Plasma lipid transport in the horse (Equus caballus)

• 1.1. Equine plasma contains lipoproteins corresponding to very low density (VLDL), low density (LDL) and high density lipoproteins (HDL).
• 2.2. HDL accounts for approximately 60% of plasma lipoprotein mass and consists of a single population of particles.
• 3.3. LDL is heterogeneous comprising three discrete subfractions.
• 4.4. Two proteins are found in the region of apolipoprotein (apo) B-100 in VLDL and LDL and a third similar to apo B-48 is in VLDL.
• 5.5. Lecithin:cholesterol acyl transferase is active in plasma and hepatic lipase and lipoprotein lipase are evident in post-heparin plasma.
• 6.6. There is no significant cholesteryl ester transfer protein activity.

     

Synthesis and secretion of lipoproteins by human hepatocytes in culture

Summary Confluent monolayers of normal human hepatocytes obtained by collagenase perfusion of liver pragments were incubated in a serum-free medium. Intracellular apolipoproteins apo AI, apo C, apo B, and apo E were detected between Day 1 and Day 6 of the culture by immunoenzymatic staining using polyclonal antibodies directed against these apoproteins and monoclonal antibodies directed against both forms of apo B (B100 and B48). Translation of mRNA isolated from these hepatocytes in an acellular system revealed that apo AI and apo E were synthesized as the precusor forms of mature plasma apo AI and apo E. Three lipoprotein fractions corresponding to the density of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) were isolated from the medium at Day 5 of culture and examined by electron microscopy after negative staining. VLDL and LDL particles are similar in size and shape to plasma lipoproteins; spherical HDL are larger than normal plasma particles isolated at the same density. Their protein represented 44, 19.5, and 36.5% respectively, of the total lipoprotein protein. The secretion rate of VLDL protein corresponded to that measured in primary cultures of rat hepatocytes. After incorporation of [³H]glycerol, more than 92% of the [³H]triglyceride secreted into the medium was recovered in the VLDL fraction. These results demonstrate that primary cultures of normal human hepatocytes are able to synthesize and secrete lipoproteins and thus could be a useful model to study lipoprotein metabolism in human liver.     

Carboxyl-terminal truncation of apolipoprotein B results in gradual loss of the ability to form buoyant lipoproteins in cultured human and rat liver cell lines

Apolipoprotein B has an obligatory role in the production of chylomicrons, VLDL, and LDL. Familial hypobetalipoproteinemia is a codominant disorder characterized by reduced levels of apo B containing lipoproteins in plasma. We have previously described mutations of the apo B gene in persons with hypobetalipoproteinemia that predict truncated forms of apo B designated apo B29 (1305 amino acid residues) and apo B39 (1799 residues). Apo B39 was present in the VLDL and LDL fractions of plasma, but apo B29 was not detected in the lipoprotein or infranatant fractions of plasma. Here we have investigated the regions of apo B necessary for apo B containing lipoprotein secretion by expression of constructs designed to express truncated forms of apo B. Apo B13 (583 residues), apo B17 (784 residues), apo B23 (1084 residues), apo B29 (1306 residues), and apo B41 (1880 residues) were transiently expressed in HepG2 cells, and apo B23 and apo B41 were stably expressed in McArdle 7777 cells. Lipoprotein (d less than 1.25 g/mL) and infranatant (d greater than 1.25 g/mL) fractions of conditioned medium were analyzed by immunoprecipitation and SDS-PAGE. The distribution between lipoprotein and infranatant fractions varied: apo B41 was found solely in the lipoprotein fraction; apo B29, apo B23, and apo B17 were present in both fractions, but with stepwise truncation, progressively more apo B was recovered in the infranatant; apo B13 was only in the infranatant. These results demonstrate that deletion from the carboxyl terminal of apo B41 results in a gradual loss of the ability of the truncated proteins to form buoyant lipoprotein particles.     

