Rat monoclonal antibodies to human apolipoprotein B: advantages and applications

Eight monoclonal antibodies (Mabs) to human serum low density lipoprotein (LDL) were derived from the fusion of spleen cells, from LOU rats immunized with human LDL, and the rat myeloma line IR983F. These Mabs were characterized in terms of isotype, specificity, and affinity. Competitive experiments indicated that the epitopes that were recognized could be grouped into three patterns depending on their apparent affinity for apoB-containing lipoprotein particles such as LDL, very low density lipoproteins (VLDL), or intermediate density lipoproteins (IDL). Six epitopes have been mapped in relation to elements of the sequence of apolipoprotein B-100 (apoB-100) and some have been assigned to the middle part of the median thrombolytic fragment T3, a region not yet well targeted by mouse Mabs. The presence of lipids for the expression of the epitopes was studied and confirmed a lipid dependence for epitopes that are close to the T2/T3 cleavage site. The capacity of binding to the LDL receptor was also tested; among the Mabs we described, one inhibited the uptake and degradation of LDL to HeLa cells receptor. Finally, some antibodies were able to precipitate LDL in gel.     

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.     

Effects of different doses of atorvastatin on human apolipoprotein B-100, B-48, and A-I metabolism

Nine hypercholesterolemic and hypertriglyceridemic subjects were enrolled in a randomized, placebo-controlled, double-blind, crossover study to test the effect of atorvastatin 20 mg/day and 80 mg/day on the kinetics of apolipoprotein B-100 (apoB-100) in triglyceride-rich lipoprotein (TRL), intermediate density lipoprotein (IDL), and LDL, of apoB-48 in TRL, and of apoA-I in HDL. Compared with placebo, atorvastatin 20 mg/day was associated with significant reductions in TRL, IDL, and LDL apoB-100 pool size as a result of significant increases in fractional catabolic rate (FCR) without changes in production rate (PR). Compared with the 20 mg/day dose, atorvastatin 80 mg/day caused a further significant reduction in the LDL apoB-100 pool size as a result of a further increase in FCR. ApoB-48 pool size was reduced significantly by both atorvastatin doses, and this reduction was associated with nonsignificant increases in FCR. The lathosterol-campesterol ratio was decreased by atorvastatin treatment, and changes in this ratio were inversely correlated with changes in TRL apoB-100 and apoB-48 PR. No significant effect on apoA-I kinetics was observed at either dose of atorvastatin. Our data indicate that atorvastatin reduces apoB-100- and apoB-48-containing lipoproteins by increasing their catabolism and has a dose-dependent effect on LDL apoB-100 kinetics. Atorvastatin-mediated changes in cholesterol homeostasis may contribute to apoB PR regulation.     

Monoclonal antibodies to human low density lipoprotein identify distinct areas on apolipoprotein B-100 relevant to the low density lipoprotein-receptor interaction.

We have characterized the epitopes for ten murine monoclonal antibodies (Mabs) to human low density lipoprotein (LDL) and studied their ability to interfere with the LDL-receptor interaction. The epitopes for the antibodies were defined by using the following approaches: 1) interaction with apoB-48; 2) interaction with apoB-100 thrombolytic fragments; and 3) interaction with beta-galactosidase-apoB fusion proteins spanning different areas of the apoB-100 sequence. The results obtained are consistent with the following map of epitopes: Mab 6E, amino acids (aa) 1-1297, Mabs 5A and 6B, aa 1480-1693, Mabs 2A, 7A, 3B, and 4B, aa 2152-2377, Mabs 8A and 9A, aa 2657-3248 and 3H, aa 4082-4306. Four Mabs (2A, 5A, 7A, and 9A) whose epitopes are located in three different areas of apoB, dramatically reduced (up to 95%) the LDL-receptor interaction on cultured human fibroblasts; Fab fragments were as effective as the whole antibodies. Mab 3H, on the other hand, increased LDL binding up to threefold. These findings are consistent with the hypothesis that several areas of apoB-100 are involved independently or in concert in modulating the apoprotein B conformation required for interaction with the LDL receptor.     

