The single proline-glutamine substitution at position 5 enhances the potency of amyloid fibril formation of murine apo A-II

The primary structure of murine apolipoprotein A-II (apo A-II) has been determined. Apo A-II consists of a single polypeptide chain of 78 amino acid residues, of which the amino-terminus is pyrrolidone carboxylic acid. Except for residues 5 and 38, the amino acid sequence is identical to that of murine senile amyloid protein (ASSAM), which has a common antigenicity with apo A-II. Substitution of glutamine (ASSAM) for proline (apo A-II) at position 5 is distinct and may possibly be related to murine senile amyloid-ogenesis.     

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.     

Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1

We used a fluorometric method to examine amyloid fibrils, in vitro. These fibrils in the case of both murine senile and secondary amyloidosis were purified to apparent homogeneity from the water-suspended fraction of the liver of senescence-accelerated mouse, using sucrose density ultracentrifugation, and then the following assays were performed. In the absence of amyloid fibrils, thioflavine T fluoresced faintly at the excitation and emission maxima of 350 and 438 nm, respectively. In the presence of amyloid fibrils, thioflavine T fluoresced brightly at the excitation and emission maxima of 450 and 482 nm, respectively, and the fluorescence change was linear from 0 to 2.0 micrograms/ml amyloid fibrils. This fluorescence was maximal around pH 9.0. Fluorescence intensity in the presence of a constant amount of amyloid fibrils reached a plateau with increase in the thioflavine T concentration. Normal high density lipoproteins which contain apo A-II, the precursor of amyloid fibrils in murine senile amyloidosis, and acute phase high density lipoproteins which contain serum amyloid protein A, the precursor of amyloid fibrils in secondary amyloidosis, showed little fluorescence. The fluorescence was considerably diminished when structure of the amyloid fibrils was disrupted by guanidine-HCl treatment. This method will be useful for the determination of amyloid fibrils in vitro.     

Lipoproteomics II: mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry

High-density lipoprotein (HDL) is the most abundant lipoprotein particle in the plasma and a negative risk factor of atherosclerosis. By using a proteomic approach it is possible to obtain detailed information about its protein content and protein modifications that may give new information about the physiological roles of HDL. In this study the two subfractions; HDL(2) and HDL(3), were isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Identified proteins in HDL were: the dominating apo A-I as six isoforms, four of them with a glycosylation pattern and one of them with retained propeptide, apolipoprotein (apo) A-II, apo A-IV, apo C-I, apo C-II, apo C-III (two isoforms), apo E (five isoforms), the recently discovered apo M (two isoforms), serum amyloid A (two isoforms) and serum amyloid A-IV (six isoforms). Furthermore, alpha-1-antitrypsin was identified in HDL for the first time. Additionally, salivary alpha-amylase was identified as two isoforms in HDL(2), and apo L and a glycosylated apo A-II were identified in HDL(3). Besides confirming the presence of different apolipoproteins, this study indicates new patterns of glycosylated apo A-I and apo A-II. Furthermore, the study reveals new proteins in HDL; alpha-1-antitrypsin and salivary alpha-amylase. Further investigations about these proteins may give new insight into the functional role of HDL in coronary artery diseases.     

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.     

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.     

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.     

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.     

Kinetics of tryptic hydrolysis as a probe of the structure of human plasma apolipoprotein A-II

As a model system to understand apolipoprotein structure-function and their relationships to proteolytic events, the kinetics of tryptic hydrolysis of apolipoprotein A-II (apo A-II) was investigated in solution and in association with phospholipid. The rates of appearance and identities of specific peptides were determined by reversed-phase high-performance liquid chromatography and amino acid analysis, respectively. For the kinetics of hydrolysis of apo A-II in solution, the carboxyl-terminal peptides of residues 55-77 and 56-77 appeared first, followed by peptides of residues 4-23, 29-39, 40-44 and 45-54, which appeared at nearly identical rates. The kinetics of hydrolysis of apo A-II associated with 1,2-dimyristoyl-sn-glycero-3-phosphocholine showed several differences. First, a 100-fold larger amount of trypsin was needed to obtain a similar rate of product formation; second, a new peptide appeared, eluting earlier than apo A-II but having a similar amino acid composition; and third, the relative rates of appearance of peptides were different. The secondary structure surrounding the bonds susceptible to trypsin cleavage was determined by several predictive algorithms. The lysine amino acid bonds were found to be in regions defined by a high helical amphipathic moment. The reduced susceptibility to tryptic hydrolysis of apo-II associated with phospholipid appears to be due to a higher free energy of stabilization of protein secondary structure. As a consequence, the lysine amino acid bonds are in folded regions of the protein where they are conformationally inaccessible to enzymatic hydrolysis. By use of structure-prediction methods, it is possible to designate which regions of apolipoproteins may be important in proteolysis.     

