Stimulation of lecithin:cholesterol acyltransferase activity by apolipoprotein A-II in the presence of apolipoprotein A-I

Various combinations of incorporation and addition of apolipoprotein A-I (apo A-I) and apolipoprotein A-II (apo A-II) individually or together to a defined lecithin-cholesterol (250/12.5 molar ratio) liposome prepared by the cholate dialysis procedure were used to study the effect of apo A-II on lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) activity of both purified enzyme preparations and plasma. When apo A-I (0.1-3.0 nmol/assay) alone was incorporated or added to the liposome, apo A-I effectively activated the enzyme. By contrast, when apo A-II (0.1-3.0 nmol/assay) alone was incorporated into or added to the liposome, apo A-II exhibited minimal activation of LCAT activity, approximately 1% of the activity obtained by an equal amount of apo A-I. Addition of apo A-II (0.1-3.0 nmol/assay) together with apo A-I (0.8 nmol/assay) to the liposome reduced the LCAT activity to approximately 30% of the level obtained with addition of apo A-I alone. On the other hand, addition of apo A-II (0.1-3.0 nmol/assay) or addition of lecithin-cholesterol liposome containing apo A-II (0.1-3.0 nmol/assay) to lecithin-cholesterol liposome containing apo A-I (0.8 nmol/assay) did not significantly alter apo A-I activation of LCAT activity. However, when the same amounts (0.1-3.0 nmol/assay) of apo A-II were incorporated together with apo A-I (0.8 nmol/assay) into the liposome, apo A-II significantly stimulated LCAT activity as compared to activity obtained with incorporation of apo A-I alone. The maximal stimulation was obtained with 0.4 nmol apo A-II/assay for both purified and plasma enzyme. At this apo A-II concentration, approximately 4-fold and 1.8-fold stimulation was observed for purified enzyme and plasma enzyme, respectively. These results indicated that apo A-II must be incorporated together with apo A-I into lecithin-cholesterol liposomes to exert its stimulatory effect on LCAT activity and that apo A-II in high-density lipoprotein may play an important role in the regulation of LCAT activity.     

Plasma factors required for human apolipoprotein A-II dimerization

Although plasma high-density lipoproteins (HDL) have been implicated in several cardioprotective pathways, the physiologic role of apolipoprotein (apo) A-II, the second most abundant of the HDL proteins, remains ambiguous. Human apo A-II is distinguished from most other species by a single cysteine (Cys6), which forms a disulfide bond with other cysteine-containing apos. In human plasma, nearly all apo A-II occurs as disulfide-linked homodimers of 17.4 kDa. Although dimerization is an important determinant of human apo A-II metabolism, its mechanism and the plasma and/or cellular sites of its dimerization are not known. Using SDS-PAGE and densitometry we investigated the kinetics of apo A-II dimerization and observed a slow (t(1/2) = approximately 10 days), second-order process in Tris-buffered saline. In 3 M guanidine hydrochloride, which disrupts apo A-II secondary structure and self-association, the rate was 3-fold slower. In contrast, lipid surfaces that promote apo A-II alpha-helix formation and lipophilic interaction profoundly enhanced the rate. Reassembled HDL increased the second-order rate constant k(2) by 7500-fold, unilamellar 1-palmitoyl-2-oleoylphosphatidylcholine vesicles increased k(2) 850-fold, and physiological concentrations of human serum albumin increased k(2) 220-fold. Thus, while dimerization of apo A-II in aqueous buffer is too slow to account for the high fraction of dimer found in plasma, lipids and proteins "catalyze" dimer formation, a process that could occur either intracellularly prior to secretion or in the plasma compartment following secretion. These data suggest that formation of disulfide links within or between polypeptide chains can be controlled, in part, by coexisting lipids and proteins.     

