoclonal antibodies

To assess the relationship of apoB structures in different species of animals, the expressions of apoB epitopes in the sera or plasmas of 23 different mammalian species and one marsupial, and on the low density lipoprotein (LDL) from three species of apes, six species of monkeys, and eight non-primates were measured in competitive radioimmunoassays. The abilities of the sera or LDL to compete with 125I-labeled human LDL for binding to seven monoclonal antihuman LDL antibodies immobilized on microtiter plates were determined. LDL of apes bound to most antibodies, while monkey LDL bound to two or three antibodies. Other mammalian LDL bound only weakly to two of the antibodies or to none. The two monoclonal antibodies binding the LDL of more species were those antibodies which also inhibited the binding to and degradation of LDL by human fibroblasts. The rank order of binding of the LDL of a given species to the antibodies correlated with the rank order inhibition of binding and degradation of 125I-labeled human LDL in the human fibroblast system. This suggests that epitopes spatially located near the recognition site of apoB for cellular receptors have a greater tendency to be conserved.     

The low density lipoprotein receptor prevents secretion of dense apoB100-containing lipoproteins from the liver

The assembly and secretion of very low density lipoproteins (VLDL) require microsomal triglyceride transfer protein (MTP). Recent evidence also suggests a role for the low density lipoprotein (LDL) receptor in this process. However, the relative importance of MTP in the two steps of VLDL assembly and the specific role of the LDL receptor still remain unclear. To further investigate the role of MTP and the LDL receptor in VLDL assembly, we bred mice harboring "floxed" Mttp alleles (Mttpflox/flox) and a Cre transgene on a low-density lipoprotein receptor-deficient background to generate mice with double deficiency in the liver (Ldlr-/- MttpDelta/Delta). In contrast to the plasma of Ldlr+/+ MttpDelta/Delta mice, the plasma of Ldlr-/- MttpDelta/Delta mice contained apoB100. Accordingly, Ldlr-/- MttpDelta/Delta but not Ldlr+/+ MttpDelta/Delta hepatocytes secreted apoB100-containing lipoprotein particles. The secreted lipoproteins were of LDL and HDL sizes but no VLDL-sized lipoproteins could be detected. These findings indicate that hepatic LDL receptors function as "gatekeepers" targeting dense apoB100-containing lipoproteins for degradation. In addition, these results suggest that very low levels of MTP are insufficient to mediate the second step but sufficient for the first step of VLDL assembly.     

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.     

Antisense inhibition of apoB synthesis with mipomersen reduces plasma apoC-III and apoC-III-containing lipoproteins

Mipomersen, an antisense oligonucleotide that reduces hepatic production of apoB, has been shown in phase 2 studies to decrease plasma apoB, LDL cholesterol (LDL-C), and triglycerides. ApoC-III inhibits VLDL and LDL clearance, and it stimulates inflammatory responses in vascular cells. Concentrations of VLDL or LDL with apoC-III independently predict cardiovascular disease. We performed an exploratory posthoc analysis on a subset of hypercholesterolemic subjects obtained from a randomized controlled dose-ranging phase 2 study of mipomersen receiving 100, 200, or 300 mg/wk, or placebo for 13 wk (n = 8 each). ApoC-III-containing lipoproteins were isolated by immuno-affinity chromatography and ultracentrifugation. Mipomersen 200 and 300 mg/wk reduced total apoC-III from baseline by 6 mg/dl (38-42%) compared with placebo group (P < 0.01), and it reduced apoC-III in both apoB lipoproteins and HDL. Mipomersen 100, 200, and 300 mg doses reduced apoB concentration of LDL with apoC-III (27%, 38%, and 46%; P < 0.05). Mipomersen reduced apoC-III concentration in HDL. The drug had no effect on apoE concentration in total plasma and in apoB lipoproteins. In summary, antisense inhibition of apoB synthesis reduced plasma concentrations of apoC-III and apoC-III-containing lipoproteins. Lower concentrations of apoC-III and LDL with apoC-III are associated with reduced risk of coronary heart disease (CHD) in epidemiologic studies independent of traditional risk factors.     

Microsomal triglyceride transfer protein -493T variant reduces IDL plus LDL apoB production and the plasma concentration of large LDL particles