Locating a low density lipoprotein-targeting domain of human apolipoprotein B-100 by expressing a minigene construct in transgenic mice

Through its interaction with the low density lipoprotein (LDL) receptor, apolipoprotein (apo) B-100 is a major determinant of LDL metabolism and plasma cholesterol. Its receptor binding ability is conformation-dependent and requires its expression on the right lipoprotein particles. The structural signal that targets apoB-100 to LDL is unknown. We have microinjected a human apoB-100 minigene construct comprising less than 25% of the apoB-100 sequence driven by the natural apoB promoter to produce transgenic mice. The transgene product was expressed at a high level and was present exclusively in the LDL of these animals. Analysis of the responsible sequence (residues 2878-3925 of apoB-100) reveals unique structural features that may be important in its role as an LDL-targeting domain.     

Binding of thyroxine to human plasma low density lipoprotein through specific interaction with apolipoprotein B (apoB-100)

Human plasma low density lipoprotein (LDL), which binds 0.2% of plasma T4, was shown to interact with the hormone through its protein moiety, apolipoprotein B-100. LDL and LDL2, the major subfraction of LDL, were found to have 3 equivalent binding sites for T4 with Ka = 2.5 x 10(6) M-1. Photoaffinity labeling of LDL with inner ring-labeled [125I]T4, followed by SDS-PAGE or agarose-SDS-PAGE of the labeled products, revealed that apoB-100 and its proteolytic cleavage products, apoB-74 and apoB-26, bound [125I]T4. In the presence of 1 or 10 microM T4, labeling was decreased in 7 separate experiments by 40-53% or 65-86%, respectively, consistent with a Ka of approximately 10(6) M-1. Binding of T4 to apoB-100 associated with VLDL was also demonstrated by photoaffinity labeling. The observed thyroid hormone binding property of lipid-complexed apoB-100 and the knowledge that receptors for the apolipoprotein exist in various tissues suggest a possible physiological role in thyroid hormone transport.     

Epitope mapping analysis of apolipoprotein B-100 using a surface plasmon resonance-based biosensor

Using a surface plasmon resonance (SPR)-based biosensor (BIA-technology), we have studied the interaction of ten different murine monoclonal antibodies (mAbs, all IgG₁), raised against the main protein constituent of human low density lipoprotein (LDL), i.e. the apolipoprotein B-100 (apoB-100). These mAbs identify distinct domains on apoB-100, relevant to LDL-receptor interaction: epitopes in the amino-terminal region (mAbs L7, L9, L10 and L11: aa 1–1297) and in the middle region (mAb 6B: aa 1480–1693; mAbs 2A, 3B: aa 2152–2377; mAbs 9A, L2 and L4: aa 2657–3248) of native apoB-100. A multisite binding analysis was performed to further characterize the epitopes recognized by all these mAbs. A rabbit anti-mouse IgG₁-Fc antibody (RAM.Fc) was first coupled to the gold surface in order to capture one anti-human apoB-100 mAb. ApoB-100 protein was subsequently injected and allowed to react with this immobilized, oriented antibody. Multisite binding assays were then performed, by sequentially flowing other mAbs, in different orders, over the sensing surface. The capacity of each mAb to interact with the entrapped apoB-100 in a multimolecular complex was monitored in real time by SPR. The results achieved were comparable to those obtained by western immunoblotting using the same reagents. However, SPR ensures a more detailed epitope identification, demonstrating that BIA-technology can be successfully used for mapping distinct epitopes on apoB-100 protein in solution dispensing with labels and secondary tracers; moreover, compared with conventional immunoassays, it is significantly time saving (CNR-P.F. MADESS 2).     

