Human apolipoprotein A-I isoprotein metabolism: proapoA-I conversion to mature apoA-I

ProapoA-I (apoA-i+2 isoform) is the major apoA-I isoprotein secreted by the liver and intestine; however, it is a minor isoprotein in plasma and lymph where the major A-I apo-lipoprotein is mature apoA-I (apoA-I0, apoA-I-1, and apoA-I-2 isoforms). In the present report we provide evidence that apoA-I is rapidly and quantitatively converted to mature apoA-I, and the mature apoA-I isoforms are catabolized at equal rates. In these studies, human proapoA-I was isolated from thoracic duct chylomicrons collected during active fat absorption and mature apoA-I was isolated from plasma high density lipoproteins. The isolated lipoproteins were delipidated, fractionated by gel permeation chromatography, and the individual apoA-I isoforms were separated by preparative isoelectrofocusing. The metabolism of apoA-I isoproteins was studied in normal volunteers (N = 6) in a metabolic ward. In the first study proapoA-I and mature apoA-I (apoA-I0 isoform) were injected simultaneously into two normal subjects and the conversion of proapoA-I to mature apoA-I and the decay of radioactivity were followed in plasma and HDL over a 14-day period. ProapoA-I was rapidly and completely converted to mature apoA-I with a fractional rate of conversion of 4.0 pools/day. The average residence times of proapoA-I and mature apoA-I were 0.23 and 6.5 days, respectively. The mature apoA-I derived from proapoA-I had a residence time which was the same as the injected mature apoA-I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzyme-linked immunosorbent assay for human proapolipoprotein A-I using specific antibodies against synthetic peptide

Apolipoprotein A-I (apoA-I), the major protein component of human high density lipoprotein, appears intracellularly as an intermediate precursor (proapoA-I) with a hexapeptide extension (Arg-His-Phe-Trp-Gln-Gln) at its amino terminus. To investigate the regulation of processes that regulate plasma apoA-I levels, a sensitive and simple assay for proapoA-I is required. We describe a specific enzyme-linked immunosorbent assay (ELISA) for quantification of proapoA-I using monospecific rabbit antibodies raised against the peptide: Arg-His-Phe-Trp-Gln-Gln-Asp-Glu-Pro. The monospecificity of antibodies to propeptide has been checked and no cross-reaction with mature apoA-I has been found although three first mature apoA-I amino acids (Asp-Glu-Pro) were included in the immunizing peptide. The assay is a non-competitive sandwich ELISA in which polystyrene microtiter plates were used with antibodies to propeptide adsorbed on the wells. After incubation with plasma samples, the bound proapoA-I was revealed by labeled rabbit polyclonal antibodies directed against mature apoA-I. The working range was 10 to 100 ng/ml, recovery of proapoA-I added to plasma was 94.6 to 106.5%, and the intra- and interassay coefficients of variation were 3.8% and 7.9%, respectively. A delipidation step using diisopropylether-n-butanol was necessary to expose antigen sites of proapoA-I in native lipoproteins. Mean level of proapoA-I in normal subjects was 87 +/- 15 micrograms/ml. It represented 7.1% of total apoA-I while in Tangier serum it represented 29%.     

Deletion of the propeptide of apolipoprotein A-I reduces protein expression but stimulates effective conversion of prebeta-high density lipoprotein to alpha-high density lipoprotein

The properties of the mature and pro-forms of recombinant apolipoprotein A-I (apoA-I) were compared with those of apoA-I isolated from human plasma. When the synthesis and secretion of pro- and mature forms of apoA-I from a baculovirus/insect cell expression system were compared in parallel experiments, the amount of the pro-form of apoA-I synthesized and secreted was severalfold higher than that of the mature form of apoA-I. A comparison of the properties of the pro- and mature forms of recombinant apoA-I and human plasma apoA-I showed no difference between all three in their secondary structure, their ability to self-associate, lipid-binding capacity, lecithin: cholesterol acyltransferase activation, and binding to the phospholipid transfer protein. The properties of reconstituted high density lipoprotein (HDL) particles formed from the proteins and their ability to promote cholesterol and phospholipid efflux from human skin fibroblasts were also similar. However, their ability to bind to plasma HDL subfractions differed, because twice as much proapoA-I associated with prebeta(1)-HDL and prebeta(2)-HDL subfractions compared with both mature recombinant and plasma apoA-I. Correspondingly, the amount of proapoA-I in alpha-HDL subfractions, especially in alpha(1)-HDL and alpha(2)-HDL, was decreased. We conclude that while the propeptide of apoA-I is required for the effective synthesis and secretion of apoA-I, cleavage of this peptide is a requisite for the effective interconversion of HDL subfractions.     

