(Received for publication, August 7, 1995; and in revised form, September 11, 1995)
From the
The effects of the size and cholesterol content on the
conformation of apolipoprotein A-I (apoA-I) have been studied in
reconstituted discoidal lipoproteins containing two apoA-I per particle
(Lp2A-I). The immunoreactivity of a series of 13 epitopes distributed
along the apoA-I sequence has been evaluated in Lp2A-I with a
phospholipid/apoA-I molar ratio ranging from 31 to 156 and in Lp2A-I
with constant phospholipids but varying in cholesterol content from 0
to 22 molecules. The results are compatible with a three domain
structure in apoA-I in which the central domain is located between
residues 99 and 143 and postulated to be a hinged domain that responds
differentially to changes in phospholipid and cholesterol contents.
Increasing the phospholipid content results in significant changes of
epitope immunoreactivity throughout the N-terminal and central domains
of apoA-I with fewer modifications in the C-terminal domain. In
contrast, increasing Lp2A-I cholesterol content modifies only the
immunoreactivity of two central epitopes, A11 (residues 99-132)
and 5F6 (residues 118-148), and an extreme N-terminal epitope,
4H1 (residues 2-8). Interestingly, the effects of increasing
cholesterol or phospholipids on these epitopes are opposite. This
suggests a specific effect of cholesterol on the central domain
tertiary structure between residues 99 and 143. Competition binding
assays among pairs of antibodies binding to apoA-I on Lp2A-I are best
explained by invoking inter- as well as intramolecular competitions.
The specificity of the intermolecular competitions suggests an N to C
termini arrangement of the two apoA-I molecules around the disc.
Increasing the phospholipid content of Lp2A-I mainly increases the
competitions between 3G10 and antibodies binding to most adjacent
epitopes. Simultaneously as Lp2A-I enlarges, several of these
antibodies also enhance the binding of 3G10. This has been interpreted
as evidence of a structural rearrangement of apoA-I as a result of the
size increase where the -helix (residues 99-121) that
contains the 3G10 epitope is increasingly interacting with lipids
resulting in the enhanced expression of this epitope. The increasing
interactions of apoA-I helices with lipids in the enlarging discs are
compatible with previous reports of a greater apoA-I stability in the
large discs. By contrast, cholesterol has limited but specific effects
on antibody competitions and decreases the interaction of the
N-terminal domain with the domain containing 3G10, either by direct
cholesterol protein interaction or by modification of the lipid phase
packing.
Apolipoprotein A-I (apoA-I) ()is the primary protein
component of plasma high density lipoproteins
(HDL)(1, 2) . Like several other related exchangeable
apolipoproteins, apoA-I contains multiple repeats of 22 amino acids,
constituting amphipathic
-helices that are the major lipid-binding
domains of the protein(3) . These repeats are interrupted by
proline or glycine residues that may act as helix-breakers by creating
-turns, providing more flexibility to the amphipathic helices, and
allowing apoA-I to conform to the surface of HDL as reviewed by Segrest et al.(4) . Differential interaction between
monoclonal antibodies and HDL support the hypothesis that the
conformation of apoA-I is not the same in all HDL subspecies, depending
on size or lipid composition (5, 6) . This is in
agreement with the demonstration that changes in reconstituted LpA-I
composition modulate the net charge and the
-helicity of apoA-I
and the stability of its helical segments(7) . More recently,
it has been demonstrated that a particular apoA-I conformation present
in pre-
-HDL could have a major role in promoting the
efflux of cellular cholesterol(8) . In general, data from the
literature suggest that apoA-I conformation is both variable and
critical for HDL metabolism, but study of its precise organization on
the surface of native HDL remains limited by the great heterogeneity of
this class of lipoproteins(9, 10) . Reconstituted
complexes of isolated apoA-I with defined molar ratios of phospholipids
and cholesterol have been used to prepare discoidal or spherical HDL in vitro(11, 12, 13) . These
complexes, similar to some HDL observed in vivo, exist as
particles of very reproducible sizes and compositions and, of
particular interest, as discretely sized particles containing a
constant number of apoA-I/particle (reviewed in (14) and (15) ). As apoA-I conformation changes between particles,
mobile regions (termed hinged domains) have been postulated to explain
and regulate the variable size, apolipoprotein composition, and shape
of HDL(10, 16) . This hypothesis is supported by
physical and chemical modification and immunochemical studies of apoA-I
within reconstituted complexes of different lipid composition (reviewed
in (17) ) and more recently, by analogy to other
apolipoproteins whose crystal structure has been
solved(18, 19) . Nevertheless, little is known about
the specific position of the domains and residues involved in the
binding of phospholipids or cholesterol in these lipoproteins, and the
existence of a hinge domain area is still a subject of speculation.