The microsomal triglyceride transfer protein (MTP) is essential for the synthesis and secretion of apolipoprotein B (apoB)-containing lipoproteins. We investigated the role the MTP -493G/T gene polymorphism in determining the apoB-100 secretion pattern and LDL heterogeneity in healthy human subjects. Groups of carriers of the T and the G variants (n = 6 each) were recruited from a cohort of healthy 50-yr-old men. Kinetic studies were performed by endogenous [(2)H(3)]leucine labeling of apoB and subsequent quantification of the stable isotope incorporation. apoB production rates, metabolic conversions, and eliminations were calculated by multicompartmental modeling (SAAM-II). LDL subfraction distribution was analyzed in the entire cohort (n = 377). Carriers of the MTP -493T allele had lower plasma LDL apoB and lower concentration of large LDL particles [LDL-I: 136 +/- 57 (TT) vs. 175 +/- 55 (GG) mg/l, P < 0.01]. Kinetic modeling suggested that MTP -493T homozygotes had a 60% lower direct production rate of intermediate-density lipoprotein (IDL) plus LDL compared with homozygotes for the G allele (P < 0.05). No differences were seen in production rates of large and small VLDL, nor were there any differences in metabolic conversion or elimination rates of apoB between the genotype groups. This study shows that a polymorphism in the MTP gene affects the spectrum of endogenous apoB-containing lipoprotein particles produced in humans. Reduced direct production of LDL plus IDL appears to be related to lower plasma concentrations of large LDL particles.     

Overexpression of SR-BI by adenoviral vector promotes clearance of apoA-I,but not apoB,in human apoB transgenic mice

Scavenger receptor BI (SR-BI) is a multi-ligand lipoprotein receptor that mediates selective lipid uptake from HDL, and plays a central role in hepatic HDL metabolism. In this report, we investigated the extent to which SR-BI selective lipid uptake contributes to LDL metabolism. As has been reported for human LDL, mouse SR-BI expressed in transfected cells mediated selective lipid uptake from mouse LDL. However, LDL-cholesteryl oleoyl ester (CE) transfer relative to LDL-CE bound to the cell surface (fractional transfer) was approximately 18-fold lower compared with HDL-CE. Adenoviral vector-mediated SR-BI overexpression in livers of human apoB transgenic mice ( approximately 10-fold increased expression) reduced plasma HDL-cholesterol (HDL-C) and apolipoprotein (apo)A-I concentrations to nearly undetectable levels 3 days after adenovirus infusion. Increased hepatic SR-BI expression resulted in only a modest depletion in LDL-C that was restricted to large LDL particles, and no change in steady-state concentrations of human apoB. Kinetic studies showed a 19% increase in the clearance rate of LDL-CE in mice with increased SR-BI expression, but no change in LDL apolipoprotein clearance. Quantification of hepatic uptake of LDL-CE and LDL-apolipoprotein showed selective uptake of LDL-CE in livers of human apo B transgenic mice. However, such uptake was not significantly increased in mice over-expressing SR-BI. We conclude that SR-BI-mediated selective uptake from LDL plays a minor role in LDL metabolism in vivo.     

Characterization of apoB, E receptor function in the luteinized ovary

Recent findings from this laboratory have led to the suggestion that the hormone-producing cells of the rat luteinized ovary in situ may obtain a large share of low density lipoprotein (LDL) cholesterol without actually internalizing the intact lipoprotein particles. We have shown that the lipoproteins are trapped at the surface of the luteal cells in a rich network of "microvillar channels" and have theorized that these channel membranes, with their large surface area for interacting with lipoprotein particles, may function in the cholesterol transfer process. In the current study, we try to establish what proportion of the human (h)LDL-cholesterol transfer in the in situ perfused tissue occurs by a classical apoB, E receptor-mediated process versus a surface extraction process. We examine the tissue for the presence of apoB, E receptors, and characterize the structural/functional interaction of hLDL with the apoB, E receptor utilizing a variety of modified hLDL particles as probes. Then, using nonmetabolizable radiolabels for both the protein and cholesteryl ester moieties of these LDL probes, we attempt to quantify the extent to which apoB, E receptors in the ovary contribute to the uptake of hLDL-cholesterol during steroidogenesis. Our experiments show that although the luteinized ovary contains apoB, E receptor protein, hLDL interacts with the tissue atypically. That is, despite modifications of LDL amino acid residues to prevent interaction with the apoB, E receptor, the modified ligands continue to contribute cholesterol for luteal cell internalization and/or steroidogenesis. We conclude, therefore, that in this tissue much of the LDL-cholesterol is not delivered by the apoB, E receptor pathway.     

Quantification of apo[a] and apoB in human atherosclerotic lesions.