Demonstration that the enzyme that converts precursor of apolipoprotein A-I to apolipoprotein A-I is secreted by the hepatocarcinoma cell line Hep G2

The conversion of the precursor of apolipoprotein A-I (proapoA-I) to apolipoprotein A-I (apoA-I) is known to occur extracellularly by an enzyme that has been shown to be present in plasma. The hepatocarcinoma-derived cell line Hep G2, when grown in culture, secretes proapoA-I. We now show that this cell line also secretes the converting enzyme that correctly processes proapoA-I to mature apoA-I as determined by radio-sequence analyses. The secreted enzyme is inhibited by EDTA and 1,10-phenanthroline, is activated by Ca2+ and is unaffected by both phenylmethylsulfonyl fluoride and diisoprophylfluorophosphate in the same way as the converting enzyme previously described in the plasma. The conversion of proapoA-I to apoA-I effected by this enzyme obeys first-order kinetics and is linear over the first 4 h with a calculated initial velocity of 3.3% conversion per hour. The converting activity is secreted in a time-dependent fashion and parallels the mass of total secreted protein.     

Identification of proapoa-I in rat lymph and plasma: metabolic conversion to "mature" apoA-I

Rat apoA-I polymorphism has been analyzed in lymph and plasma. Two major proteins were present and their relative distribution was different in lymph and plasma lipoproteins. The basic protein (pI 5.60) was quantitatively most abundant among plasma lipoproteins and the acidic protein (pI 5.50) was predominant in lymph chylomicrons and lipoproteins. Microsequence amino acid analysis of the two proteins isolated by preparative isoelectrofocusing revealed that pI 5.50 apoA-I was proapoA-I with six additional amino acids (H2N-Ser-Glu-Phe-Trp-Gln-Gln) at the N-terminal end of "mature" apoA-I (pI 5.60 apoA-I). When radioiodinated proapoA-I was injected in rats, a conversion to "mature" apoA-I was observed and the process reached 92% completion in six hours. These data demonstrate the origin of apoA-I polymorphism in vivo.     

Monoclonal antibodies specific for different regions of human apolipoprotein A-I. Characterization of an antibody that does not bind to a genetic variant of apoA-I (Glu----136 Lys)

Three monoclonal mouse hybridoma antibodies, designated 2AI, 4AI, and 5AI, specific for human plasma apolipoprotein A-I (apoA-I) were characterized. In an enzyme-linked immunosorbent assay (ELISA) each of the antibodies reacted with purified apoA-I and with A-I in normal human serum. Immunoblotting of apoA-I subjected to isoelectric focusing revealed that the three antibodies reacted with all the charge isomorphs of apoA-I and with proapoA-I. Using a solid phase competitive displacement assay, the antigenic determinant for antibody 5AI could be localized to cyanogen bromide fragment 3 of apoA-I (residues 113-148), while the epitope for antibody 4AI resided in cyanogen bromide fragment 4. Dot blot experiments and data obtained by the competitive displacement assay revealed that antibody 2AI reacts with high affinity with CNBr fragment 2 but that it also reacts with lower affinity with fragments 1 and 4. The antibody 5AI did not bind to a genetic variant of apoA-I (Glu----136 Lys), demonstrating that the substitution of a single amino acid in human apoA-I can cause the loss of an antigenic determinant.     

