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.     

Apolipoprotein B-32: a new truncated mutant of human apolipoprotein B capable of forming particles in the low density lipoprotein range.

We have identified a new species of apolipoprotein (apo) B in an individual with heterozygous hypobetalipoproteinemia. The new apo B (apo B-32) is the result of a single point mutation (1450 Gln----Stop) in the apo B gene that prevents full length translation. Apo B-32 is predicted to contain the 1449 amino-terminal amino acids of apo B-100 and is associated with a markedly decreased low density lipoprotein (LDL) cholesterol level. The density distribution of apo B-32 in the plasma lipoproteins makes it unique amongst other truncated apo B species. Normally, apo B-100 is found in both very low density lipoprotein (VLDL) and LDL particles. However, the majority of the apo B-32 protein was found in the high density lipoprotein (HDL) and lipoprotein-deplete (d greater than 1.21 g/ml) fractions, suggesting that it was mainly assembled into abnormally dense lipoprotein particles. A small amount of apo B-32 was also found in the LDL, making it the shortest known apo B variant capable of forming particles in this density range. Apo B-32 was undetected in VLDL. The apo B-32 mutation further defines the minimum length of the apo B protein that is required for the assembly of LDL.     

CD45: a leukocyte-specific member of the protein tyrosine phosphatase family.

In a recent study from this laboratory, rhesus monkeys fed a 90% palm oil/10% soybean oil-containing diet (PS), rich in 16:0 and 18:1 fatty acids, had decreased total and LDL cholesterol concentrations compared to monkeys fed a 90% coconut oil/10% soybean oil-containing diet (CS), rich in 12:0 and 14:0 fatty acids. To investigate the metabolic basis of these changes, homologous 125I-VLDL and 131I-LDL were injected simultaneously into eight monkeys (four per dietary group). Analysis of apo B specific activity curves revealed that PS monkeys had an increased pool size of VLDL apo B (P less than 0.02), a 3-fold increase in the total VLDL apo B transport rate (P less than 0.001), a decreased pool size of LDL apo B (P less than 0.01) and a 2-fold decrease in the total transport rate of LDL apo B (P less than 0.001), while the irreversible FCR for VLDL apo B and LDL apo B was similar between dietary groups. PS monkeys derived a greater percentage of LDL apo B from VLDL catabolism resulting in a greater transport rate of LDL apo B from VLDL catabolism (P less than 0.055), in comparison to CS monkeys. For CS monkeys the proportion as well as the amount of LDL apo B derived from VLDL-independent catabolism (i.e., LDL apo B derived from sources other than VLDL catabolism) was higher (P less than 0.001) than the values obtained in PS monkeys. In both dietary groups the proportion of VLDL apo B converted to LDL apo B was similar, although the absolute amount was higher for the PS monkeys (P less than 0.06). The proportion of VLDL apo B directly removed from the circulation was similar for both dietary groups, with the absolute amount being higher for the PS monkeys (P less than 0.001). Consistent with the lower pool size of LDL apo B and the higher pool size of VLDL apo B observed in PS monkeys, plasma and LDL cholesterol concentrations tended to be lower, whereas plasma triacylglycerol and VLDL cholesterol concentrations tended to be higher, but these changes were not statistically significant. Although total apo B and VLDL apo B transport rates were increased 2-3-fold in PS monkeys, LDL apo B concentration was reduced by 40% (P less than 0.02) attributed to a significant reduction in the mass and proportion of LDL apo B derived independent of VLDL catabolism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sources of interindividual variation in the quantitative levels of apolipoprotein B in pedigrees ascertained through a lipid clinic.

The quantitative level of apolipoprotein (apo) B associated with low-density lipoprotein (LDL) varies among individuals within the population. This variation in level of the LDL receptor ligand appears to have predictive value, and may have an etiologic role, in coronary artery disease. Complex segregation analysis was used to compare eight different models of transmission. This study confirms the existence of allelic variations at a single genetic locus with large effects on the interindividual variation in the level of the serum apo B associated with LDL. This is the first study to consider the possible effects of inherited polymorphic variation in the apo E molecule when analyzing the components of variation in apo B associated with LDL. Our analyses suggest that the common alleles coding for the apo E polymorphism act independently of the unmeasured single-gene locus characterized by this study.     