Insulin-stimulating protein from human plasma. Chemical characteristics and biological activity

A protein that potentiates the action of insulin in vitro was purified from human plasma. When reduced with 2-mercaptoethanol and then carboxymethylated, it yielded a single subunit, indicating that it was composed of two identical subunits connected by a single disulfide bond. This modified subunit tended to inhibit rather than stimulate insulin activity. A distinctive feature of the amino acid composition of this protein (H-ISP) was the absence of histidine, arginine, and tryptophan. The molecular mass, subunit composition, the characteristic amino acid composition and the N-terminal amino acid residue of H-ISP are very similar to those of human plasma apolipoprotein A-II (apo A-II). The isoelectric point of H-ISP was estimated to be 4.91, which is identical with that of the major apo A-II isoform. H-ISP did not itself have insulin-like activity in increasing CO2 liberation from labeled glucose and 2-deoxyglucose uptake by isolated rat adipocytes, but it potentiated the action of insulin in these parameters. It had no appreciable affect on the binding or degradation of 125I-labeled insulin by adipocytes. Like H-ISP, apo A-II isolated from human plasma also had no insulin-like activity by itself, but stimulated the effect of insulin on CO2 production from labeled glucose in isolated rat adipocytes. From these results, it is concluded that H-ISP is identical with the major apo A-II isoform. Incubation of isolated adipocytes with H-ISP resulted in marked increase in the activity of pyruvate dehydrogenase in a dose-dependent manner in the absence of added insulin. H-ISP also stimulated pyruvate dehydrogenase activity in a subcellular system consisting of plasma membranes and mitochondria from rat adipocytes. The effect of H-ISP on pyruvate dehydrogenase activity could be produced by treatment of the isolated mitochondrial fraction alone.     

Identification of an apoC-II variant (apoC-IIBethesda) in a kindred with apoC-II deficiency and type I hyperlipoproteinemia

Apolipoprotein (apo) C-II deficiency is characterized by elevated plasma triglycerides, chylomicrons, and very low density lipoproteins, as well as reduced levels of low density and high density lipoproteins. A subject with apoC-II deficiency has been identified with an apoC-II plasma level of less than 0.05 mg/dl. The plasma apoC-II in the proband was immunochemically similar to apoC-II in normal subjects when analyzed by Ouchterlony immunodiffusion, however the apoC-II had an apparently lower molecular weight and higher pI when analyzed by two-dimensional gel electrophoresis. This apoC-II variant, designated apoC-IIBethesda, was not affected by neuraminidase treatment or reduction. Two-dimensional gel electrophoresis of the plasma of the mother of the proband revealed both normal apoC-II and apoC-IIBethesda, whereas analysis of the father and two siblings revealed apoC-II of normal electrophoretic mobility. These results were interpreted as indicating that the proband was a compound heterozygote with one allele for apoC-IIBethesda inherited from the mother and an allele coding for an abnormality which results in the virtual or complete absence of plasma apoC-II from the father. This proband represents the first example of a compound heterozygote for an apolipoprotein defect associated with a dyslipoproteinemia.     

Interaction between high-density lipoprotein subpopulations in apo B-free and abetalipoproteinemic plasma.

Two populations of high-density lipoprotein (HDL) particles exist in human plasma. Both contain apolipoprotein (apo) A-I, but only one contains apo A-II: Lp(AI w AII) and Lp(AI w/o AII). To study the extent of interaction between these particles, apo B-free plasma prepared by the selective removal of apo B-containing lipoproteins (LpB) from the plasma of three normolipidemic (NL) subjects and whole plasma from two patients with abetalipoproteinemia (ABL) were incubated at 37 degrees C for 24 h. Apo B-free plasma samples were used to avoid lipid-exchange between HDL and LpB. Lp(AI w AII) and Lp(AI w/o AII) were isolated from each apo B-free plasma sample before and after incubation and their protein and lipid contents quantified. Before incubation, ABL plasma had reduced levels of Lp(AI w AII) and Lp(AI w/o AII), (40% and 70% of normals, respectively). Compared to the HDL of apo B-free NL plasma, ABL HDL had higher relative contents of free cholesterol, phospholipid and total lipid, and contained more particles with apparent hydrated Stokes diameter in the 9.2-17.0 nm region. These differences were particularly pronounced in particles without apo A-II. Despite their differences, the total cholesterol contents of Lp(AI w AII) increased, while that of Lp(AI w/o AII) decreased in all five plasma samples and the amount of apo A-I in Lp(AI w AII) increased by 6-8 mg/dl in four during the incubation. These compositional changes were accompanied by a relative reduction of particles in the 7.0-8.2 nm Stokes diameter size region and an increase of particles in the 9.2-11.2 nm region. These data are consistent with intravascular modulation between HDL particles with and without apo A-II. The observed increase in apo A-II-associated cholesterol and apo A-I, could involve either the transfer of cholesterol and apo A-I from particles without apo A-II to those with A-II, or the transfer of apo A-II from Lp(AI w AII) to Lp(AI w/o AII). The exact mechanism and direction of the transfer remain to be determined.     