Localization of two epitopes of apolipoprotein A-I that are exposed on human high density lipoproteins using monoclonal antibodies and synthetic peptides

To understand the structure of apolipoprotein A-I, we have used an immunochemical approach and identified specific regions of apoA-I that may be exposed on the apoprotein as it exists on high density lipoprotein (HDL). Twelve mouse monoclonal antibodies specific for human apoA-I were generated from six fusions. Thirteen synthetic peptides of between 5 and 16 amino acid residues in length, which span the amino-terminal two-thirds of apoA-I, were tested for their ability to react with each of the 12 antibodies. In a competitive solid-phase radioimmunoassay, a synthetic peptide, which represented residues 1-15 of mature apoA-I, inhibited the binding of antibody AI-16 to immobilized HDL. Similarly, a synthetic peptide, which represented residues 90-105 of apoA-I, inhibited the binding of antibody AI-18 to immobilized HDL. Using systematic changes in the size and sequence of the oligopeptides, the limits and essential amino acid residues of these epitopes were defined. Comparisons of the slopes of the competition curves obtained with immunoreactive peptides, isolated apoA-I, and HDL verified that these two regions of apoA-I are exposed on the surface of apoA-I as it exists on native HDL.     

The complete amino acid sequence of proapolipoprotein A-I of chicken high density lipoproteins

The complete amino acid sequence of proapolipoprotein (proapo) A-I of chicken high density lipoproteins was determined by sequencing overlapping peptides produced by trypsin, S. aureus V8 protease, and cyanogen bromide cleavage. There are 240 amino acid residues in mature chicken apoA-I. By direct sequence analysis of a cyanogen bromide peptide, we also determined the sequence of a 6-amino-acid prosegment which is present at approx. 10% the molar amount of the mature peptide in chicken plasma. Sequence comparison among apoA-I from chicken, human, rabbit, dog and rat, and secondary structure analysis indicate that while the degree of sequence homology is only moderate (less than 50% between chicken and man), there is good conservation of apoA-I secondary structure, especially in the N-terminal two-thirds of the protein in these widely separated species.     

Expression and characterization of human apolipoprotein A-I in Chinese hamster ovary cells

We produced human apolipoprotein A-I (apoA-I) in Chinese hamster ovary (CHO) cells. The CHO cells were transfected with an expression plasmid which placed the human apoA-I gene under the direction of the human metallothionein II gene promoter. Isolation of a clonal cell line resulted in high level expression of apoA-I. Greater than 30% of total protein secreted by these CHO cells was apoA-I, which enabled us to purify apoA-I with a single step purification scheme. As a result, large quantities of apoA-I can be produced and isolated without having to rely on plasma sources. Structural characterization of the recombinant apoA-I showed it to be identical to authentic apoA-I from human serum high density lipoprotein. Furthermore, we demonstrated approximately equal to 90% of the apoA-I secreted by CHO cells is processed, mature protein. A portion of the secreted recombinant apoA-I was associated with lipid and floated at a density approximately equal to 1.10 g/ml. Additional analysis identified the presence of five isoforms of apoA-I in the CHO cell conditioned medium. Processing and post-translational modification of the recombinant apoA-I occurred in the CHO cell cultures in the absence of serum components. We conclude that the human apoA-I produced by CHO cells is identical to circulating, mature apoA-I in humans and that recombinant mammalian expression offers an opportunity to investigate apoA-I processing.     

Apolipoprotein A-I directly interacts with amyloid precursor protein and inhibits A beta aggregation and toxicity

Amyloid precursor protein (APP) is the source of the neurotoxic amyloid beta (Abeta) peptide associated with Alzheimer's disease. Apolipoprotein A-I (apoA-I), a constituent of high-density lipoprotein complexes, was identified by a yeast two-hybrid system as a strong and specific binding partner of full-length APP (APPfl). This association between apoA-I and APPfl was localized to the extracellular domain of APP (APPextra). Furthermore, the interaction between apoA-I and APPfl was confirmed by coprecipitation using recombinant epitope-tagged APPextra and purified apoA-I. Several functional domains have been identified in APPextra, and we focused on a possible interaction between apoA-1 and the pathologically important Abeta peptide, because APPextra contains the nontransmembrane domain of Abeta. The binding between apoA-I and Abeta was saturable (K(d) = 6 nM), specific, and reversible. APPextra also competed with apoA-I for binding to Abeta. Direct evidence for this interaction was obtained by the formation of an SDS-resistant Abeta-apoA-I complex in polyacrylamide gels. Competitive experiments with apolipoprotein E (isoforms E2 and E4) showed that apoA-I had a higher binding affinity for Abeta. We also found that apoA-I inhibited the beta-sheet formation of Abeta with a mean inhibitory concentration close to that of alpha2-macroglobulin. Finally, we demonstrated that apoA-I attenuated Abeta-induced cytotoxicity. These results suggest apoA-I binds to at least one extracellular domain of APP and has a functional role in controlling Abeta aggregation and toxicity.     