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.     

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.     

Mass spectral analysis of pig (Sus scrofa) apo HDL: Identification of pig apoA-II, a dimeric apolipoprotein

Comparative studies of mammalian high density lipoproteins have clearly indicated that the major apolipoprotein is apoA-I and in some mammals apoA-II is the second major apolipoprotein. However, in pigs, apoA-II has been considered to be either present in trace amounts or absent. Recently, cDNA sequences for pigs A-II have been entered into the database. Translation of these sequences revealed that pig A-II consisted of 77 amino acids and that a cysteine residue was at residue 6. The A-II of three other mammals, chimpanzees, horses and humans, also has a cysteine residue at this position. As a result of a disulfide bond formed between monomers, the A-II in each of these cases circulates as a homodimer. Using electrospray-ionization mass spectrometry (ESI-MS), we obtained molecular mass data demonstrating that dimeric apoA-II is also present in pig plasma. In addition to being the first to report on the presence of apoA-II in pig plasma, we also obtained values for the molecular masses of apoA-I, apoC-III, apoD and serum amyloid A protein.     

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.     

Induction of protein conformational change in mouse senile amyloidosis

Aggregated amyloid fibrils can induce further polymerization of precursor proteins in vitro, thus providing a possible basis for propagation or transmission in the pathogenesis of amyloidoses. Previously, we postulated that the transmission of amyloid fibrils induces conformational changes of endogenous amyloid protein in mouse senile amyloidosis (Xing, Y., Nakamura, A., Chiba, T., Kogishi, K., Matsushita, T., Fu, L., Guo Z., Hosokawa, M., Mori, M., and Higuchi, K. (2001) Lab. Invest. 81, 493-499). To further characterize this transmissibility, we injected amyloid fibrils (AApoAII(C)) of amyloidogenic C type apolipoprotein A-II (APOAIIC) intravenously into 2-month-old SAMR1 mice, which have B type apolipoprotein A-II (APOAIIB), and develop few if any amyloid deposits spontaneously. 10 months after amyloid injection, deposits were detected in the tongue, stomach, intestine, lungs, heart, liver, and kidneys. The intensity of deposition increased thereafter, whereas no amyloid was detected in distilled water-injected SAMR1 mice, even after 20 months. The deposited amyloid was composed of endogenous APOAIIB with a different amyloid fibril conformation. The injection of these amyloid fibrils of APOAIIB (AApoAII(B)) induced earlier and more severe amyloidosis in SAMR1 mice than the injection of AApoAII(C) amyloid fibrils. Thus, AApoAII(C) from amyloidogenic mice could induce a conformational change of less amyloidogenic APOAIIB to a different amyloid fibril structure, which could also induce amyloidosis in the less amyloidogenic strain. These results provide important insights into the pathogenesis of amyloid diseases.     

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.     

Brain Expression of Apolipoproteins E, J, and A-I in Alzheimer's Disease

Abstract: Inheritance of the ε4 allele of apolipoprotein (apo) E is associated with increased risk of Alzheimer's disease (AD) and with increased β-amyloid peptide (Aβ) deposition in the cortex. Apo E is a member of a family of exchangeable apos, characterized by the presence of amphipathic α-helical segments that allow these molecules to act as surfactants on the surface of lipoprotein particles. Two members of this family, apo E and apo J, have been shown to bind soluble Aβ, and both are associated with senile plaques in the AD cortex. We now have studied the pattern of brain apo expression and found that five members of this class are present: apo A-I, A-IV, D, E, and J. By contrast, apos A-II, B, and C-II were not detectable. Immunohistochemistry revealed that, in addition to apo E and apo J, apo A-I immunostained occasional senile plaques in AD cortex. Immunoblot analysis showed no difference in the relative amounts of any of these apos in tissue homogenates of frontal lobe from AD or control patients. Comparison by APO E genotype showed no differences in the amount of apo E in brain among APO E ε3/3, ε3/4, or ε4/4 individuals; however, a significant decrease in the amount of apo J was associated with the APO E ε4 allele. No differences in apo J levels were detected in CSF samples of AD subjects. We propose that several members of the exchangeable apo family may interact with Aβ deposits in senile plaques through common amphipathic α-helical domains. Competition among these molecules for binding of Aβ or Aβ aggregates may influence the deposition of Aβ in senile plaques.     

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.     

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.     

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.