Apolipoproteins A-I,A-II and E are independently distributed among intracellular and newly secreted HDL of human hepatoma cells

Whereas hepatocytes secrete the major human plasma high density lipoproteins (HDL)-protein, apo A-I, as lipid-free and lipidated species, the biogenic itineraries of apo A-II and apo E are unknown. Human plasma and HepG2 cell-derived apo A-II and apo E occur as monomers, homodimers and heterodimers. Dimerization of apo A-II, which is more lipophilic than apo A-I, is catalyzed by lipid surfaces. Thus, we hypothesized that lipidation of intracellular and secreted apo A-II exceeds that of apo A-I, and once lipidated, apo A-II dimerizes. Fractionation of HepG2 cell lysate and media by size exclusion chromatography showed that intracellular apo A-II and apo E are fully lipidated and occur on nascent HDL and VLDL respectively, while only 45% of intracellular apo A-I is lipidated. Secreted apo A-II and apo E occur on small HDL and on LDL and large HDL respectively. HDL particles containing both apo A-II and apo A-I form only after secretion from both HepG2 and Huh7 hepatoma cells. Apo A-II dimerizes intracellularly while intracellular apo E is monomeric but after secretion associates with HDL and subsequently dimerizes. Thus, HDL apolipoproteins A-I, A-II and E have distinct intracellular and post-secretory pathways of hepatic lipidation and dimerization in the process of HDL formation. These early forms of HDL are expected to follow different apolipoprotein-specific pathways through plasma remodeling and reverse cholesterol transport.     

Interaction of apolipoprotein A-II with recombinant HDL containing egg phosphatidylcholine, unesterified cholesterol and apolipoprotein A-I

The preparation of discoidal, recombinant HDL (r-HDL) containing various phospholipids, apolipoproteins and a range of concentrations of unesterified cholesterol has been reported by several investigators. The present study describes the preparation of r-HDL containing both apolipoprotein (apo) A-I and apo A-II. r-HDL with 100:1 (mol:mol) egg PC.apo A-I and 0 (Series I), 5 (Series II) or 10 (Series III) mol% unesterified cholesterol were prepared by the cholate dialysis method. The resulting complexes had a Stokes' radius of 4.7 nm and contained two molecules of apo A-I per particle. When the r-HDL (2.0 mg apo A-I) were supplemented with 1.0 mg of apo A-II, one of the apo A-I molecules was replaced by two molecules of apo A-II. This modification was not accompanied by a loss of phospholipid, nor by major change in particle size. The addition of 2.5 or 4.0 mg of apo A-II resulted in the displacement of both apo A-I molecules from a proportion of the r-HDL and the formation of smaller particles (Stokes' radius 3.9 nm), which contained half the original number of egg PC molecules and three molecules of apo A-II. The amount of apo A-I displaced was dependent on the concentration of unesterified cholesterol in the r-HDL: when 2.5 mg of apo A-II was added to the Series I, II and III r-HDL, 44, 60 and 70%, respectively, of the apo A-I was displaced. Addition of 4.0 mg of apo A-II did not promote further displacement of apo A-I from any of the r-HDL. By contrast, the association of apo A-II with r-HDL was independent of the concentration of unesterified cholesterol and was a linear function of the amount of apo A-II which had been added. It is concluded that (1), the structural integrity of egg PC.unesterified cholesterol.apo A-I r-HDL, which contain two molecules of apo A-I, is not affected when one of the apo A-I molecules is replaced by two molecules of apo A-II; (2), when both apo A-I molecules are replaced by apo A-II, small particles which contain three molecules of apo A-II are formed; and (3), the displacement of apo A-I from r-HDL is facilitated by the presence of unesterified cholesterol in the particles.     