A sensitive and specific ELISA detects methionine sulfoxide-containing apolipoprotein A-I in HDL

Oxidized HDL has been proposed to play a key role in atherogenesis. A wide range of reactive intermediates oxidizes methionine residues to methionine sulfoxide (MetO) in apolipoprotein A-I (apoA-I), the major HDL protein. These reactive species include those produced by myeloperoxidase, an enzyme implicated in atherogenesis. The aim of the present study was to develop a sensitive and specific ELISA for detecting MetO residues in HDL. We therefore immunized mice with HPLC-purified human apoA-I containing MetO(86) and MetO(112) (termed apoA-I(+32)) to generate a monoclonal antibody termed MOA-I. An ELISA using MOA-I detected lipid-free apoA-I(+32), apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in buffer or human plasma. Detection was concentration dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radical or metal ions, or LDL and methionine-containing proteins other than apoA-I modified by 2e-oxidants. Because the ELISA we have developed specifically detects apoA-I containing MetO in HDL and plasma, it should provide a useful tool for investigating the relationship between oxidized HDL and coronary artery disease.     

Optimized bacterial expression of human apolipoprotein A-I

Apolipoprotein A-I (apoA-I) serves critical functions in plasma lipoprotein metabolism as a structural component of high density lipoprotein, activator of lecithin:cholesterol acyltransferase, and acceptor of cellular cholesterol as part of the reverse cholesterol transport pathway. In an effort to facilitate structure:function studies of human apoA-I, we have optimized a plasmid vector for production of recombinant wild type (WT) and mutant apoA-I in bacteria. To facilitate mutagenesis studies, subcloning, and DNA manipulation, numerous silent mutations have been introduced into the apoA-I cDNA, generating 13 unique restriction endonuclease sites. The coding sequence for human apoA-I has been modified by the introduction of additional silent mutations that eliminate 18 separate codons that employ tRNAs that are of low or moderate abundance in Escherichia coli. Yields of recombinant apoA-I achieved using the optimized cDNA were 100+/-20 mg/L bacterial culture, more than fivefold greater than yields routinely obtained with the original cDNA. Site-directed mutagenesis of the apoA-I cDNA was performed to generate a Glu2Asp mutation in the N-terminal sequence of apoA-I. This modification, which creates an acid labile Asp-Pro peptide bond between amino acids 2 and 3, permits specific chemical cleavage of an N-terminal His-Tag fusion peptide used for rapid protein purification. The product protein's primary structure is identical to WT apoA-I in all other respects. Together, these changes in apoA-I cDNA and bacterial expression protocol significantly improve the yield of apoA-I protein without compromising the relative ease of purification.     

Chicken apolipoprotein A-I: cDNA sequence,tissue expression and evolution

Using an antibody against chicken apolipoprotein (apo) A-I, we identified multiple cDNA clones for the protein in two intestinal cDNA libraries in λgtll. The complete nucleotide sequence of chicken apoA-I cDNA was determined. The sequence predicts a mature protein of 240 amino acids, a 6-amino acid propeptide and an 18-amino acid signal peptide. Using a ³²P-cDNA probe, we detected the presence of apoA-I mRNA in 21 day old chicken intestine, liver, kidney, spleen, breast muscle and brain. The primary sequence of apoA-I contains numerous tandem repeats of 11 and 22 residues in a manner similar to the mammalian proteins. Our analysis of apoA-I sequences from human, rabbit, dog, rat, and chicken indicates that the rate of amino acid substitution is considerably faster in the rat lineage than in other mammalian lineages.     