Mapping of antigenic determinants of human apolipoprotein B using monoclonal antibodies against low density lipoproteins

The reactivity of a series of monoclonal antibodies directed against human low density lipoproteins (LDL) has been tested with hepatic and intestinal apolipoprotein B (apo-B) termed B-100 and B-48, respectively (Kane, J. P., Hardman, D. A., and Paulus, H. E. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2465-2469). Whereas those antibodies that have been previously shown to recognize determinants close to the LDL receptor recognition site reacted only with B-100, two antibodies specific for other regions of apo-B reacted with both B-100 and B-48. Therefore, it is probable that sequence homologies exist between the two proteins and it must be considered that all or parts of the B-48 sequence may be contained within that of B-100. The specificity of the reaction of these antibodies with proteins designated B-74 and B-26 supports the concept that they represent complementary fragments of B-100. The present results have been incorporated in a theoretical map of the antigenic determinants recognized by these antibodies on the LDL apo-B.     

Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay as a candidate reference method for the measurement of apolipoprotein B-100

A monoclonal antibody-based direct binding enzyme-linked immunosorbent assay (ELISA) for apoprotein (apo) B-100 has been developed for use as a reference method. The assay uses the two well-characterized monoclonal antibodies, MB24 and MB47. MB47, which recognizes an epitope at the low density lipoprotein (LDL) receptor-binding domain of apoB and is specific for apoB-100, is bound to the microtiter plate as the capture antibody. MB24, which binds an epitope in the amino terminal half of the apoB-100 and identifies both apoB-100 and apoB-48, is conjugated to horseradish peroxidase and is utilized as the indicating antibody. The assay was calibrated with LDL (d 1.030-1.050 g/ml) and the LDL protein was determined by a sodium dodecyl sulfate (SDS) Lowry procedure. The working range of the assay is 0.25-1.25 micrograms/ml. Optimal dilution of whole plasma was found to be 1:2000. In the assay, MB47 bound approximately 97% of the apoB in all low density lipoprotein, and greater than 90% of the apoB in the majority of very low density lipoprotein preparations. Small dense LDL from subjects with familial combined hyperlipidemia (FCHL) and large bouyant LDL from subjects with familial hypercholesterolemia (FH) exhibited binding properties similar to LDL from healthy normolipidemic subjects when tested in the reference ELISA. The intra- and interassay coefficients of variation averaged 2.5% and 6.0%, respectively. Plasma B-100 levels were not influenced by freezing and thawing or storage at 4 degrees C for up to 3 weeks or storage at -70 degrees C for up to 11 months. Excellent agreement was obtained between the reference ELISA and a polyclonal RIA which measures total apoB (r = 0.93, n = 105, mean ELISA B-100 value = 100 mg/dl, mean RIA value = 101 mg/dl, Sy = 9.6). Reference ELISA B-100 values of samples pretreated with bacterial lipase were not significantly increased in most samples with plasma triglyceride levels below 600 mg/dl. To help reduce the large among-laboratories variability of apoB measurements, we recommend that this candidate reference direct binding ELISA be used to assign apoB target values to apoB reference pools.     

ApoB-75, a truncation of apolipoprotein B associated with familial hypobetalipoproteinemia: genetic and kinetic studies.