Apolipoprotein A-II/A-I ratio is a key determinant in vivo of HDL concentration and formation of pre-beta HDL containing apolipoprotein A-II

Overexpression of human apolipoprotein A-II (apo A-II) in mice induced postprandial hypertriglyceridemia and marked reduction in plasma HDL concentration and particle size [Boisfer et al. (1999) J. Biol. Chem. 274, 11564-11572]. We presently compared lipoprotein metabolism in three transgenic lines displaying plasma concentrations of human apo A-II ranging from normal to 4 times higher, under ad libitum feeding and after an overnight fast. Fasting dramatically decreased VLDL and lowered circulating human apo A-II in transgenic mice; conversely, plasma HDL levels increased in all genotypes. The apo A-I content of HDL was inversely related to the expression of human apo A-II, probably reflecting displacement of apo A-I by an excess of apo A-II. Thus, the molar ratios of apo A-II/A-I in HDL were significantly higher in fed as compared with fasted animals of the same transgenic line, while endogenous LCAT activity concomitantly decreased. The number and size of HDL particles decreased in direct proportion to the level of human apo A-II expression. Apo A-II was abundantly present in all HDL particles, in contrast to apo A-I mainly present in large ones. Two novel findings were the presence of pre-beta migrating HDL transporting only human apo A-II in the higher-expressing mice and the increase of plasma HDL concentrations by fasting in control and transgenic mice. These findings highlight the reciprocal modifications of VLDL and HDL induced by the feeding-fasting transition and the key role of the molar ratio of apo A-II/A-I as a determinant of HDL particle metabolism and pre-beta HDL formation.     

Coexistence of abnormalities of hepatic lipase and lipoprotein lipase in a large family.

A large family is reported with familial hepatic triglyceride lipase (HTGL) deficiency and with the coexistence of reduced lipoprotein lipase (LPL) similar to the heterozygote state of LPL deficiency. The proband was initially detected because of hypertriglyceridemia and chylomicronemia. He was later demonstrated to have beta-VLDL despite an apo E3/E3 phenotype and the lack of stigmata of type III hyperlipoproteinemia. The proband had no HTGL activity in postheparin plasma. Two of his half-sisters had very low HTGL activity (39 and 31 nmol free fatty acids/min/ml; normal adult female greater than 44). His son and daughters had decreased HTGL activity (normal male and preadolescent female greater than 102), which would be expected in obligate heterozygotes for HTGL deficiency. Low HTGL activity was associated with LDL particles which were larger and more buoyant. Several family members, including the proband, had reduced LPL activity and mass less than that circumscribed by the 95% confidence-interval ellipse for normal subjects and had hyperlipidemia similar to that described in heterozygote relatives of patients with LPL deficiency. All the sibs with hyperlipidemia had a reduced LPL activity and mass, while subjects with isolated reduced HTGL (with normal LPL activity) had normal lipid phenotypes. Analysis of genomic DNA from these subjects by restriction-enzyme digestion revealed no major abnormalities in the structure of either the HTGL or the LPL gene. Compound heterozygotes for HTGL and LPL deficiency show lipoprotein physiological characteristics typical for HTGL deficiency, while their variable lipid phenotype is typical for LPL deficiency.     

Isolation of apolipoproteins A-I, A-II and E and their estimation by rocket immunoelectrophoresis in blood plasma of dis-alpha-lipoproteinemic patients

The methods for isolation of pure apolipoproteins A-I, A-II and E from the blood plasma of donors for preparation of monospecific rabbit antisera against these apolipoproteins and their estimation in human blood plasma using immunoelectrophoresis are described. It was found that the average content of apolipoprotein A-I (apo A-I) in the blood plasma of healthy males is 126.6 mg%, that of apolipoprotein A-II (apo A-II) is 56.8 mg%, that of apolipoprotein E (apo E) is 10.2 mg%. The apo A-I content in blood plasma is increased in hyper-alpha-lipoproteinemic patients and is decreased in hypo-alpha-lipoproteinemic ones, i. e. there is a direct relationship between the changes in concentration of high density lipoproteins (HDL) and apo A-I. The concentration of apo A-II in dis-alpha-lipoproteinemias varies within a narrow range. A considerable increase of the alpha-cholesterol/apo A-I ratio suggesting an increased capacity of HDL to transport cholesterol in hyper-alpha-lipoproteinemic patients is observed. There exists an indirect correlation between the changes in the contents of apo A-I and apo E in dis-alpha-lipoproteinemic patients.     

On the heritability of serum high density lipoprotein in twins.

