Apolipoprotein A-I from normal human plasma: definition of three distinct antigenic determinants

We have prepared, selected and cloned four mouse hybridomas that secreted monoclonal antibodies against human plasma apolipoprotein A-I. These antibodies are all of the IgG-I subclass, and were named anti-A-I 6B8, 5G6, 3D4 and 5A6. We characterized the specificity of the antibodies, finding that all four of them reacted similarly, and with only the major proteins having the molecular weight and isoelectric focusing characteristics of apolipoprotein A-I. The antibodies reacted with all known charge-polymorphs of apolipoprotein A-I and pro apolipoprotein A-I. Thus, the polymorphs of apolipoprotein A-I are alike in that they all contain the antigenic sites of these four antibodies. In a solid-phase, antibody competition radioimmunoassay we found inhibition or enhancement of antibody binding to apolipoprotein A-I, according to the pair of antibodies tested. Antibodies 6B8, 5G6 and 3D4 were different from one another and reacted with different antigenic determinants, but 5A6 was similar to 3D4 and reacted at the same site. We compared the reactions of the four antibodies with CNBr-cleaved fragments of apolipoprotein A-I separated by polyacrylamide gel electrophoresis. We found three different patterns of reaction with the apolipoprotein A-I fragments; 6B8, 5G6 and 3D4 were different, but 5A6 resembled 3D4. Thus, the four antibodies reacted with at least three different antigenic sites in apolipoprotein A-I, which were present in different CNBr fragments of apolipoprotein A-I, but not on fragment 4 which forms the carboxy-terminal segment.     

Detection of immunological heterogeneity of an isolated, purified protein (vervet apolipoprotein A-I)

Using a single goat antiserum, we have identified immunological heterogeneity of purified apolipoprotein A-I from high density lipoprotein of vervet monkeys. We examined whether the apparent heterogeneity was due to separate antigenic sites within the polypeptide sequence or rather on the different isoproteins, which result in charge heterogeneity of this protein. The apolipoprotein A-I was cleaved with cyanogen bromide and the resulting three fragments were purified and characterized. By using immunodiffusion, each of the fragments was found to show a characteristic and different reaction to the antiserum. By contrast, apparent identity was found by immunodiffusion among the separate isoprotein forms of apolipoprotein A-I. We have concluded that the immunological heterogeneity of apolipoprotein A-I was due to different antigenic sites within the primary sequence of apolipoprotein A-I.     

A method to screen apolipoprotein polymorphisms in whole plasma: description of apolipoprotein A-IV variants in dyslipidemias and a reassessment of apolipoprotein A-I in Tangier disease

A sensitive and rapid immunological detection method was used to screen for apolipoprotein A-IV variants. Antibodies to human lymph chylomicron or plasma apolipoprotein A-IV, and plasma apolipoprotein A-I were raised in rabbits. Antibodies to apolipoprotein A-I or apolipoprotein A-IV were shown to be monospecific to their respective antigens by reactivity against human chylomicron apolipoproteins by immunoblot analysis. Plasma samples were obtained from dyslipidemic subjects from the Lipid Research Clinic of Columbia University. The plasma samples were isoelectrically focused (pH 4-6) on slab gels. Plasma proteins were then transferred to nitrocellulose paper for immunoblotting. Apolipoprotein A-IV polymorphism was determined by specific immunological detection of apolipoprotein A-IV. Identical apolipoprotein A-IV isoprotein patterns were observed when either antibodies to lymph or plasma apolipoprotein A-IV were used for immunoblotting. All the dyslipidemic plasma samples screened contained the two major and one or two minor isoproteins of normal plasma. In two instances, new apolipoprotein A-IV variants having an additional isoform were detected. One subject was hypertriglyceridemic (triacylglycerols = 342 mg/dl, cholesterol = 251 mg/dl) and had an additional major acidic apolipoprotein A-IV isoform. Another subject with mild hypocholesterolemia (triacylglycerols = 209 mg/dl, cholesterol = 120 mg/dl) was found to have additional major and minor basic apolipoprotein A-IV isoforms. The specificity of this technique allows detection of polymorphism of apolipoproteins of similar isoelectric points by use of a single dimension isoelectric focusing gel. This technique also demonstrated the presence of altered apolipoprotein A-I isoforms in the plasma of a patient with Tangier disease. These isoforms were previously identified as isoforms 2 and 4 of normal plasma by use of two-dimensional gel electrophoresis. However, by use of this new technique and careful evaluation of previously published two-dimensional gels, we now identify these apolipoprotein A-I isoforms as being more acidic than those of normal plasma.     