We have identified a mutation of apolipoprotein B (apoB) in a kindred with hypobetalipoproteinemia. Four affected members had plasma concentrations of total cholesterol of 115 +/- 14, low density lipoprotein (LDL)-C of 48 +/- 11, and apoB of 28 +/- 9 (mg/dl mean +/- SD). The values correspond to approximately 30% the values for unaffected relatives. Triglyceride and high density lipoprotein (HDL)-C concentrations were 92 +/- 50 and 49 +/- 4, respectively, neither significantly different from unaffected relatives. Western blots of plasma apoB of affected subjects showed two major bands: apoB-100 and an apoB-75 (mol wt of approximately 418,000). DNA sequencing of the appropriate polymerase chain reaction (PCR)-amplified genomic DNA segment revealed a deletion of the cytidine at nucleotide position 10366, resulting in a premature stop codon at amino acid residue 3387. In apoB-75/apoB-100 heterozygotes, two LDL populations containing either apoB-75 or apoB-100 could be distinguished from each other by gel permeation chromatography and by immunoblotting of nondenaturing gels using monoclonal antibodies B1B3 (epitope between apoB amino acid residues 3506-3635) and C1.4 (epitope between residues 97-526). ApoB-75 LDL were smaller and more dense than apoB-100 LDL. To determine whether the low concentration of apoB-75 was due to its enhanced LDL-receptor-mediated removal, apoB-75 LDL were isolated from the proband's d 1.063-1.090 g/ml fraction (which contained most of the apoB-75 in his plasma) by chromatography on anti-apoB and anti-apoA-I immunoaffinity columns. The resulting pure apoB-75 LDL fraction interacted with the cells 1.5-fold more effectively than apoB-100 LDL (d 1.019-1.063 g/ml). To determine the physiologic mechanism responsible for the hypobetalipoproteinemia, in vivo kinetic studies were performed in two affected subjects, using endogenous labeling of apoB-75 and apoB-100 with [13C]leucine followed by multicompartmental kinetic analyses. Fractional catabolic rates of apoB-75 VLDL and LDL were 2- and 1.3-fold those of apoB-100 very low density lipoprotein (VLDL) and LDL, respectively. Production rates of apoB-75 were approximately 30% of those for apoB-100. This differs from the behavior of apoB-89, a previously described variant, whose FCRs were also increased approximately 1.5-fold relative to apoB-100, but whose production rates were nearly identical to those of apoB-100. Thus, in contrast to the apoB-89 mutation, the apoB-75 mutation imparts two physiologic defects to apoB-75 lipoproteins that account for the hypobetalipoproteinemia, diminished production and increased catabolism.     

Modular structure of solubilized human apolipoprotein B-100. Low resolution model revealed by small angle neutron scattering

Being intimately involved in cholesterol transport and lipid metabolism human low density lipoprotein (LDL) plays a prominent role in atherogenesis and cardiovascular diseases. The receptor-mediated cellular uptake of LDL is triggered by apolipoprotein B-100 (apoB-100), which represents the single protein moiety of LDL. Due to the size and hydrophobic nature of apoB-100, its structure is not well characterized. Here we present a low resolution structure of solubilized apoB-100. We have used small angle neutron scattering in combination with advanced shape reconstruction algorithms to generate a three-dimensional model of lipid-free apoB-100. Our model clearly reveals that apoB-100 is composed of distinct domains connected by flexible regions. The apoB-100 molecule adopts a curved shape with a central cavity. In comparison to LDL-associated apoB-100, the lipid-free protein is expanded, whereas according to spectroscopic data the secondary structure is widely preserved. Finally, the low resolution model was used as a template to reconstruct a hypothetical domain organization of apoB-100 on LDL, including information derived from a secondary structure prediction.     

Human crypt intestinal epithelial cells are capable of lipid production, apolipoprotein synthesis, and lipoprotein assembly