Direct evidence for apo B-100-mediated copper reduction: studies with purified apo B-100 and detection of tryptophanyl radicals

Copper binding to apolipoprotein B-100 (apo B-100) and its reduction by endogenous components of low-density lipoprotein (LDL) represent critical steps in copper-mediated LDL oxidation, where cuprous ion (Cu(I)) generated from cupric ion (Cu(II)) reduction is the real trigger for lipid peroxidation. Although the copper-reducing capacity of the lipid components of LDL has been studied extensively, we developed a model to specifically analyze the potential copper reducing activity of its protein moiety (apo B-100). Apo B-100 was isolated after solubilization and extraction from size exclusion-HPLC purified LDL. We obtained, for the first time, direct evidence for apo B-100-mediated copper reduction in a process that involves protein-derived radical formation. Kinetics of copper reduction by isolated apo B-100 was different from that of LDL, mainly because apo B-100 showed a single phase-exponential kinetic, instead of the already described biphasic kinetics for LDL (namely alpha-tocopherol-dependent and independent phases). While at early time points, the LDL copper reducing activity was higher due to the presence of alpha-tocopherol, at longer time points kinetics of copper reduction was similar in both LDL and apo B-100 samples. Electron paramagnetic resonance studies of either LDL or apo B-100 incubated with Cu(II), in the presence of the spin trap 2-methyl-2-nitroso propane (MNP), indicated the formation of protein-tryptophanyl radicals. Our results supports that apo B-100 plays a critical role in copper-dependent LDL oxidation, due to its lipid-independent-copper reductive ability.     

A comparative study of serum lipoproteins in rabbits fed a natural ingredient diet or low-fat, cholesterol-free, semipurified diets containing casein or isolated soy protein

In rabbits fed a cholesterol-free, semipurified diet containing isolated soy protein, the average total serum cholesterol level was similar to that of rabbits fed a natural ingredient (chow) diet. However, the cholesterol and protein levels in very low density (VLDL) and low density lipoproteins (LDL) tended to increase, while the levels in high density lipoproteins (HDL) were reduced to about half of those on the chow diet, with little change in the cholesterol to protein ratio. Substitution of casein for soy protein in the semipurified diet caused a four- to five-fold increase in total serum cholesterol and a doubling of lipoprotein protein, with an increase of 1.4- to 3.0-fold in the cholesterol to protein ratio of the different lipoprotein fractions. Analysis of the apoproteins (apo) of the plasma lipoproteins indicated that apo B, E, and C all tended to increase in the VLDL and LDL of rabbits fed the soy protein diet compared with those fed chow diet. The levels of each of the apoproteins were increased further by substituting casein for soy protein in the semipurified diet. In this case, apo E showed the greatest relative increase (2.7-fold) in VLDL, while apo B and E were increased to a similar extent (about 4-fold) in LDL. Apo C was approximately doubled in each of these fractions. The apo A content in HDL of rabbits fed the semipurified diets was about half that of rabbits fed chow diet. No marked changes were noted in the apo E or C content of HDL. Separation of isoforms of the soluble apoproteins showed variations between individual animals, but these variations seemed largely unrelated to diet. The results of these studies indicate that semipurified diets produce changes in the serum lipoprotein patterns of rabbits that are only partly due to the protein component of these diets.     

Dynamic behavior of apoproteins of human plasma very low density lipoproteins and lipolysis regulation

Using the dynamic fluorescence quenching method, it was shown that very low density (VLDL) apoproteins (apo B, E and C) tryptophanyls exhibit a lower accessibility towards water-soluble quenchers as compared to apo B LDL chromophores. The efficiency of proteolytic degradation by trypsin of VLDL-associated apo E and apo C was much lower than that of apo B. These results may be due to the cluster arrangement of amphipatic apo E and apo C on the VLDL surface and/or to their partial shielding by apo B. Treatment of VLDL particles with sub-lytic concentrations of the detergent, Tween-20, did not change the relaxation characteristics of amphipatic apoprotein tryptophanyl microenvironment, but resulted in a reversible structural transition registered by a "red" shift of the emission spectrum maximum as well as by change of the iodine quenching pattern. The detergent-induced increase of the VLDL tryptophanyl accessibility to acrylamide and the decrease of the quenching constant at the partial and complete particle solubilization were related to a change of the apo B molecular package. Treatment of VLDL with Tween-20 or cow milk lipoprotein lipase resulted in the appearance of tryptophanyl population that was not involved in the resonance energy transfer to the lipid phase-localized fluorescent probe pyrene, which is indicative of the protein dissociation. Treatment of VLDL particles with sub-lytic concentrations of Tween-20 revealed a lower (compared to apo C) relative affinity of apo E for the VLDL lipid surface. Inhibition of the lipoprotein lipase activity by apoprotein C-III was found to be non-competitive. It was concluded that lipolysis is a self-regulatory process which involves changes in the effector apoprotein concentration on the surface of triglyceride-rich particles.     