Effects of apolipoprotein B-100 on the metabolism of a lipid microemulsion model in rats

In previous studies, it was shown that lipid microemulsions resembling LDL (LDE) but not containing protein, acquire apolipoprotein E when injected into the bloodstream and bind to LDL receptors (LDLR) using this protein as ligand. Aiming to evaluate the effects of apolipoprotein (apo) B-100 on the catabolism of these microemulsions, LDE with incorporated apo B-100 (LDE-apoB) and native LDL, all labeled with radioactive lipids were studied after intraarterial injection into Wistar rats. Plasma decay curves of the labels were determined in samples collected over 10 h and tissue uptake was assayed from organs excised from the animals sacrificed 24 h after injection. LDE-apo B had a fractional clearance rate (FCR) similar to native LDL (0.40 and 0.33, respectively) but both had FCR pronouncedly smaller than LDE (0.56, P<0.01). Liver was the main uptake site for LDE, LDE-apoB, and native LDL, but LDE-apoB and native LDL had lower hepatic uptake rates than LDE. Pre-treatment of the rats with 17α-ethinylestradiol, known to upregulate LDLR, accelerated the removal from plasma of both LDE and LDE-apoB, but the effect was greater upon LDE than LDE-apoB. These differences in metabolic behavior documented in vivo can be interpreted by the lower affinity of LDLR for apo B-100 than for apo E, demonstrated in in vitro studies. Therefore, our study shows in vivo that, in comparison with apo E, apo B is a less efficient ligand to remove lipid particles such as microemulsions or lipoproteins from the intravascular compartment.     

Lipoprotein secretion by the human hepatoma cell line Hep G2: differential rates of accumulation of apolipoprotein B and lipoprotein lipids in tissue culture media in response to albumin, glucose and oleate

Serum low-density lipoprotein (LDL) concentration is a major determinant of susceptibility to the development of atherosclerosis. A major component of the protein moiety of LDL and its precursor very-low-density lipoprotein is apolipoprotein B (apo B). The human hepatoma cell line, Hep G2, was used as a model for the investigation of mechanisms which control hepatic secretion of the apo B and lipid components of lipoproteins. Using a sensitive immunoradiometric assay for apo B developed in this laboratory, we showed that bovine serum albumin inhibited and glucose, and fatty acids enhanced the rate of accumulation of apo B in the culture medium of Hep G2 cells. However, these substances did not necessarily affect LDL lipids in the same way as apo B. This finding appeared to be due to Hep G2 cells expressing lipase activities which led to triacylglycerol and phospholipid hydrolysis and lipid reuptake. Reuptake of apo B also occurred, but its rate of accumulation in the culture medium suggested it was a closer reflection of its true secretory rate.     

Genetic evidence that the putative receptor binding domain of apolipoprotein B (residues 3130 to 3630) is not the only region of the protein involved in interaction with the low density lipoprotein receptor

We have searched for sequence differences in the region of the apolipoprotein B (apo B) gene encoding amino acids 3130-3630 in eight individuals with reduced affinity of low density lipoprotein (LDL) for the normal LDL-receptor. All individuals were hypercholesterolaemic and were selected either on the basis of reduced fractional catabolic rate (FCR) of autologous LDL or substantially reduced binding of their LDL to normal LDL-receptors determined by an in vitro cell growth assay using the U937 macrophage-like cell line. Segments of the apo B gene were amplified by the polymerase chain reaction. Using a combination of cloning and sequencing the amplified fragment, together with chemical cleavage mismatch analysis, no sequence differences were identified in this region of the gene. We therefore conclude that variation outside the region of the apo B gene that codes for amino acids 3130-3630 must be responsible for the reduced LDL clearance in these patients.     