Conformation and lipid binding of a C-terminal (198-243) peptide of human apolipoprotein A-I

Human apolipoprotein A-I (apoA-I) is the principle apolipoprotein of high-density lipoproteins that are critically involved in reverse cholesterol transport. The intrinsically flexibility of apoA-I has hindered studies of the structural and functional details of the protein. Our strategy is to study peptide models representing different regions of apoA-I. Our previous report on [1-44]apoA-I demonstrated that this N-terminal region is unstructured and folds into approximately 60% alpha-helix with a moderate lipid binding affinity. We now present details of the conformation and lipid interaction of a C-terminal 46-residue peptide, [198-243]apoA-I, encompassing putative helix repeats 10 and 9 and the second half of repeat 8 from the C-terminus of apoA-I. Far-ultraviolet circular dichroism spectra show that [198-243]apoA-I is also unfolded in aqueous solution. However, self-association induces approximately 50% alpha-helix in the peptide. The self-associated peptide exists mainly as a tetramer, as determined by native electrophoresis, cross-linking with glutaraldehyde, and unfolding data from circular dichroism (CD) and differential scanning calorimetry (DSC). In the presence of a number of lipid-mimicking detergents, above their CMC, approximately 60% alpha-helix was induced in the peptide. In contrast, SDS, an anionic lipid-mimicking detergent, induced helical folding in the peptide at a concentration of approximately 0.003% (approximately 100 microM), approximately 70-fold below its typical CMC (0.17-0.23% or 6-8 mM). Both monomeric and tetrameric peptide can solubilize dimyristoylphosphatidylcholine (DMPC) liposomes and fold into approximately 60% alpha-helix. Fractionation by density gradient ultracentrifugation and visualization by negative staining electromicroscopy demonstrated that the peptide binds to DMPC with a high affinity to form at least two sizes of relatively homogeneous discoidal HDL-like particles depending on the initial lipid:peptide ratio. The characteristics (lipid:peptide weight ratio, diameter, and density) of both complexes are similar to those of plasma A-I/DMPC complexes formed under similar conditions: small discoidal complexes (approximately 3:1 weight ratio, approximately 110 A, and approximately 1.10 g/cm3) formed at an initial 1:1 weight ratio and larger discoidal complexes (approximately 4.6:1 weight ratio, approximately 165 A, and approximately 1.085 g/cm3) formed at initial 4:1 weight ratio. The cross-linking data for the peptide on the complexes of two sizes is consistent with the calculated peptide numbers per particle. Compared to the approximately 100 A disk-like complex formed by the N-terminal peptide in which helical structure was insufficient to cover the disk edge by a single belt, the compositions of these two types of complexes formed by the C-terminal peptide are more consistent with a "double belt" model, similar to that proposed for full-length apoA-I. Thus, our data provide direct evidence that this C-terminal region of apoA-I is responsible for the self-association of apoA-I, and this C-terminal peptide model can mimic the interaction with the phospholipid of plasma apoA-I to form two sizes of homogeneous discoidal complexes and thus may be responsible for apoA-I function in the formation and maintenance of HDL subspecies in plasma.     

Apolipoprotein A-I isoforms in human lymph: effect of fat absorption

The effect of fat feeding (100 g of cream) on the apoA-I isoproteins distribution has been analyzed by two-dimensional gel electrophoresis in the chylomicrons, VLDL, LDL, and HDL isolated from the thoracic duct lymph of patients undergoing lymph drainage for immunosuppression, Isoforms apoA-I3 and apoA-I4 are the most abundant apoA-I isoproteins in plasma lipoproteins as well as in lymph lipoproteins collected in the fasting state. Fat feeding, on the other hand, results in a marked change in the apoA-I isoform pattern in lymph chylomicrons and VLDL, with a significant increase in the relative concentration of the apoA-I1 isoform. As a result the total concentration of this isoprotein in the lymph increased. The data indicate that fat feeding is associated with major changes in the distribution of the apoA-I isoforms in the lymph (d less than 1.006 g/ml lipoproteins), which may be of significance in their plasma catabolism.     