Metabolism of apoB-100 in lipoproteins separated by density gradient ultracentrifugation in normal and Watanabe heritable hyperlipidemic rabbits

We have examined the capability of a previously developed compartmental model to explain the kinetics of radioiodinated apolipoprotein (apo) B-100 in very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL) separated by density gradient ultracentrifugation after intravenous injection of radioiodinated VLDL into New Zealand white (NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits. Our model was developed primarily from kinetics in whole blood plasma of apoB-100 in particles with and without apoE after intravenous injection of large VLDL, total VLDL, IDL, and LDL. When the initial conditions for this model were assumed to be an intravenous injection of radiolabeled VLDL, the plasma VLDL and LDL simulations for NZW rabbits and the VLDL, IDL, and LDL simulations for WHHL rabbits were found to be inconsistent with the observed density gradient data. By adding a new pathway in the VLDL portion of the model for NZW rabbits and a new compartment in VLDL for WHHL rabbits, and by assuming some cross-contamination in the density gradient ultracentrifugal separations, it was possible to bring our model, which was based upon measurements of 125I-labeled apoB-100 in whole plasma, into conformity with the data obtained by density gradient ultracentrifugation. The relatively modest changes required in the model to fit the gradient ultracentrifugation data support the suitability of our approach to the kinetic analysis of the metabolism of apoB-100 in VLDL and its conversion to IDL and LDL based upon measurements of 125I-labeled apoB-100 in whole plasma after injection of radiolabeled VLDL, IDL, and LDL. Furthermore, the differences in kinetics observed by us between data from whole plasma and data from plasma submitted to ultracentrifugal separation from the same or similar animals highlight the fact that small variations that can occur in the separation of lipoprotein classes by buoyant density can lead to confusing results.     

The very low density lipoprotein (VLDL) receptor – a peripheral lipoprotein receptor for remnant lipoproteins into fatty acid active tissues

The VLDL (very low density lipoprotein) receptor is a member of the LDL (low density lipoprotein) receptor family. The VLDL receptor binds apolipoprotein (apo) E but not apo B, and is expressed in fatty acid active tissues (heart, muscle, adipose) and macrophages abundantly. Lipoprotein lipase (LPL) modulates the binding of triglyceride (TG)-rich lipoprotein particles to the VLDL receptor. By the unique ligand specificity, VLDL receptor practically appeared to function as IDL (intermediate density lipoprotein) and chylomicron remnant receptor in peripheral tissues in concert with LPL. In contrast to LDL receptor, the VLDL receptor expression is not down regulated by lipoproteins. Recently several possible functions of the VLDL receptor have been reported in lipoprotein metabolism, atherosclerosis, obesity/insulin resistance, cardiac fatty acid metabolism and neuronal migration. The gene therapy of VLDL receptor into the LDL receptor knockout mice liver showed a benefit effect for lipoprotein metabolism and atherosclerosis. Further researches about the VLDL receptor function will be needed in the future.     

Comparison of the lipoprotein profiles obtained from rat, bovine, horse, dog, rabbit and pig serum by a new two-step ultracentrifugal gradient procedure

A new two-step gradient technique has been used in the separation of the different classes of lipoproteins from the serum of cows, horses, dogs, pigs, rabbits and rats. Total lipoproteins were first isolated at d 1.21 then floated through a d 1.006 to d 1.21 gradient. Collection by mean of a gradient fractionator provided directly comparable lipoprotein profiles, allowed the determination of the exact density range of each lipoprotein class and the fraction by fraction analysis of composition. Cholesterol and apo AI recoveries were high. Horse, dog, rabbit and pig exhibited three distinct lipoprotein classes: VLDL, LDL and HDL. LDL were polydisperse in the pig (three components), light in the rabbit and scarce in the horse. In the Sprague-Dawley rat, LDL could not be individualized from HDL. In the bovine, LDL overlapped with a light form of HDL. Although AI was the main apoprotein in the HDL of all species, it ranged in proportion from 35% in the rat to 75% in the bovine. Apo AII was dimeric in the dog, as already known, but also in the horse, rabbit and bovine (MW:17,000) and in the pig MW:13,000). Apo AIV was present in the heavier HDL of all species. Rabbit, horse and pig HDL contained only one species of apo C which, in the pig was identified as apo CII.     

