(Received for publication, January 2, 1996)
From the
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
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
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.
High density lipoproteins (HDL) ()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.
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.
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
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
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
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 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 (-
) and
nontransgenic (
-
) mice; B, murine
apoA-I in transgenic (
-
) and nontransgenic
(
-
) mice; C, murine apoA-II in
transgenic (
-
) and nontransgenic
(
-
) 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.