A quantitative assay for the non-covalent association between apolipoprotein[a] and apolipoprotein B: an alternative measure of Lp[a] assembly

Increasing evidence suggests that the assembly of lipoprotein[a] (Lp[a]) proceeds in two steps. In the first step, non-covalent interactions between apolipoprotein[a] (apo[a]) and apolipoprotein B (apoB) of low density lipoprotein (LDL) form a dissociable apo[a]:LDL complex. In the second step, a covalent disulfide linkage forms the stable Lp[a] particle. Several methods are currently used to study the assembly of Lp[a], however, these methods are laborious, time-consuming, and not suitable for a high throughput screening. We report here the development of a rapid and simple assay based on the binding of labeled LDL to a Lp[a]/apo[a] substrate which is immobilized on the surface of a microtiter plate. Quantification of bound LDL provides a measure of the extent of complex formation. Labeled LDL bound to both Lp[a] and apo[a] substrates with similar affinity. Plasma lipoproteins containing apoB as well as free apo[a] were capable of competing with LDL binding. The binding of LDL to Lp[a]/apo[a] was inhibited by L-proline and lysine analogs, which are known to inhibit the non-covalent association between apo[a] and apoB. Using this method we have found that nicotinic acid and captopril are able to inhibit the association of apo[a] with apoB. This method is compatible with automation and can be applied to a high throughput screening of inhibitors of Lp[a] formation.     

In vitro oxidative footprinting provides insight into apolipoprotein B‐100 structure in low‐density lipoprotein

Low‐density lipoprotein (LDL) is a major cholesterol carrier in human blood. Oxidations of apolipoprotein B‐100 (apo B‐100, LDL protein) could be proatherogenic and play critical roles in early stages of plaque formation in the arterial wall. The structure of apo B‐100 is still poorly understood, partially due to its size (550 KDa, 4563 amino acids). To gain an insight into LDL structure, we mapped the regions of apo B‐100 in human LDL that were prone to oxidation using peroxynitrite and hypochlorite as probes. In this study, LDL was incubated with various concentrations of peroxynitrite and sodium hypochlorite in bicarbonate buffer. The LDL protein apo B‐100 was delipidated, denatured, alkylated, and subjected to tryptic digestion. Tryptic peptides were analyzed employing LC‐MS/MS. Database search was performed against the apo B‐100 database (SwissProt accession #P04114) using “SEQUEST” algorithm to identify peroxynitrite and hypochlorite‐mediated oxidations markers nitrotyrosine, nitrotryptophan, hydroxy‐tryptophan, and 3‐chlorotyrosine. Several site‐specific oxidations were identified in apo B‐100 after treatment of intact LDL particles with the oxidants. We hypothesize that these regions could be accessible to oxidant and critical for early events in atherosclerotic plaque deposition.     

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.     

Investigation of Lipid Peroxidation in Human Low Density Lipoprotein

Human plasma low density lipoprotein (LDL) exposed to oxygen saturated buffer becomes depleted of alpha-tocopherol within 3 to 6 hours. Thereafter, lipid peroxidation commences as evidenced by the loss of 18:2 (67nmol/mg LDL) and 20:4 (12nmol/mg LDL) and the concomitant formation of 4-hydroxy-nonenal (0.28 nmol/mg LDL) and fluorescent compounds. The major fluorophor in apo B of oxidized LDL has an excitation maximum at 355 nm and an emission maximum at 430 nm. A fluorophor with the same spectral properties is produced in apo B, if LDL is incubated with 4-hydroxynonenal, whereas malonal-dehyde gives a fluorophor with excitation and emission maxima at 400/470nm. Three-dimensional fluorescence spcetroscopy proved to be an useful tool in analysing the complex fluorescence of apo B.     

一次密度梯度超速离心分离人血清四种主要脂蛋白的某些理化性质的研究

 本文对一次密度梯度超速离心获得的四种脂蛋白(VLDL、LDL、HDL_2、HDL_3)进行了理化性质的研究。超速离心分析LDL、HDL_2,HDL_3均呈现一个单一尖锐的上浮峰,上浮速率分别为S_1 6.9和F~0_(1.21) 5.7及2.6。等电点聚焦电泳显示,VLDL主要含载脂蛋白C族,E族和少量A-I,B。HDL_2、HDL_3二者载脂蛋白的种类很相似,但量上略有差异,均以载脂蛋白A-Ⅰ,A-Ⅱ为主,Apoc’s,E次之。VLDL、LDL、HDL_2和HDL_3的化学组分分析与文献报道相似。 作者用本法初步分析了不同性别的各脂蛋白分布图,获得有意义的结果。     

Low-density-lipoprotein receptors in different rabbit liver cells.