Apolipoprotein A-I: structure-function relationships

The inverse relationship between high density lipoprotein (HDL) plasma levels and coronary heart disease has been attributed to the role that HDL and its major constituent, apolipoprotein A-I (apoA-I), play in reverse cholesterol transport (RCT). The efficiency of RCT depends on the specific ability of apoA-I to promote cellular cholesterol efflux, bind lipids, activate lecithin:cholesterol acyltransferase (LCAT), and form mature HDL that interact with specific receptors and lipid transfer proteins. From the intensive analysis of apoA-I secondary structure has emerged our current understanding of its different classes of amphipathic alpha-helices, which control lipid-binding specificity. The main challenge now is to define apoA-I tertiary structure in its lipid-free and lipid-bound forms. Two models are considered for discoidal lipoproteins formed by association of two apoA-I with phospholipids. In the first or picket fence model, each apoA-I wraps around the disc with antiparallel adjacent alpha-helices and with little intermolecular interactions. In the second or belt model, two antiparallel apoA-I are paired by their C-terminal alpha-helices, wrap around the lipoprotein, and are stabilized by multiple intermolecular interactions. While recent evidence supports the belt model, other models, including hybrid models, cannot be excluded. ApoA-I alpha-helices control lipid binding and association with varying levels of lipids. The N-terminal helix 44-65 and the C-terminal helix 210-241 are recognized as important for the initial association with lipids. In the central domain, helix 100-121 and, to a lesser extent, helix 122-143, are also very important for lipid binding and the formation of mature HDL, whereas helices between residues 144 and 186 contribute little. The LCAT activation domain has now been clearly assigned to helix 144-165 with secondary contribution by helix 166-186. The lower lipid binding affinity of the region 144-186 may be important to the activation mechanism allowing displacement of these apoA-I helices by LCAT and presentation of the lipid substrates. No specific sequence has been found that affects diffusional efflux to lipid-bound apoA-I. In contrast, the C-terminal helices, known to be important for lipid binding and maintenance of HDL in circulation, are also involved in the interaction of lipid-free apoA-I with macrophages and specific lipid efflux. While much progress has been made, other aspects of apoA-I structure-function relationships still need to be studied, particularly its lipoprotein topology and its interaction with other enzymes, lipid transfer proteins and receptors important for HDL metabolism.     

Phospholipid transfer protein (PLTP) causes proteolytic cleavage of apolipoprotein A-I

Plasma phospholipid transfer protein (PLTP) is a factor that plays an important role in HDL metabolism. In this study we present data suggesting that PLTP has an inherent protease activity. After incubation of HDL3 in the presence of purified plasma PLTP, the d < 1.25 g/ml particles (fusion particles) contained intact 28.2 kDa apoA-I while the d > 1.25 g/ml fraction (apoA-I-PL complexes) contained, in addition to intact apoA-I, a cleaved 23 kDa form of apoA-I. Purified apoA-I was also cleaved by PLTP and produced a similar 23 kDa apoA-I fragment. The cleavage of apoA-I increased as a function of incubation time and the amount of PLTP added. The process displayed typically an 8-10 h lag or induction period, after which the cleavage proceeded in a time-dependent manner. This lag-phase was necessary for the development of the cleavage activity during incubation at 37 degrees C. The specific apoA-I cleavage activity of different PLTP preparations varied between 0.4-0.8 microg apoA-I degraded/h per 1000 nmol per h of PLTP activity. The 23 kDa apoA-I fragment reacted with monoclonal antibodies specific for the N-terminal part of apoA-I, indicating that the apoA-I cleavage occurred in the C-terminal portion. The apoA-I cleavage products were further characterized by mass spectrometry. The 23 kDa fragment yielded a mass of 22.924 kDa, demonstrating that the cleavage occurs in the C-terminal portion of apoA-I between amino acid residues 196 (alanine) and 197 (threonine). The intact apoA-I and the 23 kDa fragment revealed identical N-terminal amino acid sequences. The cleavage of apoA-I could be inhibited with APMSF and chymostatin, suggesting that it is due to a serine esterase-type of proteolytic activity. Recombinant PLTP produced in CHO cells or using the baculovirus-insect cell system caused an apoA-I cleavage pattern identical to that obtained with plasma PLTP. The present results raise the question of whether PLTP-mediated proteolytic cleavage of apoA-I might affect plasma HDL metabolism by generating a novel kinetic compartment of apoA-I with an increased turnover rate.     

