©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Contrasting in Vivo Effects of Murine and Human Apolipoprotein A-II
ROLE OF MONOMER VERSUS DIMER (*)

(Received for publication, January 2, 1996)

Elaine L. Gong Lori J. Stoltzfus Catherine M. Brion Deepa Murugesh Edward M. Rubin (§)

From the Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The role of apolipoprotein A-II (apoA-II) in high density lipoprotein (HDL) structure and metabolism has been studied previously in transgenic mice overexpressing either human or murine apoA-II. These studies have shown differences between these two groups of transgenic animals in the levels of very low density, low density, and high density lipoproteins, in the HDL particle size distribution, and in the relationship between apoA-II levels and lipoprotein levels. To determine whether these differences are due to the fact that human apoA-II is dimeric and murine apoA-II monomeric, we have examined the effects of monomeric human apoA-II (hA-II) in transgenic mice. Site-directed mutagenesis (Cys^6 Ser) was used to generate 15 transgenic founder lines of hA-II mice, that contained plasma hA-II concentrations over a 10-fold range (11 mg/dl to 185 mg/dl). The hA-II floated in the d leq 1.21 g/ml fraction and migrated as an apoA-II monomer by nonreducing SDS-polyacrylamide gel electrophoresis. HDL levels were not correlated with hA-II levels (r = -0.26); HDL particle size and size distribution, as well as very low density and low density lipoprotein levels and sizes, were unchanged compared to nontransgenic control mice. These results suggest that differences between mice overexpressing human dimeric apoA-II and those overexpressing murine apoA-II are the result of sequence differences between these two apoA-II molecules and are not solely due to the fact that human apoA-II exists as a dimer.


INTRODUCTION

High density lipoproteins (HDL) (^1)are macromolecules composed of approximately equal amounts of protein and lipid. HDL contains two major protein constituents: apolipoprotein A-I (apoA-I), comprising 70% of HDL protein, and apolipoprotein A-II (apoA-II), comprising 20% of HDL protein. The remaining 10% of HDL protein includes apolipoproteins E and A-IV (apoE, apoA-IV). HDL particles contain either apoA-I (Lp(AI)) or apoA-I and apoA-II (Lp(AI + AII)) as the major protein constituents(1) . These two major classes of HDL have been studied extensively in humans and have been shown to differ in both size and lipid composition. Numerous studies have also investigated the biological properties of these two forms of HDL. Human clinical studies have tested for an association between protein composition of HDL and atherosclerosis susceptibility, and in vitro studies have examined the ability of Lp(AI) versus Lp(AI + AII) particles to promote cholesterol efflux from tissue culture cells. Conflicting results from these studies have spurred interest in the defined in vivo system provided by the mouse and its ability to be genetically manipulated to address issues concerning properties of lipoproteins Lp(AI) and Lp(AI + AII).

Overexpression of human and murine apoA-II in transgenic mice has revealed significant differences between the effects of apoA-II from these two species. A high concentration of human apoA-II in mice has no significant effect on either total or HDL-cholesterol plasma levels and no effect on the particle size of the major murine HDL species(2) . Human apoA-II overexpression does result, however, in the appearance of a small 8 nm HDL particle containing human apoA-II, the concentration of which correlated positively with the human apoA-II concentration(2, 3) . By contrast, overexpression of mouse apoA-II results in an increase in both total and HDL-cholesterol levels and an increase in HDL particle size(4, 5) . In addition, human apoA-II transgenic mice develop smaller aortic atherogenic lesions than their nontransgenic littermates when fed an atherogenic high fat diet. This differs from murine apoA-II transgenic mice which develop larger aortic lesions than the nontransgenic control mice on both atherogenic and low fat (chow) diets(4) .

These marked differences in the effects of human and murine apoA-II on plasma lipoproteins and atherogenicity in mice is not surprising in light of the significant differences in apoA-II from these two species. The primary amino acid sequences of apoA-II from the two organisms differ by approximately 40%(6, 7) . In addition, human apoA-II exists as a dimer in plasma, while murine apoA-II exists exclusively as a monomer. No naturally occurring mutation of apoA-II has been found that results in monomeric human apoA-II. Thus, it has been difficult to determine whether the differences in the in vivo effects of human and murine apoA-II are due to the fact that one exists as a dimer and one as a monomer or are due to sequence differences between these two molecules.

In order to investigate the effects of dimeric and monomeric human apoA-II in vivo, we have altered, by site-specific mutagenesis, the codon for the single cystine present in mature human apoA-II. Multiple lines of transgenic mice have been engineered that express this altered gene at various levels. The effects of high level expression of monomeric human apoA-II (hA-II) on plasma lipoproteins were examined.