To estimate the relative contributions of hereditary vs. environmental factors in the variation of high density lipoprotein, we measured the concentrations of its main apoprotein components, apoprotein A-I (apo A-I) and apoprotein A-II (apo A-II), in serum samples from 65 monozygotic (MZ) and 70 dizygotic (DZ) like-sexed twin pairs. Evidence for a genetic component of variance was found for apo A-II, giving heritability (h2) estimates of .35 and .30 for males and females, respectively. No genetic contribution to the variance of apo A-I could be demonstrated. Additionally, males had lower concentrations of apo A-I, but higher of apo A-II, than females.     

A mutation in the human apolipoprotein A-I gene. Dominant effect on the level and characteristics of plasma high density lipoproteins

Epidemiologic and genetic data suggest an inverse relationship between plasma high density lipoprotein (HDL) cholesterol and the incidence of premature coronary artery disease. Some of the defects leading to low levels of HDL may be a consequence of mutations in the genes coding for HDL apolipoproteins A-I and A-II or for enzymes that modify these particles. A proband with plasma apoA-I and HDL cholesterol that are below 15% of normal levels and with marked bilateral arcus senilis was shown to be heterozygous for a 45-base pair deletion in exon four of the apoA-I gene. This most likely represents a de novo mutation since neither parent carries the mutant allele. The protein product of this allele is predicted to be missing 15 (Glu146-Arg160) of the 22 amino acids comprising the third amphipathic helical domain. The HDL of the proband and his family were studied. Using anti-A-I and anti-A-II immunosorbents we found three populations of HDL particles in the proband. One contained both apoA-I and A-II, Lp(A-I w A-II); one contained apoA-I but no A-II, Lp(A-I w/o A-II); and the third (an unusual one) contained apoA-II but no A-I. Only Lp(A-I w A-II) and (A-I w/o A-II) were present in the plasma of the proband's parents and brother. Analysis of the HDL particles of the proband by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two protein bands with a molecular mass differing by 6% in the vicinity of 28 kDa whereas the HDL particles of the family members exhibited only a single apoA-I band. The largely dominant effect of this mutant allele (designated apoA-ISeattle) on HDL levels suggests that HDL particles containing any number of mutant apoA-I polypeptides are catabolized rapidly.     

MspI polymorphism of the apolipoprotein A-II gene, serum lipids and apolipoproteins in Chinese from Singapore.

The effect of a DNA polymorphism (MspI) of the apolipoprotein (apo) A-II gene on serum lipid and apo levels was studied in a group of 125 healthy Chinese of both sexes. The frequency of the 3.7-kb rarer allele (M2) was found to be significantly higher in the Chinese (0.30) than in Caucasians (0.16; p < 0.025). The distribution of apo A-II genotypes was in Hardy-Weinberg equilibrium in the Chinese population. The presence of a polymorphic site (MspI) within an Alu sequence at the 3' end of the gene, the 3.0 kb (M1) allele, was associated with significantly higher levels of serum apo A-I and A-II (p < 0.05 and < 0.01, respectively). Serum high-density Lipoprotein cholesterol levels were also correspondingly higher in individuals with M1, but did not reach a significant level. Male heterozygotes of the apo A-II polymorphism had significantly higher levels of serum triglycerides compared to homozygotes (p < 0.05). Thus the MspI polymorphism of the apo A-II gene appears to be associated with altered levels of lipids and apos in the Chinese population.     

Spectroscopic studies of the tyrosine residues of human plasma apolipoprotein A-II

Human apolipoprotein A-II (apo A-II) in solution and associated with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was investigated by a combination of absorbance and fluorescence methods. Each apo A-II polypeptide chain contains four tyrosine residues but no tryptophan residues. Two and three tyrosine residues, respectively, appear to be buried for apo A-II in aqueous solution and in the lipid-associated protein. The spectroscopic properties of the tyrosine residues of lipid-associated apo A-II were also investigated. Plots of fluorescence intensity against temperature revealed a discontinuity in the region of the phase transition; however, over the same temperature range, there was no change in the exposure of tyrosine residues to the aqueous environment or in their mobility as measured by fluorescence polarization. Near-ultraviolet circular dichroic measurements demonstrated that the environments of the tyrosine residues of lipid-associated apo A-II and nitrated apo A-II were different from that of the apo A-II in solution or in a denatured state. Similar measurements also revealed that the microenvironments around tyrosines of apo A-II bound to DMPC in the gel phase are different from those observed in the liquid crystalline phase. Using environmentally sensitive fluorescence lipid probes, we have previously demonstrated that the polarity of the lipid/water interface of DMPC changes through a phase transition. The observations presented here indicate that these environmental changes also occur at the lipid/protein interface.     