The recent availability of spontaneously proliferating, non-transformed human crypt intestinal epithelial cells (HIEC) affords an opportunity to investigate lipid metabolism in undifferentiated enterocytes. The major purpose of this study was to explore the capability of undifferentiated crypt cells to synthesize, assemble, and secrete lipids and apolipoproteins. HIEC were cultured in medium with 5% fetal bovine serum for 5 to 21 d. The cells were clearly able to incorporate [(14)C]oleic acid (dpm/mg protein) into triglycerides (128,279 +/- 16,988), phospholipids (30, 278 +/- 2,107), and cholesteryl esters (2,180 +/- 207). Although improvement in lipid secretion was noted with prolongation of cell culture periods, low efficiency of lipid export (10.3 +/- 2.2% of intracellular content) characterized the HIEC. All phospholipid classes were elaborated, with phosphatidylcholine accounting for 79. 3 +/- 1.3% of cellular phospholipids. Chylomicrons were the dominant (46.4%) lipoproteins secreted, followed by high, low, and very low density lipoproteins (HDL, LDL, and VLDL) comprising 22.5, 20.2, and 10.8% of the total, respectively. HIEC elaborated most of the major apolipoprotein (apo) classes (A-I, A-IV, B-100, C, and E), but were less efficient in producing apoB-48. In contrast to the production of apoA-I and C as early as 5 days after confluence, apoA-I and A-IV were maximally expressed at 11 d. Culture media accumulated much more apoB-100 than apoB-48 (B-48/B-100 ratio 0.21 +/- 0.03), reflecting limited apoB mRNA editing. HIEC demonstrated both endogenous cholesterol synthesis and LDL receptor expression. Cholesterol synthesis was sensitive to 25-hydroxycholesterol and mevinolin, but unresponsive to LDL treatment, suggesting independent regulation pathways. In contrast, LDL inhibited receptor activity. The present findings provide the first solid evidence that immature HIEC are capable of key fat absorptive functions of well-differentiated enterocytes. The intracellular mechanisms required for lipid and apolipoprotein synthesis as well as for lipoprotein assembly are already present in intestinal crypt cells. These cells also retain the capacity for sterol enzyme and receptor expression. However, certain limitations, especially apoB-48 production and lipoprotein secretion as well as unresponsiveness of cholesterol synthesis to LDL, may be ascribed to the lack of differentiation.     

Evidence of increased secretion of apolipoprotein B-48-containing lipoproteins in subjects with type 2 diabetes

Patients with type 2 diabetes have high levels of triglyceride-rich lipoproteins (TRLs), including apolipoprotein B-48 (apoB-48)-containing TRLs of intestinal origin, but the mechanism leading to overaccumulation of these lipoproteins remains to be fully elucidated. Therefore, the objective of this study was to examine the in vivo kinetics of TRL apoB-48 and VLDL, intermediate density lipoprotein (IDL), and LDL apoB-100 in type 2 diabetic subjects (n = 11) and nondiabetic controls (n = 13) using a primed-constant infusion of l-[5,5,5-D(3)]leucine for 12 h in the fed state. Diabetic subjects had significantly higher fasting glycemia, higher fasting insulinemia, higher plasma triglyceride, and lower HDL-cholesterol levels than controls. Compared with controls, diabetic subjects had increased TRL apoB-48, VLDL apoB-100, and IDL apoB-100 pool sizes as a result of increased production rates (PRs) and reduced fractional catabolic rates of these lipoprotein subfractions. Furthermore, multiple linear regression analyses revealed that the diabetic/control status was an independent predictor of TRL apoB-48 PR and represented nearly 35% of its variance. These results suggest that the overaccumulation of TRLs seen in patients with type 2 diabetes is attributable to increased PRs of both intestinally derived apoB-48-containing lipoproteins and TRL apoB-100 of hepatic origin and to decreased catabolism of these subfractions.     

Genotype rs8099917 near the IL28B gene and amino acid substitution at position 70 in the core region of the hepatitis C virus are determinants of serum apolipoprotein B-100 concentration in chronic hepatitis C