Specific docking of apolipoprotein A-I at the cell surface requires a functional ABCA1 transporter

The identification of defects in ABCA1 as the molecular basis of Tangier disease has highlighted its crucial role in the loading with phospholipids and cholesterol of nascent apolipoprotein particles. Indeed the expression of ABCA1 affects apolipoprotein A-I (apoA-I)-mediated removal of lipids from cell membranes, and the possible role of ABCA1 as an apoA-I surface receptor has been recently suggested. In the present study, we have investigated the role of the ABCA1 transporter as an apoA-I receptor with the analysis of a panel of transfectants expressing functional or mutant forms of ABCA1. We provide experimental evidence that the forced expression of a functional ABCA1 transporter confers surface competence for apoA-I binding. This, however, appears to be dependent on ABCA1 function. Structurally intact but ATPase-deficient forms of the transporter fail to elicit a specific cell association of the ligand. In addition the diffusion parameters of membrane-associated apoA-I indicate an interaction with membrane lipids rather than proteins. These results do not support a direct molecular interaction between ABCA1 and apoA-I, but rather suggest that the ABCA1-induced modification of the lipid distribution in the membrane, evidenced by the phosphatidylserine exofacial flopping, generates a biophysical microenvironment required for the docking of apoA-I at the cell surface.     

Quantitative determination of apolipoprotein A-I in high-density lipoproteins and 'free' apolipoprotein A-I by two-dimensional agarose gel lipoprotein-'rocket' immunoelectrophoresis of human serum

A method has been developed for quantitative analysis of 'free' apolipoprotein A-I and apolipoprotein A-I associated with high-density lipoprotein (HDL) in serum. The method utilizes the difference between the rate of electrophoretic migration of apolipoprotein A-I associated with HDL (alpha) and 'free' apolipoprotein A-I (pre-beta) in agarose gel. Apolipoprotein A-I is subsequently quantitated by electrophoresis in a second dimensional gel containing anti-apolipoprotein A-I antibodies. Using this method all apolipoprotein A-I of normal fasting serum was found associated with HDL (n = 16). By contrast, 'free' apolipoprotein A-I accounted for up to 12% of the total in the serum of patients with isolated hypertriglyceridemia (n = 8) or mixed hyperlipoproteinemia (n = 8). Between 30 and 35% of 'free' apolipoprotein A-I was found in one patient afflicted with the apolipoprotein C-II deficiency syndrome. Also, 'free' apolipoprotein A-I could be detected in normal postabsorptive serum. 30 and 90 min following heparin-enhanced lipolysis 'free' apolipoprotein A-I accounted for 23 and 20%, respectively, of the total apolipoprotein A-I of serum. Apolipoprotein A-I associated with HDL remained unaltered. It appears, therefore, that 'free' apolipoprotein A-I is liberated from triglyceride-rich lipoproteins during lipolysis.     

Studies on the polymorphism of human apolipoprotein A-I

Upon preparative isoelectric focussing of human apo-HDL, four major forms of apolipoprotein A-I have been isolated. As identified by the following nomenclature and pI, they comprise: apolipoprotein A-I1, pI 5.62; apolipoprotein A-I2, pI 5.53; apolipoprotein A-I3, pI 5.45; apolipoprotein A-I4 pI 5.36. These forms of apolipoprotein A-I were shown to have identical migration on polyacrylamide gel electrophoresis, molecular weights of 26 000 on sodium dodecyl sulfate gel electrophoresis and a common antigenicity with antisera against apolipoprotein A-I or A-I1. Each form had very similar amino acid compositions with the exception of form apolipoprotein A-I4 which contained one isoleucine residue per mol. All forms but apolipoprotein A-I4 were activators of lecithin:cholesterol acyltransferase, the latter was inhibitory to the reaction. From these results, it was concluded that apolipoprotein A-I1, A-I2 and A-I3 are equivalent forms of apolipoprotein A-I whereas apolipoprotein A-I4 is different or heterogeneous. Upon refocussing, the polymorphs were shown to be stable at their pI and not affected by changes in concentration and by the presence of urea or ampholytes. Exposure of a form of apolipoprotein A-I to alkaline pH partially regenerated the original heterogeneity; however, apolipoprotein A-I4 regenerated from apolipoprotein A-I1 did not contain isoleucine, which further demonstrates form apolipoprotein A-I4 heterogeneity.     

