Expression of human apolipoprotein A-I epitopes in high density lipoproteins and in serum

The expression and immunoreactivity of apolipoprotein (apo) A-I epitopes in high density lipoproteins (HDL) and serum has been investigated using two series of monoclonal antibodies (Mabs) which have been described elsewhere. Series 1 Mabs, identified as 3D4, 6B8, and 5G6, were obtained by immunization and screening with apoA-I, and series 2 Mabs, identified as 2F1, 4H1, 3G10, 4F7, and 5F6, were obtained by immunization and screening with HDL. These Mabs were characterized with respect to their binding to HDL particles in solution. In series 2 Mabs, 2F1, 3G10, and 4F7, which react with apoA-I CNBr-fragments 1 and 2, could precipitate 100% of 125I-labeled HDL, while 4H1 and 5F6, which react with CNBr fragments 1 and 3, precipitated 90 and 60% of 125I-labeled HDL, respectively. Therefore, three distinct epitopes mapped to CNBr fragments 1 and 2 have been identified which are expressed on all HDL particles, indicating that several antigenic do mains exist on apoA-I which have the same conformation on all apoA-I-containing lipoproteins. The Mabs reacting at these sites have significantly higher affinity constants for 125I-labeled HDL than those that failed to precipitate 100% of HDL. This suggests that the high affinity Mabs react with apoA-I epitopes that are both expressed on all lipoproteins and located in thermo-dynamically stable regions of the molecules. All Mabs from series 1 precipitated 35% or less of 125I-labeled HDL prepared from freshly collected serum, but the proportion of HDL particles expressing the epitopes for these Mabs doubled or more upon serum storage at 4 degrees C. The time course of the alteration of apoA-I antigen in vitro was measured in three normolipemic donors. Upon storage of serum at 4 degrees C, the immunoreactivity of series 2 Mabs (4H1, 3G10) remained unchanged. However, the immunoreactivity of series 1 Mab 3D4 increased linearly at 38%/day for 4 weeks and by 12 weeks had plateaued at about 280-fold compared to day 1. The immunoreactivity of other series 1 Mabs also increased significantly with time in vitro. This process was partially inhibited in the presence of EDTA and by addition of antioxidants, however, the exact molecular nature of this in vitro alteration of apoA-I antigen was not identified.     

Isolation and characterization of the major apolipoprotein from chicken high density lipoproteins

High density lipoproteins were isolated from plasma of white Leghorn hens by ultracentrifugal flotation between densities 1.063 and 1.210 g/ml. After delipidation, the lipid-free proteins were fractionated by chromatography on Sephadex G-150 in urea; one major apolipoprotein was isolated and characterized. From its chemical, physical and immunochemical properties, the major apoprotein from hen high-density lipoproteins has characteristics similar to the major apoprotein of human high density lipoproteins, apoA-I. Thus the hen protein has been designated hen apoA-I.Hen apoA-I has a molecular weight of approximately 28 000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Its calculated molecular weight from its 234 constituent amino acids is 26 674. Hen apoA-I differed from its human counterpart by containing isoleucine. Treatment of hen apoA-I with carboxypeptidase A yielded a COOH-terminal sequence of Leu-Val-Ala-Gln. Automatic Edman degradation of the apoprotein gave an NH₂-terminal sequence of Asp-Glu-Pro-Gln-Pro-Glu-Leu. Hen apoA-I had a circular dichroic spectrum typical of α-helical structures; the calculated helicity was 90%. Goat antisera prepared to hen apoA-I formed precipitin lines of complete identity to the hen apoprotein but lines of only partial identity to human apoA-I. These studies show that the major apoprotein from hen and human high-density lipoproteins have similar properties to each other suggesting a common physiologic function.     

Isolation and characterization of the major apolipoprotein from chicken high density lipoproteins.

High density lipoproteins were isolated from plasma of white Leghorn hens by ultracentrifugal flotation between densities 1.063 and 1.210 g/ml. After delipidation, the lipid-free proteins were fractionated by chromatography on Sephadex G-150 in urea; one major apolipoprotein was isolated and characterized. From its chemical, physical and immunochemical properties, the major apoprotein from hen high-density lipoproteins has characteristics similar to the major apoprotein of human high density lipoproteins, apoA-I. Thus the hen protein has been designated hen apoA-I. Hen apoA-I has a molecular weight of approximately 28 000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Its calculated molecular weight from its 234 constituent amino acids is 26 674. Hen apoA-I differed from its human counterpart by containing isoleucine. Treatment of hen apoA-I with carboxypeptidase A yielded a COOH-terminal sequence of Leu-Val-Ala-Gln. Automatic Edman degradation of the apoprotein gave an NH2-terminal sequence of Asp-Glu-Pro-Gln-Pro-Glu-Leu. Hen apoA-I had a circular dichroic spectrum typical of alpha-helical structures; the calculated helicity was 90%. Goat antisera prepared to hen apoA-I formed precipitin lines of complete identity to the hen apoprotein but lines of only partial identity to human apoA-I. These studies show that the major apoprotein from hen and human high-density lipoproteins have similar properties to each other suggesting a common physiologic function.     