Protein composition of Lp(a) lipoprotein from human plasma

The apolipoprotein composition of purified human Lp(a) lipoprotein was investigated by SDS--polyacrylamide gel electrophoresis and immunochemically. The lipoprotein contains two different polypeptides. One is identical by its app. Mr of approximately 250 000 and immunologically with apolipoprotein B of LDL (B-100). The other polypeptide has a higher app. Mr (approximately 350 000) and stains strongly with the periodate-Schiff's reagent. This high-Mr glycoprotein contains the specific Lp(a) immunoreactivity but does not react with antibodies against apo B. Apo B and Lp(a)-protein seem to be linked by disulfide bonds in the native lipoprotein. The unreduced detergent delipidized protein moiety from Lp(a) lipoprotein shows a single band of Mr approximately 700 000 in SDS--polyacrylamide gel electrophoresis and the immunoprecipitates formed against anti-Lp(a) and anti-apo B by the unreduced protein show a reaction of immunological identity.     

Characterization of nascent lipoproteins produced by perfused rat liver: evidence of hepatic secretion and post-secretory modification of nascent lipoproteins

We characterized the lipoproteins produced by perfused rat liver in recirculating and non-recirculating systems. The apolipoprotein (apo) B of the perfusate very low density lipoprotein (VLDL) and low density lipoprotein (LDL) were labeled with a radioactive precursor amino acid in both systems, suggesting that newly synthesized apo B was secreted in association with VLDL and LDL. When the lipoproteins obtained from the non-recirculating perfusate were injected into rats in vivo, the half life of the VLDL was 13 min and most of it was converted to LDL, while that of the LDL was 5.2 h, indicating that the perfusate LDL was different from the VLDL with respect to its metabolic fate. These observations suggest that both VLDL and LDL are produced as independent primary products in the liver, although the majority of LDL is derived from VLDL in vivo. The nascent lipoproteins in the non-recirculating perfusate were richer in apo E than those in the recirculating perfusate, and a part of the apo E disappeared when the VLDL was added to the recirculating perfusate. The particle sizes of the VLDL and LDL were examined by electron microscopy, which revealed that those in the non-recirculating perfusate were more homogeneous and smaller than the plasma counterparts, while those in the recirculating perfusate were more heterogeneous and their mean diameter was closer to that of the plasma lipoproteins, than in the case of non-recirculating perfusate. These observations suggest that apo E secreted with the nascent lipoproteins may be picked up by the liver just after secretion, causing the heterogeneity in size, as observed in the case of plasma lipoproteins.     

Cellular metabolism of human plasma intermediate-density lipoprotein (IDL)

The cellular metabolism of human plasma intermediate-density lipoprotein (IDL) was investigated in cultured human skin fibroblasts and hepG-2 cell in the absence and presence of exogenous recombinant or plasmatic apo E-3. IDL (d 1.006-1.019 g/ml) and LDL (d 1.019-1.063 g/ml) were prepared by centrifugation from the plasma of apo E-3/3 or 4/3 normolipidemic human subjects. Without added apo E-3, IDL binding and cell association are similar or slightly reduced while their degradation is one third to one half. This results in degradation to binding ratios for IDL that are half those for LDL. Exogenous apo E-3 enhances binding, association and degradation of IDL by 50-150%, but the degradation to binding ratio remains low. Exogenous apo E-3 also increased the ability of IDL but not LDL, to down-regulate the incorporation of [14C]acetate to sterol by the cells. The optimal concentration of apo E-3 is 4 micrograms protein/10 micrograms IDL protein and at that concentration appreciable amounts of the apo E are found associated with the lipoprotein. Apo E-2 has no effect on the cellular metabolism of IDL and apo E-3 is not effective in receptor-negative human fibroblasts. Monoclonal antibodies that block apo E binding to B,E (LDL) receptor (1D7) abolish the cellular metabolism of IDL while antibodies against B-100 (4G3) are ineffective. In competitive binding experiments, IDL is slightly more effective than LDL in displacing 125I-LDL from receptors in hepG-2 cells and appreciably more effective than LDL when tested against 125I-IDL. Apo E-3 increases the capacity of IDL to compete with either 125I-LDL or 125I-IDL. Addition of apo E-3 also increases the binding affinity of IDL to hepG-2 receptors, with Kd values of 2.50, 0.93 micrograms protein/ml, respectively. The study demonstrates the essential role that functional apo E molecules play in the interaction of human IDL with cellular receptors. Yet, in spite of presence of apo E in IDL (2-3 molecules/particle) and enrichment of IDL with apo E-3 (to 4-5 molecules/particle) the proteolytic degradation of the lipoprotein by specific cellular receptor is similar to LDL.     