Familial apolipoprotein A-I and C-III deficiency, variant II

The biochemical, clinical, and genetic features were examined in the proband (homozygote) and heterozygotes (n = 17) affected with familial apolipoprotein A-I and C-III deficiency, variant II (previously described as apolipoprotein A-I absence). The proband was a 45-year-old white female with mild corneal opacification and significant three-vessel coronary artery disease (CAD), who died shortly after bypass surgery. Autopsy findings included significant atherosclerosis in the coronary and pulmonary arteries and the abdominal aorta as well as extracellular stromal lipid deposition in the cornea. No reticuloendothelial lipid deposits in the liver, bone marrow, or spleen were noted (unlike Tangier disease). Laboratory features included marked high density lipoprotein (HDL) deficiency and undetectable plasma apolipoproteins (apo) A-I and C-III. The percentage of plasma cholesterol in the unesterified form was normal at 30%. The activity and mass of lecithin:cholesterol acyltransferase (LCAT) were 42% and 36% of normal, respectively, and the cholesterol esterification rate was 43% of normal. Deficiencies of plasma vitamin E and essential fatty acid (linoleic, C18:2) were also noted. Evaluation of plasma lipoproteins and apolipoproteins in 37 kindred members revealed 17 heterozygotes with HDL cholesterol values below the 10th percentile of normal. Of these, all had apoA-I levels more than one standard deviation below the normal mean, and 37.5% had a similar decrease in apoC-III values. Mean (+/- SD) plasma HDL cholesterol, apoA-I, and apoC-III values (mg/dl) in heterozygotes were 54.0%, 62.4%, and 79.2% of normal, respectively. No evidence of CAD was observed in 10 heterozygotes 40 years of age or less; however, CAD was detected in 3 of 7 heterozygotes over 40 years of age, one of whom died at age 56 years of complications of myocardial infarction and stroke. The inheritance pattern in this kindred was autosomal codominant. ApoA-I isolated from a heterozygote had an isoelectric focusing pattern and amino acid composition similar to normal. Utilizing DNA isolated from two obligate heterozygotes, no abnormalities in the apoA-I or apoC-III genes were detected by Southern blot analysis utilizing specific probes following restriction enzyme digestion. The data indicate that familial apolipoprotein A-I and C-III deficiency, variant II, is similar to variant I (described by Norum et al. 1982. N. Engl. J. Med. 306: 1513-1519), but differs at the clinical level (lack of xanthomas), the biochemical level (lack of detectable apoA-I, lower apoA-II level), and at the gene level.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and characterization of human apolipoprotein A-I Fukuoka (110 Glu----Lys). A novel apolipoprotein variant

A novel genetic variant of apolipoprotein(apo) A-I Fukuoka, has been identified in a Japanese family. This variant has a relative charge of +2 compared to normal apolipoprotein A-I (A-I4), on the isoelectric focusing gels and the same molecular mass and immunologic characteristics as normal apolipoprotein A-I. This variant, transmitted as an autosomal co-dominant inheritance was purified by preparative Immobiline isoelectric focusing. Sequence analysis after cleavage with lysyl endopeptidase and CNBr, followed by high-performance liquid chromatography revealed a single substitution of lysine at position 110, instead of the usual glutamic acid. This mutant apolipoprotein A-I has much the same potential as to activate lecithin-cholesterol acyltransferase.     