Rapid chromatographic purification of apolipoproteins A-I and A-II from human plasma

Rapid, large-scale isolation of human apolipoproteins A-I and A-II has been accomplished using two chromatographic procedures. The apolipoproteins adsorbed from plasma onto a column of phenyl-Sepharose are eluted with increasing propylene glycol concentrations. Apolipoproteins A-I and A-II can be resolved by elution with a linear 0 to 80% propylene glycol gradient. Homogeneous preparations of apo A-I and A-II are obtained following gel filtration in 3M guanidinium chloride.     

Characterization of human high-density lipoprotein subclasses LP A-I and LP A-I/A-II and binding to HepG2 cells

Plasma HDL can be classified according to their apolipoprotein content into at least two types of lipoprotein particles: lipoproteins containing both apo A-I and apo A-II (LP A-I/A-II) and lipoproteins with apo A-I but without apo A-II (LP A-I). LP A-I and LP A-I/A-II were isolated by immuno-affinity chromatography. LP A-I has a higher cholesterol content and less protein compared to LP A-I/A-II. The average particle mass of LP A-I is higher (379 kDa) than the average particle weight of LP A-I/A-II (269 kDa). The binding of 125I-LP A-I to HepG2 cells at 4 degrees C, as well as the uptake of [3H]cholesteryl ether-labelled LP A-I by HepG2 cells at 37 degrees C, was significantly higher than the binding and uptake of LP A-I/A-II. It is likely that both binding and uptake are mediated by apo A-I. Our results do not provide evidence in favor of a specific role for apo A-II in the binding and uptake of HDL by HepG2 cells.     

Self-association of human apolipoproteins A-I and A-II and interactions of apolipoprotein A-I with bile salts: quasi-elastic light scattering studies

We employed quasi-elastic light scattering (QLS) to systematically study the aqueous self-association of human apolipoproteins A-I and A-II (apo A-I and apo A-II) and the interactions of apo A-I with common taurine-conjugated bile salts. Self-association of apo A-I was promoted by increases in apolipoprotein concentration (0.09-2.2 mg/mL) and ionic strength (0.15-2.0 M NaCl), inhibited by increases in temperature (5-50 degrees C) and guanidine hydrochloride concentration (0-2.0 M), and unaffected by hydrostatic pressures up to 500 atm. The mean hydrodynamic radius (Rh) of apo A-I micelles ranged from 38 A to a maximum asymptotic value of 68 A. We examined several possible models of apo A-I self-association; the model that best fitted the Rh values assumed that apo A-I monomers first interacted at low concentrations to form dimers, which then further associated to form ring-shaped limiting octamers. Comparison of the temperature-dependent and ionic strength dependent free energy changes for the formation of octamers from apo A-I dimers suggested that hydrophobic forces strongly favored self-association and that electrostatic repulsive forces were only weakly counteractive. Apo A-II self-association was also promoted by increases in apolipoprotein concentration (0.2-1.8 mg/mL) and inhibited by increases in guanidine hydrochloride concentration (0-1.0 M) but was unaffected by variations in temperature (10-37 degrees C): the largest Rh values observed were consistent with limiting tetramers. As demonstrated by equilibrium dialysis, bile salts in concentrations below their critical micellar concentrations (cmc) bound to apo A-I micelles but had no effect upon apo A-I self-association, as inferred from constant Rh values. When bile salt concentrations exceeded their aqueous cmc values, a dissociation of apo A-I micelles resulted with the formation of mixed bile salt/apo A-I micelles. These studies support the concepts that apo A-I and apo A-II form small dimeric micelles at low concentrations that grow sharply to reach limiting sizes over a narrow concentration range. The influences of bile salt concentration and species upon these micelles have relevance to the plasma transport of bile salts in high-density lipoproteins and to the physical-chemical state of apo A-I and apo A-II molecules in native biles.     