Proposed mechanisms for binding of apo[a] kringle type 9 to apo B-100 in human lipoprotein[a].

The protein component of human lipoprotein[a] consists primarily of two apolipoproteins, apo[a] and apo B-100, linked through a cystine disulfide(s). In the amino acid sequence of apo bd, Cys4057 located within a plasminogen kringle 4-like repeat sequence (3991-4068) is believed to form a disulfide bond with a specific cysteine residue in apo B-100. Our fluorescence-labeling experiments and molecular modeling studies have provided evidence for possible interactions between this apo[a] kringle type and apo B-100. The fluorescent probe, fluorescein-5-maleimide, was used in parallel experiments to label free sulfhydryl moieties in lipoprotein[a] and low-density lipoprotein (LDL). In apo B-100 of LDL, Cys3734 was labeled with the probe, but this site was not labeled in autologous lipoprotein[a]. The result strongly implicates Cys3734 of apo B-100 as the residue forming the disulfide linkage with Cys4057 of apo[a]. To explore possible noncovalent interactions between apo B-100 and apo[a], the crystallographic coordinates for plasminogen kringle 4 were used to generate molecular models of the apo[a] kringle-repeat sequence (3991-4068, LPaK9), the only plasminogen kringle 4 type repeat in apo[a] having an extra cysteine residue not involved in an intramolecular disulfide bond. The Cys4057 residue (henceforth designated as Cys67 in the LPaK9 sequence) is believed to form an intermolecular disulfide bond with a cysteine of apo B-100. In computer graphics molecular models of LPaK9, Cys67 is located on the surface of the kringle near the lysine ligand binding site. Selected segments of the LDL apo B-100 sequence that contain free sulfhydryl cysteines were subjected to energy minimization and docking with the ligand binding site and adjacent regions of the LPaK9 model. In the docking experiments, apo B-100 segment 3732-3745 (PSCKLDFREIQIYK) displayed the best fit and the largest number of van der Waals contacts with models of LPaK9. Other apo B-100 peptides with sulfhydryl cysteine were found to be less compatible when minimized with this kringle. These results support and extend previously suggested mechanisms for a complex interaction between apo[a] and apo B-100 that involve more than a simple covalent disulfide bond.     

Insulin suppression of VLDL apo B secretion is not mediated by the LDL receptor

Insulin inhibits hepatic very low density lipoprotein (VLDL) apo B secretion in rats. Current studies test whether the insulin effect is LDL receptor-mediated by examining the effect of insulin on VLDL apo B secretion in hepatocytes derived from Ldlr-/- and control mice. Primary hepatocytes were incubated overnight with media containing 14C-leucine and either 0.1nM (basal) or 200nM insulin. Afterwards, secreted VLDL B100 and B48 were quantitated. Insulin reduced 14C-labeled B100 and B48 comparably in control and Ldlr-/- hepatocytes with a 62+/-12% vs. 59+/-12% decrease in B100, and a 56+/-11% vs. 61+/-9% decrease in B48. Results indicate: (1) mouse hepatocytes respond to insulin by reducing VLDL apo B output; (2) both VLDL B100 and B48 secretion are suppressed; and (3) insulin inhibition of VLDL apo B secretion is retained in Ldlr-/- hepatocytes.     