Human proapolipoprotein A-I: development of an antibody to the propeptide as a probe of apolipoprotein A-I biosynthesis and processing

In human plasma, apolipoprotein A-I is present as pro and mature isoproteins. The development of a highly specific antibody to the pro isoprotein of apoA-I has been difficult due to the close structural similarity between the pro and mature isoforms of apoA-I. To sermount this difficulty, a peptide was synthesized by the solid phase method which corresponded to the amino acid sequence present in the pro region of apoA-I. The synthetic peptide was coupled to serum albumin and the conjugate utilized to immunize rabbits for antibody production. Immunoblot analysis of purified proapoA-I and mature apoA-I revealed that the antibody was specific for the propeptide of apoA-I. Analysis of apoA-I in the plasma from a Tangier disease patient and newly secreted apoA-I from HepG2 cells clearly demonstrated the isoforms which contained the proisoprotein. The proapoA-I specific antibody should prove to be a useful tool in developing a radioimmunoassay for quantitation of the proisoprotein in plasma, isolation of proapoA-I from normal and dyslipoproteinemic subjects by immunoaffinity chromatography and in studies related to the synthesis and processing of apoA-I.     

Proteolytic processing of human preproapolipoprotein A-I. A proposed defect in the conversion of pro A-I to A-I in Tangier's disease

The primary translation product of human intestinal apolipoprotein A-I mRNA was isolated from wheat germ and ascites cell-free translation systems. Comparison of its NH2-terminal sequence with that of plasma high density lipoprotein-associated A-I showed that it is initially synthesized as a preproprotein. Like rat preproapolipoprotein A-I, it contains an 18-amino acid prepeptide and a 6-amino acid propeptide. The highly unusual COOH-terminal Gln-Gln dipeptide present in the rat pro-segment is also represented at the same position in the human sequence. The functional division of the 24-amino acid NH2-terminal extention into pro- and presegments was verified by finding that the stable intracellular form of A-I in a human hepatoma cell line was the proprotein. Edman degradation of radiolabeled intracellular and extracellular A-I indicated that this apolipoprotein was secreted without proteolytic cleavage of its hexapeptide prosegment. Therefore, it appears that apolipoprotein A-I undergoes an additional proteolytic processing step before it is fully integrated into plasma high density lipoprotein. Two-dimensional gel electrophoresis of purified proapolipoprotein A-I isolated from the hepatocyte cell culture media indicated that it corresponds to isoforms 2 and 3, the basic A-I isoproteins which are the precursors of plasma A-I and the predominant plasma A-I isoforms found in patients with Tangier's disease (Zannis, V. I., Lees, A. M., Lees, R. S., and Breslow, J. L. (1982) J. Biol. Chem., 257, 4978-4986). Therefore this pathologic state probably arises from a defect in the conversion of proapolipoprotein A-I to apolipoprotein A-I.     

Characterization of A-I-containing lipoproteins in subjects with A-I Milano variant

The A-I Milano variant of apolipoprotein A-I (A-IM), by virtue of its Arg-173----Cys substitution, is capable of forming a disulfide bond with the 77-amino-acid apolipoprotein A-II polypeptide (A-IIS) as well as with itself to produce dimers, A-IM/A-IIS and A-IM/A-IM, respectively. A-I-containing lipoproteins (Lp): particles with A-II (Lp(A-I with A-11)) and particles without A-II (Lp(A-I without A-II)) in the plasma of two nonhyperlipidemic A-IM carriers were investigated to determine the effect of A-IM on these lipoproteins. Despite the existence of abnormal apolipoprotein dimers and the unusually low HDL cholesterol (17 and 14 mg/dl), A-I (67 and 75 mg/dl), and A-II (18 and 18 mg/dl) levels in the two carriers, the plasma A-I of the carriers was distributed between Lp(A-I with A-II) and Lp(A-I without A-II) in a proportion comparable to that observed in normals. As expected, A-IM/A-IIS mixed dimer was found in carrier Lp(A-I with A-II). However, A-IM/A-IM dimer was located almost exclusively in carrier Lp(A-I without A-II). Chemical (dimethylsuberimidate) crosslinking of the protein moieties of the major subpopulations of Lp(A-I with A-II) and Lp(A-I without A-II) of normal and A-IM carriers showed that Lp(A-I with A-II), which is located predominantly in the 7.8-9.7 nm interval ((HDL2a + 3a + 3b)gge), had an apparent protein molecular weight equivalent to two molecules of A-I and one to two molecules of A-II per particle. Most of the Lp(A-I without A-II) particles, located predominantly in the size intervals of 9.7-12.9 nm (designated (HDL2b)gge) and 8.2-8.8 nm (HDL3a)gge) had protein moieties exhibiting a molecular weight equivalence predominantly of four and three molecules of A-I, respectively. A small quantity of particles with apparent protein content of two molecules of A-I in the 7.2-8.2 nm interval ((HDL3b + 3c)gge) was also detected. These studies showed that in nonhyperlipidemic A-IM carriers, the occurrence of apolipoprotein dimers had not markedly affected the protein stoichiometry of Lp(A-I with A-II) and Lp(A-I without A-II).     