Role of apolipoprotein A-I in the structure of human serum high density lipoproteins. Reconstitution studies.

For a better definition of the role of human serum apolipoprotein A-I (apo A-I) in high density lipoprotein structure, a systematic investigation was carried out on factors influencing the in vitro association of this apoprotein with lipids obtained from the parent high density lipoprotein (HDL); these lipids include phospholipids, free cholesterol, cholesteryl esters, and triglycerides. Following equilibration, mixtures of apo A-I and lipids in varying stoichiometric amounts were fractionated by sequential flotation, CsCl density gradient ultracentrifugation, or gel-permeation chromatography, and the isolated complexes were characterized by physicochemical means. As defined by operational criteria (flotation at density 1,063 to 1.21 g/ml), only two types of HDL complexes were reassembled; one, reconstituted HDLS, small with a radius of 31 A, and the other, reconstituted HDLL, large with a radius of 39 A. The two types incorporated all of the lipid constituents of native HDL and contained 2 and 3 mol of apo A-I, respectively. A maximal yield of reconstituted HDL (R-HDL) was observed at an initial protein concentration of 0.1 muM, where apo A-I is predominantly monomeric. At increasing protein concentrations, the amount of apo A-I recovered in R-HDL was found to be proportional to the initial concentration of monomer and dimer in solution. The composition and yield of the complexes were independent of ionic strength and pH within the ranges studied. Both simple incubation and cosonication of apo A-I with HDL phospholipids produced complexes of identical composition, although the yeild of complexes was higher with co-sonication. When the comparison of the same methods was extended to mixtures of apo A-I and whole HDL lipids, the results confirmed previous observations that co-sonication is essential for the incorporation of the neutral lipid into the R-HDL complexes. The results indicate that (a) in vitro complexation of apo A-I with lipids is under kinetic control; (b) apo A-I can generate a lipid-protein complex with properties similar to those of the parent lipoprotein; (c) the process requires well defined experimental conditions and, most importantly, the presence in solution of monomers and dimers of apo A-I; (d) the number of apo A-I molecules incorporated into R-HDL determines the size and structure of the reassembled particle. All of these observations strongly support the essential role of apo A-I in the structure of human HDL.     

Studies on human serum high density lipoproteins. Self-association of apolipoprotein A-I in aqueous solutions.

Human serum apolipoprotein A-I (apo-A-I), the major protein component of the human serum high density lipoproteins, was studied in aqueous solutions of differing ionic strength and pH by the techniques of sedimentation equilibrium ultracentrifugation and frontal analysis gel chromatography. The ultracentrifugal studies indicate the apo-A-I is a self-associating system that is dependent upon protein concentration, but relatively independent of the nature of the medium. The apparent weight average molecular weights obtained from solutions of initial apo-A-I concentration between 0.2 and 0.9 mg/ml were in the range of 3.0 to 16.7 x 10(4) (monomer molecular weight = 28,014). Of the several models of self-associated examined, that which gave the best theoretical fit was for the monomer-dimertetramer-octamer model. The self-association of apo-A-I in aqueous solutions was further documented by frontal analysis gel chromatography, which not only corroborated the ultracentrifugal results, but also indicated that the multiple species of apo-A-I in solution attain equilibrium rather rapidly. Besides having intrinsic importance, these results indicate that the solution properties of apo-A-I must be established before ligand binding studies are conducted and interpreted.     

Particle distribution of human serum high density lipoproteins.

Density gradient ultracentrifugation of human serum high density lipoproteins (HDL) from both normolipemic males and females results in a distribution of HDL concentration versus subfraction hydrated density which has three maxima. Gradient gel electrophoresis of total HDL is characterized by three banding maxima, the positions of which suggest the presence of three particle size ranges: I. 10.8-12.0 nm, II. 9.7-10.7 nm, and III. 8.5-9.6 nm. Gradient gel electrophoresis of density gradient subfractions established an inverse relationship between particle size and particle hydrated density which was corroborated by electron microscopy and analytic ultracentrifugation. Comparison of male HDL from size ranges I, II, and III with female HDL from the same size ranges showed only small differences in the mean value of the peak F degrees 1.20 rate, size, molecular weight, protein weight percent, and weight protein/weight phospholipid. Major differences between males and females were seen in the relative amounts of HDL in density gradient subfractions 1-3 (size range I material) and 11-12 (size range III material); the percent total HDL in the group of subfractions 1-3 was greatly increased in female HDL while that of the group of subfractions 11-12 was increased in the male HDL. These studies indicate the presence of at least three major components in HDL instead of two (HDL2 and HDL3) and that peak F degrees 1.20 rate differences in HDL schlieren patterns between males and females are a function of the relative levels of these three components.     