The life cycle of the hepatitis C virus (HCV) is closely related to host lipoprotein metabolism. Serum levels of lipid are associated with the response to pegylated interferon plus ribavirin (PEG-IFN/RBV) therapy, while single nucleotide polymorphisms (SNPs) around the human interleukin 28B (IL28B) gene locus and amino acid substitutions in the core region of the HCV have been reported to affect the efficacy of PEG-IFN/RBV therapy in chronic hepatitis with HCV genotype 1b infection. The aim of this study was to elucidate the relationship between serum lipid and factors that are able to predict the efficacy of PEG-IFN/RB therapy, with specific focus on apolipoprotein B-100 (apoB-100) in 148 subjects with chronic HCV G1b infection. Our results demonstrated that both the aa 70 substitution in the core region of the HCV and the rs8099917 SNP located proximal to the IL28B were independent factors in determining serum apoB-100 and low-density lipoprotein (LDL) cholesterol levels. A significant association was noted between higher levels of apoB-100 (P = 1.1 × 10(-3)) and LDL cholesterol (P = 0.02) and the subjects having Arg70. A significant association was also observed between subjects carrying the rs8099917 TT responder genotype and higher levels of apoB-100 (P = 6.4 × 10(-3)) and LDL cholesterol (P = 4.2 × 10(-3)). Our results suggest that apoB-100 and LDL cholesterol are markers of impaired cellular lipoprotein pathways and/or host endogenous interferon response to HCV in chronic HCV infection. In particular, serum apoB-100 concentration might be an informative marker for judging changes in HCV-associated intracellular lipoprotein metabolism in patients carrying the rs8099917 responder genotype.     

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.     

The peroxisome proliferator-activated receptor alpha (PPARalpha) agonist ciprofibrate inhibits apolipoprotein B mRNA editing in low density lipoprotein receptor-deficient mice: effects on plasma lipoproteins and the development of atherosclerotic lesions

Low density lipoprotein receptor (LDLR)-deficient mice fed a chow diet have a mild hypercholesterolemia caused by the abnormal accumulation in the plasma of apolipoprotein B (apoB)-100- and apoB-48-carrying intermediate density lipoproteins (IDL) and low density lipoproteins (LDL). Treatment of LDLR-deficient mice with ciprofibrate caused a marked decrease in plasma apoB-48-carrying IDL and LDL but at the same time caused a large accumulation of triglyceride-depleted apoB-100-carrying IDL and LDL, resulting in a significant increase in plasma cholesterol levels. These plasma lipoprotein changes were associated with an increase in the hepatic secretion of apoB-100-carrying very low density lipoproteins (VLDL) and a decrease in the secretion of apoB-48-carrying VLDL, accompanied by a significant decrease in hepatic apoB mRNA editing. Hepatic apobec-1 complementation factor mRNA and protein abundance were significantly decreased, whereas apobec-1 mRNA and protein abundance remained unchanged. No changes in apoB mRNA editing occurred in the intestine of the treated animals. After 150 days of treatment with ciprofibrate, consistent with the increased plasma accumulation of apoB-100-carrying IDL and LDL, the LDLR-deficient mice displayed severe atherosclerotic lesions in the aorta. These findings demonstrate that ciprofibrate treatment decreases hepatic apoB mRNA editing and alters the pattern of hepatic lipoprotein secretion toward apoB-100-associated VLDL, changes that in turn lead to increased atherosclerosis.     

Scission of human apolipoprotein B-100 by kallikrein: characterization of the cleavage site

Low density lipoprotein (LDL) from human plasma was digested with the specific endoprotease, kallikrein. Apolipoprotein B-100, the protein moiety of LDL, was cleaved by kallikrein into two fragments (K1 and K2) which we have compared to the naturally occurring fragments, B-74 and B-26. We have found that K1 and K2 precisely match B-74 and B-26 with respect to molecular weight, stoichiometry, and amino terminal amino acid sequence. These findings provide strong evidence that kallikrein is the agent responsible for the formation of B-74 and B-26 in human LDL.     