Characterization and measurement of human apolipoprotein A-II by radioimmunoassay

The development of a radioimmunoassay for apolipoprotein A-II (apo A-II) is described. Initial studies revealed a lack of immunological identity between purified apo A-II used as the standard and serum or HDL. Extensive testing of different buffers, standards, antisera, tracers, utilization of a detergent, and heating of sera failed to resolve the problem. Gel filtration of iodinated and non-iodinated apo A-II on Sephadex G-100 columns showed that apo A-II, in dilute solution, elutes in a higher molecular zone than expected with a broad, assymetrical profile. The use of a subfraction of the tracer in the assay resulted in parallelism in the serum and standard dilution curves. The apo A-II assay was sensitive, specific, and reproducible. Apo A-II added to sera was fully recovered and delipidation did not affect the immunoreactivity of either serum or HDL. Apo A-II contributed approximately 20% to the protein mass of HDL. Comparison of these results with those obtained by radial immunodiffusion, and with previously reported data, indicates that the reactivity of apo A-II in its native and delipidated forms may be markedly influenced by different immunologic methodologies and their specific reagents. Caution should thus be shown at present in assigning absolute concentrations to apo A-II in serum or HDL.     

Molecular cloning and nucleotide sequence of cDNA for murine senile amyloid protein: nucleotide substitutions found in apolipoprotein A-II cDNA of senescence accelerated mouse (SAM).

cDNA clones encoding the murine senile amyloid protein (ASSAM) have been isolated from animal models of accelerated senescence (SAM-P/1) and from normal aging (SAM-R/1). Immunochemical and protein sequence studies revealed that apolipoprotein (apo) A-II is a serum precursor of ASSAM. A 17-base synthetic oligonucleotide based on residues 39-44 of ASSAM was used as a hybridization probe for screening newly constructed SAM-P/1 and SAM-R/1 liver cDNA libraries. The structure of murine apo A-II cDNA is of interest because of the amino acid substitution found in ASSAM and serum apo A-II of SAM-P; in SAM-R or other random bred slc:ICR mice, amino acid residue 5 of mature apo A-II is proline but, in SAM-P, this amino acid is changed to glutamine. This amino acid replacement is caused by two nucleotide substitutions (CCA for proline codon to CAG for glutamine codon). The third base mutation may not be relevant to the substitution of amino acid. Attention is directed to the relation of this amino acid substitution to the specific deposition of apo A-II, as a tissue amyloid fibril.     

Apo A-II participates in HDL–liposome interaction by the formation of new pre-β mobility particles and the modification of liposomes

Interaction between high density lipoproteins (HDL) and liposomes results in both a structural modification of HDL and the generation of new pre-β HDL-like particles. Here, phosphatidylcholine liposomes and human HDL were incubated at liposomal phospholipid/HDL phospholipid (L-PL/HDL-PL) ratios of 1:1, 3:1 and 5:1 with a subsequent assessment of the distribution of apolipoprotein (apo) A-I, apo A-II, free cholesterol (FC) and PL between newly generated pre-β mobility lipoproteins and non-disrupted liposomes. Both at L-PL/HDL-PL ratios of 3:1 and 5:1 the fraction of liposomal-derived PL associated with pre-β fraction was significantly higher than those accepted by α-HDL. We found that 78% of apo A-I released from HDL was incorporated into pre-β mobility fraction. The relative contents of PL and apo A-I in pre-β fraction were constant irrespective of the initial L-PL/HDL-PL ratio in the incubation mixture and accounted for approximately 83 and 11%, respectively. Apo A-II was detached from HDL to a similar extent as apo A-I and distributed evenly between pre-β fraction and non-disrupted liposomes. Apo A-II constituted approximately 1%, by weight, in these fractions at all L-PL/HDL-PL ratios investigated. It corresponded approximately to 10% of pre-β fraction protein mass. Both liposomes and pre-β fraction accepted comparable amounts of FC released from HDL. This data indicated that during the interaction between human HDL and phosphatidylcholine liposome apo A-II participates both in structural modification of liposomes and in the generation of pre-β mobility fraction of constant content of PL, apo A-I and apo A-II. Involvement of apo A-II in HDL–liposome interaction may influence the anti-atherogenic properties of liposomes.