EXPERIMENTAL PROCEDURES

Generation of Transgenic Mice

The 1.3-kilobase PstI fragment within human apoA-II was subcloned into the PstI site of M13mp18. Site-directed mutagenesis was performed (Mutagene Kit, Bio-Rad) using an oligonucleotide complementary to sequences surrounding the Cys codon (5` AGG CTC TCC ACA GAT GGC TCC TTT G). The presence of the desired Gly Cys mutation (resulting in Cys Ser) was verified by sequence analysis. The mutated PstI fragment was subcloned back into its location in the original human apoA-II-containing plasmid. The 3-kilobase HindIII fragment containing the mutated apoA-II construct was isolated and purified by GeneClean (Bio101) and microinjected into fertilized eggs of the inbred mouse strain FVB as described(8) . Mice containing the transgene were identified by polymerase chain reaction amplification of tail DNA using human apoA-II-specific primers (5` TGA CTC TAT TCC TAC CTA GGG GC and 5` GTT CCA AGT TCC ACG AAA TAG within intron 3 and exon 4, respectively).

SDS-PAGE of Plasma Lipoproteins and Immunoblotting

SDS-PAGE (10-20% Tris/Tricine polyacrylamide gels, EP Mini-Electrophoresis System, Schleicher & Schuell) was performed according to the manufacturer's instructions on total plasma lipoproteins isolated from human apoA-II transgenic, hA-II transgenic, and nontransgenic FVB mice and from human controls. Samples were mixed with 2.5% beta-mercaptoethanol for reducing conditions. Proteins were transferred electrophoretically to nitrocellulose (EP Mini-Blotter, 200-mA constant current for 1 h) according to manufacturer's instructions and immunoblotted with goat antiserum to human apoA-II (International Immunology Corp., Murrieta, CA). The blots were developed using rabbit anti-goat serum conjugated with alkaline phosphatase and detection reagents (Bio-Rad) according to supplier's instructions. Goat anti-human apoA-II did not cross-react with normal mouse apoA-II. Isolated human HDL was prepared as described previously (9) ; purified human apoA-II was obtained from Sigma.

SDS-PAGE (4-20% Tris/glycine polyacrylamide gradient gels, Schleicher & Schuell) was also performed on 10-µl aliquots of FPLC fractions (see below) of total mouse plasma. Immunoblot analysis using rabbit anti-mouse apoE (kindly provided by Dr. Brian Ishida) followed by development with goat anti-rabbit serum conjugated with alkaline phosphatase (Vecta Stain Inc.) was carried out.

Nondenaturing Gradient Gel Electrophoresis of Plasma Lipoproteins

Lipoprotein particle size distribution was determined by nondenaturing gradient gel electrophoresis of the d leq 1.21 g/ml ultracentrifugal fraction from plasma(9) . Polyacrylamide gradient gels (2-10% and 4-30%(10) ) were stained with Coomassie G-250 following electrophoresis. Protein standards (HMW Calibration Kit, Pharmacia Biotech Inc.) and computer-assisted scanning densitometry were used to determine lipoprotein particle sizes as described previously(9) .

Plasma Lipid, Lipoprotein, and Apolipoprotein Measurements

Mice were fasted overnight prior to blood collection; plasma was separated and stored at 4 °C prior to analysis. Total plasma cholesterol and HDL-cholesterol were measured enzymatically (Boehringer Mannheim, High Performance Cholesterol Kit). Non-HDL cholesterol (representing very low density lipoprotein (VLDL)-cholesterol plus low density lipoprotein (LDL)-cholesterol) was calculated by subtracting HDL-cholesterol from the total cholesterol concentration. Human apoA-II, mouse apoA-II, and mouse apoA-I plasma concentrations were measured by radial immunodiffusion (RID) assay using goat anti-human apoA-II and rabbit anti-mouse apoA-II and rabbit anti-mouse apoA-I, respectively(11) . Purified human or murine apoA-II or murine apoA-I was included on appropriate plates as calibration standard. RID immunoplates were stained with Coomassie Blue R-250(11) . No cross-reactivity was observed between human and murine apoA-II.

FPLC Analysis of Plasma Lipoproteins

Plasma (50 µl) was chromatographed with the SMART-FPLC system (Pharmacia Biotech Inc.) equipped with a Superose 6 PC 3.2/30 column, using Tris buffer (20 mM Tris, 0.27 mM EDTA, 150 mM NaCl, pH 8). Fractions of 50 µl were collected at a rate of 40 µl/min. Elution profiles of total protein (represented by absorbance at 280 nm) were generated; profiles from duplicate runs appeared identical. Lipid and apolipoprotein measurements were made as described above.