Apolipoprotein B, apolipoprotein A-I, insulin resistance and the metabolic syndrome

PURPOSE OF REVIEW: The goal of identifying subjects with metabolic syndrome is to detect those at higher risk of developing cardiovascular disease. Evidence continues to accumulate as to the superiority of apolipoprotein B and apolipoprotein A-I over the conventional lipoprotein lipids as markers of vascular risk. It would seem reasonable, therefore, to redefine the dyslipidemia of the metabolic syndrome incorporating apolipoproteins. Therefore, our objective is to elucidate how apolipoprotein B and apolipoprotein A-I amplify evidence of the interactions amongst metabolic syndrome, insulin resistance, abdominal obesity, and vascular risk. RECENT FINDINGS: In several large epidemiological studies, including the NHANES III database, apolipoprotein B/apolipoprotein A-I ratio was tightly linked to the metabolic syndrome and each of its components, the descending order being: low HDL cholesterol, high triglyceride, high waist circumference, high glucose, and high blood pressure. Moreover, apolipoprotein B associates more closely with inflammatory markers and insulin resistance than triglyceride and all cholesterol markers. Yet despite close association of the apolipoprotein B/apolipoprotein A-I ratio to metabolic syndrome, both are independent predictors of future myocardial infarction. SUMMARY: We believe that the dyslipidemia of the metabolic syndrome should be redefined to include apolipoprotein B and apolipoprotein A-I.     

A purification method for apolipoprotein A-I and A-II

Apolipoproteins A-I and A-II were isolated from precipitates obtained by cold ethanol fractionation of human plasma. The starting material used in this report was precipitate B of the Kistler and Nitschmann method which corresponds approximately to fraction III of the Cohn and Oncley procedure. Through the use of urea, chloroform, and ethanol in appropriate concentrations, apolipoproteins A-I and A-II were isolated by a simple extraction technique avoiding time-consuming ultracentrifugation. Starting from 10 g of centrifuged precipitate B, approximately 100 mg of apolipoprotein A-I and 10 mg of apolipoprotein A-II were obtained. When incubated with normal human or rabbit plasma, both apolipoproteins were readily incorporated into high-density lipoproteins. Apolipoprotein A-I obtained by the cold ethanol method activated lecithin-cholesterol acyltransferase to the same extent as apolipoprotein A-I prepared by the classical flotation method. Apolipoprotein A-II had no such properties by itself, but was capable of potentiating lecithin-cholesterol acyltransferase activity of apolipoprotein A-I.     

Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I

The ABCA1 transporter contains two large domains into which many of the genetic mutations in individuals with Tangier disease fall. To investigate the structural requirements for the cellular cholesterol efflux mediated by ABCA1, we have determined the topology of these two domains and generated transporters harboring five naturally occurring missense mutations in them. These mutants, unlike wild type ABCA1, produced little or no apoA-I-stimulated cholesterol efflux when transfected into 293 cells, establishing their causality in Tangier disease. Because all five mutant proteins were well expressed and detectable on the plasma membrane, their interaction with the ABCA1 ligand, apolipoprotein (apo) A-I, was measured using bifunctional cross-linking agents. Four of five mutants had a marked decline in cross-linking to apoA-I, whereas one (W590S) retained full cross-linking activity. Cross-linking of apoA-I was temperature-dependent, rapid in onset, and detectable with both lipid- and water-soluble cross-linking agents. These results suggest that apoA-I-stimulated cholesterol efflux cannot occur without a direct interaction between the apoprotein and critical residues in two extracellular loops of ABCA1. The behavior of the W590S mutant indicates that although binding of apoA-I by ABCA1 may be necessary, it is not sufficient for stimulation of cholesterol efflux.     