Alterations in plasma lipoproteins and apolipoproteins associated with estrogen-induced hyperlipidemia in the laying hen

The laying hen represents a physiological model in which the mechanisms of action of estrogens on lipid transport can be evaluated. The plasma lipoproteins in the laying hen were subfractionated into discrete particle species by isopycnic density gradient ultracentrifugation and the physicochemical properties and apolipoprotein contents of individual subfractions evaluated. The qualitative and quantitative aspects of this estrogen-specific profile were then compared to those of the immature chicken. As observed earlier, estrogens induced dramatic elevation in very-low-density lipoproteins (VLDL) (up to 900 mg/dl). Indeed, triglyceride-rich lipoproteins with densities up to 1.035 g/ml, i.e. VLDL and their remnants, behaved as a continuum which displayed little variation in size (20.5-21 nm), electrophoretic mobility (beta-like) and apolipoprotein content; apo B-100 (540 kDa) predominated while apo A-I (27 kDa), apo VLDL-II (19 kDa) and an apo-C-like protein (13 kDa) were present as minor components. The typical high-density lipoproteins (HDL) in the immature chicken were replaced by a lipoprotein population whose physicochemical properties were quite distinct. Thus these particles were distributed as a single, asymmetric peak over the density range 1.030-1.158 g/ml, a wide interval which overlapped that of apo-B-rich particles at its lower limit. The rho 1.030-1.158 g/ml lipoproteins were present at concentrations (approximately equal to 200 mg/dl) some twofold to threefold lower than those of HDL in immature birds. Furthermore, they displayed physical and chemical properties in common with both low-density lipoproteins (LDL) and HDL and were LDL-like in exhibiting beta mobility but HDL-like in size (9-15 nm diameter). Their protein moiety was also HDL-like in its predominant content of apo A-I; small amounts of apo VLDL-II and the apo-C-like protein were also detected. Substantial amounts of lipid were found at rho greater than 1.195 g/ml: such substances are absent in the immature chicken and may reflect the presence of vitellogenins. The hyperestrogenic state in the laying hen is therefore associated with major modifications in lipoprotein and apolipoprotein profile. Such modifications may be of relevance to clinical disorders involving estrogen-induced hyperlipidemia.     

Structural heterogeneity of apoB-containing serum lipoproteins visualized using cryo-electron microscopy.

Cryo-electron microscopy was used to analyze the structure of lipoprotein particles in density gradient subfractions of human very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL). Lipoproteins from a normolipidemic subject with relatively large and buoyant LDL (pattern A) and from a subject with a predominance of small dense LDL (pattern B) were compared. Projections of VLDL in vitreous ice were heterogeneous in size, but all were circular with a relatively even distribution of contrast. Selected projections of LDL, on the other hand, were circular with a high density ring or rectangular with two high density bands. Both circular and rectangular LDL projections decreased in average size with increasing subfraction density, but were found in all of 10 density gradient subfractions, both in pattern A and in pattern B profiles. Preparations of total IDL contained particles with the structural features of VLDL as well as particles resembling LDL. IDL particles resembling LDL were observed in specific density gradient subfractions in the denser region of the VLDL;-IDL density range. Within the group of IDL particles resembling LDL considerable heterogeneity was observed, but no structural features specific for the pattern A or pattern B lipoprotein profile were recognized.The observed structural heterogeneity of the apolipoprotein B-containing serum lipoproteins may reflect differences in the composition of these particles that may also influence their metabolic and pathologic properties.     

Evaluation of the secondary structure of apo B-100 in low-density lipoprotein (LDL) by infrared spectroscopy

The secondary structure of the apo B-100 protein present in human low density lipoprotein has been investigated by transmission and attenuated total reflection infrared spectroscopy. The amount of beta-sheet (41%) is significantly higher than that determined by CD spectroscopy in the present study (12%) and elsewhere (15-16%). The high percentage of beta-sheet structure in apo B-100 supports the importance of such segments in maintaining the lipid-protein assembly in LDL. Polarized infrared spectroscopy indicates that the beta-sheet component of apo B-100 adopts a preferential orientation with respect to the phospholipid monolayer surrounding the LDL, whereas no such orientation is observed for the other secondary structure components.     