RESULTS AND DISCUSSION

Transgenic mice containing the hA-II gene were generated by injection of fertilized eggs with a 3-kilobase genomic HindIII fragment containing the mutated (Cys Ser) human apoA-II gene. Fifteen lines of transgenic mice, designated hA-II, were identified by polymerase chain reaction analysis.

Expression of monomeric human apoA-II in these transgenic mice was confirmed by SDS-PAGE followed by immunoblotting with human apoA-II antibody on nonreduced and reduced plasma lipoprotein samples from hA-II transgenic mice (Fig. 1). Human apoA-II has been shown previously to exist primarily as a homodimer in human apoA-II transgenic mice(3) , and murine apoA-II exists as a monomer in the mouse(12) . The immunoblot of the nonreducing SDS-PAGE gel (Fig. 1A) shows that human apoA-II from plasma of hA-II transgenic mice was present exclusively at the position of monomeric apoA-II (8.6 kDa). In contrast, the immunoblot shows that human apoA-II from the plasma of normal (dimeric) human apoA-II transgenic mice is present at the position of dimeric apoA-II (17.2 kDa). The immunoblot of the reduced gel (Fig. 1B) shows the reduced human apoA-II migrating to the same position as the hA-II, thus confirming the presence of monomeric human apoA-II in these transgenic mice. In order to determine how much of the hA-II was lipoprotein-bound, and thus seen in these SDS-PAGE gels, we subjected plasma from hA-II transgenic mice and human dimeric apoA-II transgenic mice to preparative ultracentrifugation at d = 1.21 g/ml (42.2 Ti rotor, 195,000 times g, 18 h, 10 °C). By RID analysis, >90% of the human apoA-II (either monomeric or dimeric) was detected at the top of the centrifuge tube (d = 1.21 g/ml), with the remainder detected at the bottom of the tube (data not shown). Thus, most of the hA-II is lipoprotein-bound.


Figure 1: Immunoblots of plasma lipoproteins from hA-II transgenic mice and nontransgenic FVB control. Plasma lipoproteins (d leq 1.21 g/ml) were electrophoresed under nonreducing (A) and reducing (B) conditions as described under ``Experimental Procedures.'' Proteins were transferred to nitrocellulose for immunodetection using goat anti-human apoA-II antibody. A (nonreduced), lane 1, isolated human apoA-II; lane 2, isolated human HDL; lane 3, human apoA-II transgenic mouse; lanes 4 and 5, hA-II mice; lane 6, control FVB mouse. B (reduced), lane 1, isolated human apoA-II; lane 2, isolated human HDL; lane 3, human apoA-II transgenic mouse; lanes 4 and 5, hA-II transgenic mice.



Table 1shows apolipoprotein and cholesterol values for founder mice and nontransgenic control animals. Plasma concentrations of hA-II ranged from 11 to 185 mg/dl in the transgenic founder animals. Total cholesterol and HDL-cholesterol values measured in the transgenic mice were within the range of the nontransgenic control animals. No significant correlation existed between concentration of hA-II and either total cholesterol (r = -0.097), HDL-cholesterol (r = -0.262) or VLDL-/LDL-cholesterol (r = -0.055). These results are in contrast to those reported for mice overexpressing mouse apoA-II which demonstrated a 2-fold increase in HDL level and a 2- to 4-fold increase in plasma VLDL-/LDL-cholesterol levels(5) . Murine apoA-II values were equal to or slightly greater than those for the FVB control animals, showing a lack of effect on the expression of mouse apoA-II by hA-II (data not shown).



The effect of hA-II on lipoprotein distribution and particle size was determined by GGE analysis of d leq 1.21 g/ml fractions isolated from hA-II and control mice (Fig. 2). In the present study, hA-II transgenic mice exhibited a major peak of 9.6 nm particles similar to that observed in the FVB control animals. HDL from dimeric human apoA-II transgenic mice consists of a major peak of 9.5 nm particles plus a peak of 8 nm particles which has been shown previously to consist almost exclusively of human apoA-II(3) . The hA-II transgenic mice did not exhibit 8 nm particles. The gradient gel profiles of VLDL and LDL were identical in both the nontransgenic and transgenic samples (data not shown).


Figure 2: Densitometric scans from nondenaturing gradient gel electrophoresis of d leq 1.21 g/ml fraction. 4-30% gradient gels were stained for protein with Coomassie G-250 and densitometrically scanned as described under ``Experimental Procedures.'' Top, control FVB mouse; middle, hA-II mouse; bottom, human apoA-II transgenic mouse. Size of peak particles (in nm) are shown for each peak. Scale at the bottom indicates positions of human HDL subpopulations(9) .