Predicting the structure of apolipoprotein A-I in reconstituted high-density lipoprotein disks.

In reconstituted high-density lipoproteins, apolipoprotein A-I and phosphatidylcholines combine to form disks in which the amphipathic alpha-helices of apolipoprotein A-1 bind to the edge of a lipid bilayer core, shielding the hydrophic lipid tails from the aqueous environment. We have employed experimental data, sequence analysis, and molecular modeling to construct an atomic model of such a reconstituted high-density lipoprotein disk consisting of two apolipoprotein A-I proteins and 160 palmitoyloleoylphosphatidylcholine lipids. The initial globular domain (1-47) of apolipoprotein A-I was excluded from the model, which was hydrated with an 8-A shell of water molecules. Molecular dynamics and simulated annealing were used to test the stability of the model. Both head-to-tail and head-to-head forms of a reconstituted high-density lipoprotein were simulated. In our simulations the protein contained and adhered to the lipid bilayer while providing good coverage of the lipid tails.     

High level expression of human apolipoprotein A-I in transgenic rats raises total serum high density lipoprotein cholesterol and lowers rat apolipoprotein A-I

To examine the consequences of increased apolipoprotein A-I production on cholesterol and lipoprotein metabolism, we have produced two lines of transgenic rats; one expressing moderate and one very high levels of human apolipoprotein A-I. The rats were produced by microinjection of a 13 kbp DNA fragment containing the human apolipoprotein A-I gene plus 10 kbp of its 5′ flanking sequence and 1 kbp of its 3′ flanking sequence. Both lines of transgenic rats express human apolipoprotein A-I mRNA in liver and human apolipoprotein A-I in plasma. Sera from these rats contain significantly higher levels of total apolipoprotein A-I, high density lipoprotein cholesterol and phospholipid than sera from non-transgenic littermates. Transgenic rats expressing high levels of human apolipoprotein A-I have reduced levels of serum rat apolipoprotein A-I suggesting a mechanism exists to down-regulate apolipoprotein A-I production. These transgenic rats provide a unique animal model to examine the effects of increased apolipoprotein A-I production on lipid and lipoprotein metabolism.     

ABCA1-mediated transport of cellular cholesterol and phospholipids to HDL apolipoproteins

Lipid-poor apolipoproteins remove cellular cholesterol and phospholipids by an active transport pathway controlled by an ATP binding cassette transporter called ABCA1 (formerly ABC1). Mutations in ABCA1 cause Tangier disease, a severe HDL deficiency syndrome characterized by a rapid turnover of plasma apolipoprotein A-I, accumulation of sterol in tissue macrophages, and prevalent atherosclerosis. This implies that lipidation of apolipoprotein A-I by the ABCA1 pathway is required for generating HDL particles and clearing sterol from macrophages. Thus, the ABCA1 pathway has become an important therapeutic target for mobilizing excess cholesterol from tissue macrophages and protecting against atherosclerosis.     

The intracellular quality control system down-regulates the secretion of amyloidogenic apolipoprotein A-I variants: A possible impact on the natural history of the disease