The linkage with apolipoprotein (a) in lipoprotein (a) modifies the immunochemical and functional properties of apolipoprotein B

Lipoprotein (a) [Lp(a)] was isolated from several donors and its apolipoprotein (a) [apo(a)] dissociated by a reductive treatment, generating the apo(a)-free form of Lp(a) [Lp(a--)] that contains apolipoprotein B (apo B) as its sole protein. Using anti-apo B monoclonal antibodies, the properties of apo B in Lp(a), Lp(a--), and autologous low-density lipoprotein (LDL) were compared. Marked differences in apo B immunoreactivity were found between these lipoproteins, due to the presence of apo(a) in Lp(a). Apo(a) enhanced the expression of two epitopes in the amino-terminal part of apo B while it diminished the immunoreactivity of three other epitopes in the LDL receptor binding domain. Accordingly, the binding of the lipoproteins to the LDL receptor was also decreased in the presence of apo(a). In a different experimental system, the incubation of antibodies that react with 27 distinct epitopes distributed along the whole length of apo B sequence with plastic-bound Lp(a) and Lp(a--) failed to reveal any epitope of apo B that is sterically hindered by the presence of apo(a). Our results demonstrate that the presence of apo(a) modified the organization and function of apo B in Lp(a) particles. The data presented indicate that most likely the modification is not due to a steric hindrance but that some more profound conformational changes are involved. We suggest that the formation of the disulfide bridge between apo B and apo(a) in Lp(a) alters the system of disulfide bonds present in apo B and thereby modifies apo B structure.     

Molecular parameters that control the association of low density lipoprotein apo B-100 with chondroitin sulphate

The association of low density lipoprotein (LDL) with proteoglycans of the intima, in particular chondroitin 6-sulphate proteoglycans, may contribute to LDL accumulation during atherogenesis. We studied the interactions of apolipoprotein B-100 (apo B-100) peptide segments and model peptides with chondroitin 6-sulphate. The ability of these peptides to inhibit complex formation between LDL and chondroitin 6-sulphate was used as a measurement of the interaction. Results from earlier studies suggest that surface located segments of apo B-100 are responsible for the interaction of LDL with heparin and chondroitin sulphate-rich arterial proteoglycans. Therefore 16 hydrophilic apo B-100 peptides were selected for studies and synthesized with a peptide synthesizer. These synthetic peptides were 7 to 26 amino acids long. Four of the peptides inhibited the association of LDL with chondroitin 6-sulphate, namely apo B segments 4230–4254, 3359–3377, 3145–3157 and 2106–2121. The 3359–3377 segment was the most efficient. A common feature betweeb the interacting peptides was an excess of positively charged side chains and based on these results we synthesized nine model peptides that shared sequence characateristics with the interacting apo B-100 peptides. Five of these: RSGRKRSGK, RSSRKRSGK, RGGRKRGGK, RSRSRSRSR AND RGRGRGRGR were shown to block the LDL-chrondroitin-6-sulphate association, RSRSRSRSR being the most effective. The results suggest that the optimal association of the peptides with chrondroitin 6-sulphate is obtained with a minimal chain length of nine amino acids and a minimum of five positive charges and that flexibility in the binding region is important.     

Effects of chronic glucagon administration on rat lipoprotein composition

Male adult rats of the Wistar strain received daily at 9 a.m. and 5 p.m. 10 micrograms of Zn-protamine glucagon (Novo) for 21 days by subcutaneous injections. Plasma levels of cholesterol, triacylglycerol and phospholipids were decreased by 47, 40 and 21%, respectively. Lipoproteins were separated by sequential ultracentrifugation. Concentrations of cholesterol, phospholipids and proteins were decreased in chylomicrons, VLDL, LDL2 (1.040-1.063 g/ml) and HDL, LDL2 being the most affected by glucagon treatment (-70%). Triacylglycerol levels were decreased only in chylomicrons and VLDL. The relative proportions of cholesterol, triacylglycerol, phospholipids and proteins in lipoproteins were virtually unchanged by glucagon, suggesting a reduced number of some lipoprotein particles in plasma. However, lipoproteins of glucagon-treated rats were depleted in cholesteryl esters, while the proportion of triacylglycerol increased in LDL and HDL. Apo E contents were decreased in plasma, LDL1 (1.006-1.040 g/ml), LDL2 and HDL, whereas apo B100 proportions increased in VLDL and LDL1 in glucagon-treated rats. Glucagon appeared to be a potent hypolipidemic agent affecting mainly the apo-E-rich lipoproteins.