Plasma from transgenic and control FVB mice was fractionated by FPLC, and fractions were analyzed for total cholesterol (Fig. 3A). All fractions with detectable total cholesterol were further assayed for mouse apoA-I (Fig. 3B) and apoA-II (Fig. 3C). Fractions from transgenic mouse plasma were also assayed for hA-II (Fig. 3C). Mouse apoA-II appeared in a slightly broader HDL size distribution range in the nontransgenic mouse, but the HDL peak position was identical in the transgenic and nontransgenic mice. The distribution of hA-II was identical with that of mouse apoA-II, with the concentration peak at fraction 21 (Fig. 3C). In spite of significant amounts of hA-II protein in the transgenic mouse plasma, the concentration of mouse apoA-II does not decrease with the increase of hA-II. This observation is in contrast to that in the human apoA-I transgenic mice, in which the murine apoA-I concentration decreases dramatically when the human apoA-I is expressed (11, 13) . Mouse apoA-I distribution (Fig. 3B) had the peak of concentration at the same fraction in both the transgenic and nontransgenic animals with a somewhat broader distribution range in the nontransgenic mice. The murine apoA-II distribution matches that of murine apoA-I. An increase in the cholesterol concentration of fractions in the VLDL elution range (fractions 6-12) and the LDL elution range (fractions 13-16) is seen in the transgenic mice. Studies of mice overexpressing murine apoA-II have reported the apparent accumulation of a cholesteryl ester-rich particle in the LDL density range which contains both apoA-II and apoE(4) . We performed SDS-PAGE and immunoblotting for mouse apoE on FPLC fractions from hA-II transgenic and FVB control mice. ApoE was identified in the VLDL and HDL fractions from the control FVB mice, but was detected only in the VLDL and LDL fractions from the transgenic mice (data not shown). Thus, apoE normally present in HDL may be displaced by the increased total apoA-II (murine apoA-II plus hA-II) present in the hA-II mice, resulting in the appearance of apoE in the LDL elution region. This change in apoE distribution is similar to that seen in mice overexpressing murine apoA-II(4) .


Figure 3: Distribution of cholesterol and apolipoproteins in fractions from plasma chromatographed by FPLC. Plasma (50 µl) was fractionated on the SMART FPLC System as described under ``Experimental Procedures.'' Fraction numbers represent 40-µl fractions collected throughout the lipoprotein elution range. Elution fraction range for individual lipoproteins was determined previously using human plasma and gradient gel electrophoresis. VLDL elutes in fractions 6-12, LDL in fractions 13-16, and HDL in fractions 17-27. A, total cholesterol in transgenic (bullet-bullet) and nontransgenic (circle-circle) mice; B, murine apoA-I in transgenic (bullet-bullet) and nontransgenic (circle-circle) mice; C, murine apoA-II in transgenic (bullet-bullet) and nontransgenic (circle-circle) mice; hA-II in transgenic (-) mice.



The analysis of FPLC fractions confirms the gradient gel electrophoresis observations that there is no apparent change in VLDL, LDL, or HDL particle size distribution in the hA-II transgenic mice. These mice do not have increased total cholesterol or HDL-cholesterol or any changes in HDL particle size or distribution. Thus, the differences in in vivo properties of human and mouse apoA-II overexpression are not only due to the fact that one exists as a dimer and one as a monomer, but must be linked to additional effects of sequence differences between these two apolipoproteins.


FOOTNOTES

*
This work was supported by National Institutes of Health NHLBI Grants PPG HL 18574 and HL 46281-04 and a grant funded by the National Dairy Promotion and Research Board and administered in cooperation with the National Dairy Council. Research was conducted at the Lawrence Berkeley National Laboratory under U. S. Department of Energy Contract DE-AC0376SF00098, University of California, Berkeley. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Human Genome Center-M/S 74-157, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720. Tel: 510-486-5072; Fax: 510-486-6746.

(^1)
The abbreviations used are: HDL, high density lipoprotein; apoA-I, apolipoprotein A-I; apoA-II, apolipoprotein A-II; Lp(AI), lipoprotein containing apolipoprotein A-I; Lp(AI + AII), lipoprotein containing apolipoproteins A-I and A-II; hA-II, human monomeric apolipoprotein A-II; VLDL, very low density lipoprotein; LDL, low density lipoprotein; PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography; RID, radial immunodiffusion; Tricine, N-tris(hydroxymethyl)methylglycine.


ACKNOWLEDGEMENTS

We thank Laura Holl, Phil Cooper, Junli Zhang, and Pat Blanche for excellent technical assistance.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.