Lipoprotein[a] or Lp[a] is a cholesterol-rich plasma lipoprotein that is associated with increased risk for cardiovascular disease. To better understand this association we determined the amount of apo[a] and apoB as possible estimates for Lp[a] and low density lipoprotein (LDL) accumulation in atherosclerotic lesions and in plasma, from patients undergoing vascular surgery, using specific radioimmunoassays for apolipoprotein[a] and apolipoprotein B. Apo[a] and apoB were operationally divided into a loosely bound fraction obtained by extracting minced samples of plaque with phosphate-buffered saline (PBS), and a tightly bound fraction obtained by extracting the residual tissue with 6 M guanidine-HCl (GuHCl). We found that 83% of all apo[a] but only 32% of all apoB in lesions was in the tightly bound fraction. When normalized for corresponding plasma levels, apo[a] accumulation in plaques was more than twice that of apoB. All fractions of tissue apo[a], loosely bound, tightly bound, and total, correlated significantly with plasma apo[a]. However, no significant correlations were found between any of the tissue fractions and plasma apoB. If all apo[a] and apoB had been associated with intact Lp[a] or LDL particles, the calculated mass of tightly bound Lp[a] would actually have exceeded that of tightly bound LDL in five cases with plasma Lp[a] levels above 5 mg apo[a] protein/dl. When PBS and GuHCl extracts of lesions were subjected to one-dimensional electrophoresis, the major band stained for lipid and immunoblotted positively for apo[a] and apoB, suggesting the presence of some intact Lp[a] in these extracts. These results suggest that Lp[a] accumulates preferentially to LDL in plaques, and that plaque apo[a] is directly associated with plasma apo[a] levels and is in a form that is less easily removable than most of the apoB. This preferential accumulation of apo[a] as a tightly bound fraction in lesions, could be responsible for the independent association of Lp[a] with cardiovascular disease in humans.     

Cloning of apoB intrabodies: specific knockdown of apoB in HepG2 cells

Apolipoprotein (apo) B is essential for the assembly and secretion of triglyceride-rich lipoproteins made by the liver. As the sole protein component in LDL, apoB is an important determinant of atherosclerosis susceptibility and a potential pharmaceutical target. Single-chain antibodies (sFvs) are the smallest fragment of an IgG molecule capable of maintaining the antigen binding specificity of the parental antibody. In the present study, we describe the cloning and construction of two intracellular antibodies (intrabodies) to human apoB. We targeted these intrabodies to the endoplasmic reticulum for the purpose of retaining nascent apoB within the ER, thereby preventing its secretion. Expression of the 1D1 intrabody in the apoB-secreting human hepatoma cell line HepG2 resulted in marked reduction of apoB secretion. This study demonstrates the utility of an intrabody to specifically block the secretion of a protein determinant of plasma LDL as a therapeutic strategy for the treatment of hyperlipidemia.     

ELISA quantitation of apolipoproteins in plasma lipoprotein fractions: ApoE in apoB-containing lipoproteins (Lp B:E) and apoB in apoE-containing lipoproteins (Lp E:B)

Growing clinical evidence suggests that metabolic behavior and atherogenic potential vary within lipoprotein subclasses that can be defined by apolipoprotein variation. Variant constituency of apolipoproteins B and E (apoB and apoE) may be particularly important because of the central roles of these apolipoproteins in the endogeneous lipid delivery cascade. ApoB is the sole protein of low-density lipoprotein (LDL), and like LDL cholesterol, the plasma apoB level has been positively correlated with risk for atherosclerotic disease. ApoE is a major functional lipoprotein in the triglyceride-rich lipoproteins, and may be crucial in the conversion of very low density lipoprotein (VLDL) to LDL. Based on work by others that enabled the quantititation of apoB-containing particles by content of up to two other types of apolipoprotein, we have developed a method for determining the amount of apoE in apoB-containing lipoproteins (Lp B:E) and the amount of apoB in apoE-containing lipoproteins (Lp E:B). From the Lp B:E and Lp E:B concentrations, the molar ratio of apoE to apoB in lipoproteins containing apoB and/or apoE in plasma can be determined. The methodology is fast, specific, and sensitive and should prove extremely useful in further categorizing lipoproteins and characterizing their behavior. In applying this method to clinical groupings of normo- and hyperlipidemia, we found that the plasma triglyceride level correlated with the apoE and Lp B:E concentrations in plasma, while the total cholesterol level correlated with the apoB and Lp E:B levels.     

Immuno-electron cryo-microscopy imaging reveals a looped topology of apoB at the surface of human LDL

A single copy of apoB is the sole protein component of human LDL. ApoB is crucial for LDL particle stabilization and is the ligand for LDL receptor, through which cholesterol is delivered to cells. Dysregulation of the pathways of LDL metabolism is well documented in the pathophysiology of atherosclerosis. However, an understanding of the structure of LDL and apoB underlying these biological processes remains limited. In this study, we derived a 22 Å-resolution three-dimensional (3D) density map of LDL using cryo-electron microscopy and image reconstruction, which showed a backbone of high-density regions that encircle the LDL particle. Additional high-density belts complemented this backbone high density to enclose the edge of the LDL particle. Image reconstructions of monoclonal antibody-labeled LDL located six epitopes in five putative domains of apoB in 3D. Epitopes in the LDL receptor binding domain were located on one side of the LDL particle, and epitopes in the N-terminal and C-terminal domains of apoB were in close proximity at the front side of the particle. Such image information revealed a looped topology of apoB on the LDL surface and demonstrated the active role of apoB in maintaining the shape of the LDL particle.     

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.