Receptor-dependent uptake mechanisms for low-density lipoprotein (LDL) were studied in rabbit liver parenchymal and non-parenchymal cells. Hybridization studies with a cDNA probe revealed that mRNA for the apo (apolipoprotein) B,E receptor was present in endothelial and Kupffer cells as well as in parenchymal cells. By ligand-blotting experiments we showed that apo B,E-receptor protein was present in both parenchymal and non-parenchymal cells. Studies of binding of homologous LDL in cultured rabbit parenchymal cells suggested that about 63% of the specific LDL binding was mediated via the apo B,E receptor. Approx. 47% of the specific LDL binding was dependent on Ca2+, suggesting that specific Ca2+-dependent as well as Ca2+-independent LDL-binding sites exist in liver parenchymal cells. Methylated LDL bound to the parenchymal cells in a saturable manner. Taken together, our results showed that apo B,E receptors are present in rabbit liver endothelial and Kupffer cells as well as in the parenchymal cells, and that an additional saturable binding activity for LDL may exist on rabbit liver parenchymal cells. This binding activity was not inhibited by EGTA or reductive methylation of lysine residues in apo B. LDL degradation in parenchymal cells was mainly mediated via the apo B,E receptor.     

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.     

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.     

Isolation and partial characterization of apolipoprotein (a) from human lipoprotein (a)

A procedure was developed for the dissociation of apolipoprotein (a) (apo (a)) from pure human lipoprotein (a) (Lp(a)) prepared by density gradient ultracentrifugation and gel filtration. Lp(a) was ultracentrifuged through a layer of saline which was adjusted to a density of 1.182 g/mL and contained 30 mM dithiothreitol (50 mM) and phenylmethylsulfonyl fluoride (1.25 mM). Following centrifugation, the lipid and apolipoprotein B (apo B) were recovered as a lipoprotein (Lp(a) B) in the supernatant fraction, while the apo (a) was recovered as a lipid-poor protein pellet. An investigation of the supernatant lipoprotein by electron microscopy and compositional analysis revealed that it was similar in size and composition to low density lipoprotein (LDL) isolated from the same density range and contained apo B100 with an amino acid and carbohydrate composition which was similar to apo B from LDL. Estimates of the apparent molecular weight of the apo (a) varied amongst individuals but was always greater than apo B100 (congruent to 450,000). The amino acid composition of apo (a), which was very distinct from apo B, was characterized by a higher content of serine, threonine, proline, and tyrosine, but lower amounts of isoleucine, phenylalanine, and lysine when compared with apo B of Lp(a) or LDL. The apo (a) contained a much higher proportion of carbohydrate, in particular N-acetylgalactosamine, galactose, and N-acetylneuraminic acid (which were three- to six-fold higher) than the apo B of Lp(a). It is concluded that apo (a) is distinct from other apolipoproteins owing to its low avidity for lipid and the nature of the interaction with apo B. Lp(a) consists of an LDL-like particle with a carbohydrate-rich apo (a) attached to the surface of apo B.     

Structure of the lipopolysaccharide/human plasma low density lipoprotein complex. 31P-NMR and fluorescence spectroscopy studies

Complexes of Salmonella typhimurium lipopolysaccharide toxin (LPS) with low density lipoproteins (LDL) containing various amounts of LPS were prepared in vitro. The 31P-NMR spectra showed that in the LDL-LPS complexes as well as in native LDL all phosphate groups of phospholipids are accessible to the paramagnetic shift reagent, Pr3+. Besides, the low frequency mobility of phospholipid phosphates in the complex is diminished. It was supposed that the phospholipid molecules in the LDL/LPS complex as in native LDL form a monolayer structure on the surface of LDL. The intrinsic fluorescence spectra of tryptophan residues of the apoprotein (apo B-100) revealed that the incorporation of LPS molecules into LDL particles is accompanied by minor changes in the conformation and orientation of the apo B molecule. As a result of these changes, certain fragments become exposed to a more hydrophilic environment and become more accessible to fluorescence quenchers. The use of charged (I-, Cs+) and uncharged (acrylamide) quenchers permitted to identify in the apo B molecule different tryptophan residues, some of which are localized in the vicinity of negatively charged groups, whereas others are neighbouring positively charged groups. It is suggested that the LPS molecules incorporated into LDL particle do not screen the apo B molecule to such an extent that it would hinder the LDL/LPS complex binding to apo B/E cellular receptors.     