Transthyretin in high density lipoproteins: association with apolipoprotein A-I

Previous studies have revealed the presence of transthyretin (TTR) on lipoproteins. To further address this issue, we fractionated plasma lipoproteins from 9 normal individuals, 10 familial amyloidotic polyneuropathy (FAP) patients, and 19 hyperlipidemic subjects using gel filtration. In the majority of the subjects, as well as in 9 of the 10 FAP patients and 14 of the 19 patients with hyperlipidemia, TTR was detected by ELISA in the high density lipoprotein (HDL) fraction. The presence of TTR in HDL was confirmed by direct sequencing and by immunoblotting; using non-reducing conditions, TTR was found by immunoblotting in a high molecular weight complex, which reacted also for apolipoprotein A-I (apoA-I). The amount of TTR present in HDL (HDL-TTR), as quantified by ELISA corresponded to 1;-2% of total plasma TTR. However, no detectable TTR levels were found in HDL fraction from 6 of the hyperlipidemic subjects. No correlation was found between the lack of TTR in HDL and plasma levels of total, LDL-, or HDL-associated cholesterol as well as levels of apoA-I and total plasma TTR. Ligand binding experiments showed that radiolabeled TTR binds to the HDL fraction of individuals with HDL-TTR but not to the corresponding fractions of individuals devoid of HDL-TTR, suggesting that HDL composition may interfere with TTR binding. The component(s) to which TTR binds in the HDL fraction were investigated. Polyclonal antibody against apoA-I was able to block the interaction of TTR with HDL, suggesting that the interaction of TTR with the HDL particle occurs via apoA-I. This hypothesis was further demonstrated by showing the formation of a complex of TTR with HDL and apoA-I by crosslinking experiments. Furthermore, anti-apoA-I immunoblot under native conditions suggested the existence of differences in HDL particle properties and/or stability between individuals with and without HDL-TTR.     

A gel-based method for purification of apolipoprotein A-I from small volumes of plasma

We present here a gel-based method for rapid purification of apolipoprotein A-I (apoA-I) from small volumes of human plasma. After isolation of high density lipoprotein from plasma, the apoA-I protein was separated by electrophoresis and the apoA-I band excised from the gel. The apoA-I was then eluted from the gel strip, concentrated, and delipidated ready for use. The structure and function of the gel-purified apoA-I protein was compared against apoA-I purified by the traditional size-exclusion chromatography method. The α-helical content of the gel-purified apoA-I as determined by circular dichroism was similar to chromatography-purified apoA-I. The functional activity of gel-purified apoA-I, as determined by cholesterol efflux assays in primary human fibroblasts and RAW264.7 macrophages, was also comparable with chromatography-purified apoA-I. This method is a valid alternative for apoA-I purification with some advantages over traditional chromatography purification including a much reduced plasma volume requirement, less time and cost, and a higher percentage protein recovery. The method is particularly suitable for applications requiring the purification of apoA-I from multiple human or animal samples of interest.     

A key point mutation (V156E) affects the structure and functions of human apolipoprotein A-I

A naturally occurring point mutant of human apolipoprotein A-I (apoA-I), V156E, which is associated with extremely low plasma apoA-I and high density lipoprotein (HDL) levels, and coronary artery disease (Huang, W., Sasaki, J., Matsunaga, A., Nanimatsu, H., Moriyama, K., Han, H. Kugi, M., Koga, T., Yamaguchi, K., and Arakawa, K. (1998) Arterioscler. Throm. Vasc. Biol. 18, 389-396), was produced in an Escherichia coli expression system. The purified recombinant proapoA-I V156E mutant was examined in its structural and functional properties, both, in the lipid-free and lipid-bound states. In the lipid-free form the mutant protein exhibited small changes in conformation, but was more stable, and quite resistant to self-association, compared with control apoA-I. The V156E mutant was able to interact with phospholipid (PL) at high PL:protein ratios (95:1, mol/mol), but was inefficient in forming reconstituted HDL (rHDL) complexes at lower PL:protein ratios (40:1). In the lipid-bound, rHDL state, the mutant protein was somewhat more alpha-helical and formed a larger complex (110 A) than control apoA-I (97 A). Furthermore, the rHDL particles containing the V156E mutant did not rearrange to smaller particles in the presence of low density lipoproteins, and had minimal reactivity with lecithin-cholesterol acyltransferase (LCAT), compared with rHDL particles made with control apoA-I. These results suggest a key role for Val-156, or the adjacent central region of apoA-I in the modulation of apoA-I conformation, stability, and self-association in solution, and in the formation of small HDL, the conformational adaptability of apoA-I leading to structural rearrangements of HDL, and the activation of LCAT.