Evidence that endothelial lipase remodels high density lipoproteins without mediating the dissociation of apolipoprotein A-I

Endothelial lipase (EL) is a triglyceride lipase gene family member that has high phospholipase and low triglyceride lipase activity. The aim of this study was to determine whether the phospholipase activity of EL is sufficient to remodel HDLs into small particles and mediate the dissociation of apolipoprotein A-I (apoA-I). Spherical, reconstituted HDLs (rHDLs) containing apoA-I only [(A-I)rHDLs], apoA-II only [(A-II)rHDLs], or both apoA-I and apoA-II [(A-I/A-II) rHDLs] were prepared. The rHDLs, which contained only cholesteryl esters in their core and POPC on the surface, were incubated with EL. As the rHDLs did not contain triacylglycerol, only the POPC was hydrolyzed. Hydrolysis was greater in the (A-I/A-II)rHDLs than in the (A-I)rHDLs. The (A-II)rHDL phospholipids were not hydrolyzed by EL. EL remodeled the (A-I)rHDLs and (A-I/A-II)rHDLs, but not the (A-II)rHDLs, into smaller particles. The reduction in particle size was related to the amount of phospholipid hydrolysis, with the diameter of the (A-I/A-II)rHDLs decreasing more than that of the (A-I)rHDLs. These changes did not affect the conformation of apoA-I, and neither apoA-I nor apoA-II dissociated from the rHDLs. Comparable results were obtained when human plasma HDLs were incubated with EL. These results establish that the phospholipase activity of EL remodels plasma HDLs and rHDLs into smaller particles without mediating the dissociation of apolipoproteins.     

Crystallization effects in serum high density lipoproteins

Peculiarities of aggregation in the samples of high density serum lipoproteins LHD2 and LHD3 obtained from healthy donors and patients with ishaemic heart disease were studied under isothermal exposition. It was found that lipoprotein samples from defective serum were characterized by chemical and structural inhomogeneity. It is reflected in the formation of spherolites of various types and of well-grown dendrite forms. Growth of stepped lens-shaped crystals of cholesterol takes place in the normal serum LHD3 preparations after long exposition. Crystal growth is thought to begin according to dislocational mechanism on linear defects existing in the liquid crystalline phase; then transformation to kinetically rough surface and to normal growth of the face occurs with the concentration increase.     

Human apolipoprotein A-I kinetics within triglyceride-rich lipoproteins and high density lipoproteins.

Stable isotope methodology was used to determine the kinetic behavior of apolipoprotein (apo) A-I within the triglyceride-rich lipoprotein (TRL) fraction and to compare TRL apoA-I kinetics with that of apoA-I in high density lipoprotein (HDL) and TRL apoB-48. Eight subjects (5 males and 3 females) over the age of 40 were placed on a baseline average American diet and after 6 weeks received a primed-constant infusion of [5,5,5-(2)H(3)]-l-leucine for 15 h while consuming small hourly meals of identical composition. HDL and TRL apoA-I and TRL apoB-48 tracer/tracee enrichment curves were obtained by gas chromatography;-mass spectrometry. Data were fitted to a compartmental model to determine the fractional secretion rates of apoA-I and apoB-48 within each lipoprotein fraction. Mean plasma apoA-I levels in TRL and HDL fractions were 0. 204 +/- 0.057 and 134 +/- 15 mg/dl, respectively. The mean fractional catabolic rate (FCR) of TRL apoA-I was 0.250 +/- 0.069 and HDL apoA-I was 0.239 +/- 0.054 pools/day, with mean estimated residence times (RT) of 4.27 and 4.37 days, respectively. The mean TRL apoB-48 FCR was 5.2 +/- 2.0 pools/day and the estimated mean RT was 5.1 +/- 1.8 h. Our results indicate that apoA-I is catabolized at a slower rate than apoB-48 within TRL, and that apoA-I within TRL and HDL fractions are catabolized at similar rates.     