Isolation and characterization of three monoclonal antibodies to human serum low density lipoprotein apoprotein B

Human serum low density lipoprotein (LDL) is a large (Mr = 2-3 X 10(6), complex particle composed of lipid, protein and carbohydrate. We obtained about 40 mouse spleen-myeloma hybrid cell lines which produce antibodies against LDL. Three of them, SC2, SC3 and SC10, have been cloned and subcloned and their antibody products characterized. They recognize three non-overlapping epitopes in native LDL. Two of them, SC3 and SC10, also are capable of recognizing very low density lipoprotein, (VLDL), whereas SC2 reacts only weakly with VLDL. All three antigenic determinants remain intact, and accessible to antibodies on the LDL protein apo B, prepared by delipidation in a 'non-denaturing' detergent, sodium deoxycholate. However, apo B prepared by organic solvent, ether-ethanol, or sodium dodecyl sulfate (SDS) delipidation, while reacting strongly with SC10, is only poorly recognized by SC2 or SC3. Proteolysis of LDL with trypsin, chymotrypsin, Staphylococcus aureus protease, papain or thermolysin gives, in each case, several non-identical protein fragments which are separable by SDS-polyacrylamide gel electrophoresis. Upon immunoblotting, some of these fragments are now recognized by either SC3 or SC10 but not SC2, some are recognized by both SC3 and SC10, and others are immunologically unreactive. The protein bands that are separated by SDS gel electrophoresis are composed of several non-identical fragments and contain the antigenic sites to differing degrees. Some of the immunologically reactive fragments do not appear to contain carbohydrate. Reduction and carboxymethylation do not destroy the immunoreactivity of LDL toward any of the antibodies; however, modification of lysine residues by citraconic anhydride markedly diminishes the reactivity of LDL toward SC3. It is likely that the two antibodies SC3 and SC10 are directed against different linear amino acid sequences or very stable domains, whereas the third, SC2, is directed against a more fragile conformational domain of apo B.     

A 13C NMR characterization of lysine residues in apolipoprotein B and their role in binding to the low density lipoprotein receptor

NMR spectroscopy of 13C-labeled human low density lipoprotein (LDL) has been employed to characterize the lysine (Lys) residues in apo B-100. Reductive methylation with [13C]formaldehyde converts up to two-thirds of the Lys to the dimethylamino derivative; this pool of Lys is exposed at the surface of the LDL particle. The [13C]dimethyl-Lys which are visualized exhibit resonances at chemical shifts of 42.8 and 43.2 ppm (pH 7.6) indicating that they exist in two different microenvironments; this is a reflection of the native conformation of apo B associated with lipid, because the labeled, reduced, and alkylated protein gives a single resonance when dissolved in 7 M guanidine hydrochloride. The pH dependences of the Lys chemical shifts indicate that the two types of Lys titrate with different pK values; "active" Lys have a pK of 8.9, while "normal" Lys have a pK of 10.5. About 53 active Lys and 172 normal Lys are exposed on the surface of LDL with the remaining 132 Lys which are present in the human apo B-100 molecule being buried and unavailable for methylation. Addition of paramagnetic ions indicates that the active and normal Lys have different exposures to the aqueous phase; apparently this is a reflection of folding of the apo B molecule. The relative involvement of active and normal Lys in binding of apo B-100 to the LDL receptor on fibroblasts was explored by measuring the decrease in receptor binding as a function of the degree of methylation of the two types of Lys. Upper limits of 21 active and 31 normal Lys in the entire apo B-100 molecule are involved in the binding of LDL to the receptor. It is likely that these Lys are located in domains of apo B which contain clusters of basic amino acid residues and also bind heparin. If the sequence corresponding to apo B-48 (residues 1-2151) which does not bind to the receptor is excluded, then the above limits are halved; an upper limit of 10 active Lys may be particularly involved in receptor binding.     

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.