Polarized secretion of newly synthesized lipoproteins by the Caco-2 human intestinal cell line

Lipoprotein secretion by Caco-2 cells, a human intestinal cell line, was studied in cells grown on inserts containing a Millipore filter (0.45 micron), separating secretory products from the apical and basolateral membranes into separate chambers. Under these conditions, as observed by electron microscopy, the cells formed a monolayer of columnar epithelial cells with microvilli on the apical surface and tight junctions between cells. The electrical resistances of the cell monolayers were 250-500 ohms/cm2. Both 14C-labeled lipids and 35S-labeled proteins were used to assess lipoprotein secretion. After a 24-hr incubation with [14C]oleic acid, 60-80% of the secreted triglyceride (TG) was in the basolateral chamber; 40% of the TG was present in the d less than 1.006 g/ml (chylomicron + VLDL) fraction and 50% in the 1.006 less than d less than 1.063 g/ml (LDL) fraction. After a 4-hr incubation with [35S]methionine, apolipoproteins were found to be major secretory products with 75-100% secreted to the basolateral chamber. Apolipoproteins B-100, B-48, E, A-I, A-IV, and C-III were identified by immunoprecipitation. The d less than 1.006 g/ml fraction was found to contain all of the major apolipoproteins, while the LDL fraction contained primarily apoB-100 and apoE; the HDL (1.063 less than d less than 1.21 g/ml) fraction principally contained apoA-I and apoA-IV. Mn-heparin precipitated all of the [35S]methionine-labeled apoB-100 and B-48 and a majority of the other apolipoproteins, and 80% of the [14C]oleic acid-labeled triglyceride, but only 15% of the phospholipid, demonstrating that Caco-2 cells secrete triglyceride-rich lipoproteins containing apoB. Secretion of lipoproteins was dependent on the lipid content of the medium; prior incubation with lipoprotein-depleted serum specifically reduced the secretion of lipoproteins, while addition of both LDL and oleic acid to the medium maintained the level of apoB-100, B-48, and A-IV secretion to that observed in the control cultures.     

Primary sequence mapping of human apolipoprotein B-100 epitopes. Comparisons of trypsin accessibility and immunoreactivity and implication for apoB conformation

Differential trypsin-accessibility and monoclonal antibodies (Mabs) to human apolipoprotein (apo) B-100 are both important tools for probing apoB structure and conformation on low-density lipoproteins (LDL). In this study, we have mapped greater than 80% of the C-terminal region (720 residues) of LDL apoB-100 using trypsin digestion. Our results extend our previous data [Yang et al. (1986) Nature (Lond.) 323, 738-742] confirming that the C-terminal region of about 420 residues of apoB-100 is largely inaccessible to trypsin, whereas the part just preceding this region has interspersed trypsin-accessible and inaccessible peptides. We have determined the amino acid sequence of specific apoB-100 peptides containing epitopes recognized by four separate Mabs: two epitopes have been mapped to within 20 residues, one has been mapped to 36 residues, and the last to 80 residues. We used polyclonal antisera to identify 16 overlapping clones of varying lengths of apoB-100 cDNAs extending from the C-terminus of apoB-100 cloned in the expression vector, lambda gt11. These clones were then tested against individual Mabs. By nucleotide sequence analysis of overlapping clones that show differential reactivities to different Mabs, we have mapped the individual epitopes of each Mab to within about 50-150 amino acid residues predicted from the DNA sequences. Confirmation and further fine mapping were accomplished by competition for LDL binding using partially purified fusion proteins and chemically synthesized oligopeptides. Two epitopes (Mabs 7 and 22) were mapped to the C-terminal 20 amino acids of apoB-100, one (Mab 16) to residues 4154-4189, and another (Mab 20) to residues 3926-4005. Mab 16 precipitates more than 80% of LDL particles. Mab 20 precipitates only denatured apoB but not native LDL apoB [Milne et al. (1987) Mol. Immunol. 24, 435]. Mabs 7 and 22 are unique in that they precipitate LDL apoB modified by storage much better than freshly isolated LDL-apoB. Although epitope expression and trypsin-accessibility represent two useful probes for the study of protein conformation, there was no obvious correlation between these two parameters when applied to LDL apoB for the antibodies we have examined.