Cholesteryl ester and apolipoprotein E transfer between human high density lipoproteins and chylomicrons

The transfer of cholesteryl esters and apolipoprotein E has been studied between plasma HDL and chylomicrons isolated either from ascitic fluid or from the plasma of a patient with type V hyperlipoproteinemia. Whereas apolipoprotein E transfer was rapid and occurred at low temperature, cholesteryl ester transfer was suppressed at 4 degrees C. Apolipoprotein E transfer did not depend upon the presence of cholesteryl ester transfer protein and was in fact inhibited by the partially purified preparation of this protein. Apolipoprotein E transfer was not increased by reduction with dithiothreitol. The transfer of cholesteryl esters increased sharply at a chylomicron to HDL ratio of cholesteryl ester above 1/10, a value which may be of physiological significance at the peak of postprandial lipemia. At this ratio, the transfer of apolipoprotein E was minimal and increased only at ratios above 2/1. From these results, it is concluded that there is no connection between apolipoprotein E and cholesteryl ester transfer from HDL to chylomicrons. It is, therefore, proposed that whereas chylomicron apolipoprotein E is acquired rapidly and mostly in the lymphatic system, the concentration of chylomicron cholesteryl esters increases significantly and independently in the circulation.     

The structure of apolipoprotein A-II in discoidal high density lipoproteins

It is well accepted that high levels of high density lipoproteins (HDL) reduce the risk of atherosclerosis in humans. Apolipoprotein A-I (apoA-I) and apoA-II are the first and second most common protein constituents of HDL. Unlike apoA-I, detailed structural models for apoA-II in HDL are not available. Here, we present a structural model of apoA-II in reconstituted HDL (rHDL) based on two well established experimental approaches: chemical cross-linking/mass spectrometry (MS) and internal reflection infrared spectroscopy. Homogeneous apoA-II rHDL were reacted with a cross-linking agent to link proximal lysine residues. Upon tryptic digestion, cross-linked peptides were identified by electrospray mass spectrometry. 14 cross-links were identified and confirmed by tandem mass spectrometry (MS/MS). Infrared spectroscopy indicated a beltlike molecular arrangement for apoA-II in which the protein helices wrap around the lipid bilayer rHDL disc. The cross-links were then evaluated on three potential belt arrangements. The data clearly refute a parallel model but support two antiparallel models, especially a "double hairpin" form. These models form the basis for understanding apoA-II structure in more complex HDL particles.     

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.     

Lipid transfer between human serum high density lipoproteins and egg yolk lipoproteins in incubation mixtures

Ultracentrifugally isolated human serum high density lipoproteins of d 1.063-1.21 (HDL) were incubated with egg yolk lipoproteins of d < 1.006 for up to 24 hr at various concentrations. Transfer of HDL cholesterol esters to egg yolk lipoproteins occurred simultaneously with transfer of glycerides from egg yolk lipoproteins to HDL. These observations show that exchange of lipids can take place between lipoproteins in the absence of other serum proteins and enzymes. The mole ratios of HDL cholesterol esters to glycerides approached an integral value of 1 : 1 during the course of the incubation. These results suggest that lipid components form complexes within the HDL structure.     

Immunochemical evidence for the presence in human plasma of lipoproteins with apolipoprotein A-II as the major protein constituent

Apolipoprotein-A-containing lipoproteins have been studied by means of crossed immunoelectrophoresis with intermediate gels. The experiments confirmed the presence in human plasma of lipoprotein particles with both apoA-I and apoA-II (LpA) and of those with apoA-I but no apoA-II (LpAI). Furthermore, they obtained evidence for the occurrence in human plasma of small amounts of lipoproteins containing apoA-II but not apoA-I, apoB, apoC-II, apoC-III or apoE.     

Apoproteins of human serum high density lipoproteins. Isolation and characterization of the peptides of Sephadex fraction V from normal subjects and patients with abeta-lipoproteinemia.

1. Sephadex fraction V, obtained from human serum high density lipoprotein apoprotein (HDL apoprotein) of normal subjects and of patients with abetalipoproteinemia, was resolved by DEAE-cellulose ion exchange column chromatography into several fractions which were defined in terms of amino acid composition, NH2- and COOH-terminsls, sialic acid content, immunologic and electrophoretic properties, and in vitro activation of purified lipoprotein lipase from rat adipose tissue. 2. Fraction V of HDL apoprotein of both normal and abetalipoproteinemic subjects was found to contain polypeptides corresponding to apolipoproteins C-I, C-II, C-III-1, and C-III-2, which had been described previously in very low-density lipoproteins (VLDL). The content of apo C-III-1 in abetalipoproteinemia-HDL was very low, whereas the percentage, by weight, of apo C-I was about twice as high as that in the normal subjects studied. Furthermore, both normal and abetalipoproteinemia-HDL apoprotein contained a previously unreported peptide which had a molecular weight of about 7 000 and electrophoretic, chemical, and immunological properties distinct from those of the known C apolipoproteins. Of all of the peptides comprising fraction V, only apo C-II activated a purified preparation of rat adipose tissue lipoprotein lipase. This was the case for both normal and abetalipoproteinemic subjects.