Hereditary systemic amyloidosis caused by apolipoprotein A-I variants is a dominantly inherited disease characterised by fibrillar deposits mainly localized in the kidneys, liver, testis and heart. We have previously shown that the apolipoprotein A-I variant circulates in plasma at lower levels than the wild-type form (Mangione et al., 2001; Obici et al., 2004) thus raising the possibility that the amyloid deposits could sequester the circulating amyloidogenic chain or that the intracellular quality control can catch and capture the misfolded amyloidogenic chain before the secretion. In this study we have measured plasma levels of the wild-type and the variant Leu75Pro apolipoprotein A-I in two young heterozygous carriers in which tissue amyloid deposition was still absent. In both cases, the mutant was present at significantly lower levels than the wild-type form, thus indicating that the low plasma concentration of the apolipoprotein A-I variant is not a consequence of the protein entrapment in the amyloid deposits. In order to explore the cell secretion of amyloidogenic apolipoprotein A-I variants, we have studied COS-7 cells expressing either wild-type apolipoprotein A-I or two amyloidogenic mutants: Leu75Pro and Leu174Ser. Quantification of intracellular and extracellular apolipoprotein A-I alongside the intra-cytoplasmatic localization indicates that, unlike the wild-type protein, both variants are retained within the cells and mainly accumulate in the endoplasmic reticulum. The low plasma concentration of amyloidogenic apolipoprotein A-I may therefore be ascribed to the activity of the intracellular quality control that represents a first line of defence against the secretion of pathogenic variants.     

The role of apolipoprotein A-I and apolipoprotein A-II in high-density lipoprotein binding to human adipocyte plasma membranes

Adipocyte plasma membranes purified from omental fat tissue biopsies of massively obese subjects possess specific binding sites for high-density lipoprotein (HDL3). This binding was independent of apolipoprotein E as HDL3 isolated from plasma of an apolipoprotein E-deficient individual was bound to a level comparable to that of normal HDL3. To examine the importance of apolipoprotein A-I, the major HDL3 apolipoprotein, in the specific binding of HDL3 to human adipocytes, HDL3 modified to contain varying proportions of apolipoproteins A-I and A-II was prepared by incubating normal HDL3 particles with different amounts of purified apolipoprotein A-II. As the apolipoproteins A-I-to-A-II ratio in HDL3 decreased, the binding of these particles to adipocyte plasma membranes was reduced. Compared to control HDL3, a 92 +/- 3.1% reduction (mean +/- S.E., n = 3) in maximum binding capacity was observed along with an increased binding affinity for HDL3 particles in which almost all of the apolipoprotein A-I had been replaced by A-II. The uptake of HDL cholesteryl ester by intact adipocytes as monitored by [3H]cholesteryl ether labeled HDL3, was also significantly reduced (about 35% reduction, P less than 0.005) by substituting apolipoprotein A-II for A-I in HDL3. These data suggest that HDL binding to human adipocyte membranes is mediated primarily by apolipoprotein A-I and that optimal delivery of cholesteryl ester from HDL to human adipocytes is also dependent on apolipoprotein A-I.     

Apolipoprotein A-I(Milano): current perspectives

PURPOSE OF REVIEW: Strategies to increase HDL are among the major targets of clinical research in atherosclerosis prevention. The mutant apolipoprotein A-I(Milano) has been associated with a reduced incidence of coronary disease in carriers. Furthermore, recombinant apolipoprotein A-I(Milano) has displayed remarkable atheroprotective activities and the possibility of directly reducing the burden of atherosclerosis in experimental models. This review is aimed at providing an update on the experimental studies in which apolipoprotein A-I(Milano), produced as a recombinant protein, has displayed important effects in the treatment of vascular diseases. RECENT FINDINGS: In the past year, two reports have appeared, indicating that a single-dose administration of recombinant apolipoprotein A-I(Milano) dimers formulated into liposomes can reduce atheromas in models such as the apolipoprotein E-deficient mice and a rabbit model of carotid focal lesion, in which a direct 90 min infusion of the product reduced atheroma up to 30%. This finding was associated with an increase in HDL free cholesterol and the permanence of the recombinant product in the lesion for over 72 h. SUMMARY: Recombinant apolipoprotein A-I(Milano), formulated as synthetic HDL with phospholipids, appears to exert a direct removing effect on arterial cholesterol. This is well evident in experimental animals and, more recently in clinical findings, as indicated by a dramatic increase in HDL free cholesterol after the infusion of different doses of the agent. As the product appears to be well tolerated and non-immunogenic, ongoing phase II studies in patients are being awaited with interest to obtain a 'proof of principle' for 'HDL therapy'.