Epitope expression studies using reconstituted LpA-I have shown that
binding to lipids produces drastic changes in apoA-I conformation and
that particle size can also modify specific domains, such as a central
hinged domain constituted by two adjacent antiparallel
-helices(16, 20, 21) . The inhibitory or
enhancing effects of anti-apoA-I mAbs on lecithin:cholesterol
acyltransferase reaction with LpA-I also demonstrate the importance of
the central domain of apoA-I(22) . Although characterized by
well defined molar ratios, the particles used in previous studies often
vary in more than one component at a time, making the interpretation
difficult. Recently, a new approach to prepare reconstituted HDL has
been published(23) . Simpler than previous methods, this
approach is particularly accommodating for compositional manipulations
of the particles and gives a larger scale for varying some components
than that previously obtained(23) . Using this method, we have
undertaken a study of the immunoreactivity of apoA-I epitopes in order
to define the changes of apoA-I conformation specifically associated
with variations in either phospholipid or cholesterol contents of the
particles. This is the first study to relate the contribution of
individual lipids in reconstituted HDL to apoA-I epitope expression.
The results confirm the mobility of the central area of apoA-I, which
responds differentially and specifically to changes in phospholipid or
cholesterol concentration. An unexpected implication of the N terminus
region of the protein during size changes has also been observed, a
region previously described by a complex tertiary
structure(20) .
Figure 1: Epitope map of apolipoprotein A-I. The positions of epitopes recognized by these mAbs have been previously defined(20) . The names of mAbs used in this study are placed above the solid bars, which represent the sequences recognized by these mAbs. The dashed lines on either side of the bars indicate that the antigenic recognition at this site may extend further.
Five other particles, varying in cholesterol content, have also been prepared (Table 1). They show homogeneity on gradient gel electrophoresis (not illustrated). As shown in Table 1, only the cholesterol composition is significantly different, ranging from 0 to 11 molecules/apoA-I; their sizes are not statistically different from what is obtained with similar POPC content alone.
Figure 2:
Immunoreactivity of apoA-I epitopes in
Lp2A-I as a function of increasing POPC content (A) or
increasing cholesterol content (B). The composition of each
Lp2A-I preparation is given in the insets, and the epitopes
are positioned on the x axis as they appear in apoA-I
sequence. They can be grouped as representative of the N-terminal (4H1
to 2G11), the central (3G10 to A03), and the C-terminal regions (A07 to
4A12). Each bar represents the ED (inversely
proportional to immunoreactivity) with its standard deviation (a, b, c, and d indicate the
decreasing degree of significance relative to ED
for the
first particle: a, p < 0.05; b, p < 0.01; c, p < 0.005; d, p < 0.001).
In the N-terminal domain, the epitope for mAb 4H1 (residues 2-8) is progressively less reactive as the particle size of Lp2A-I increases. In contrast, four antibodies A16, A51, A05, and 2G11, which are specific for epitopes further in the N-terminal region and which overlap differently between residues 8 and 100 (Fig. 1), are more immunoreactive with large than small Lp2A-I.
In the middle of apoA-I, out of four epitopes tested (3G10, A11, 5F6, and A03), only the two central ones are modified when the particle size increases. These are the epitopes for mAbs A11 and 5F6, which overlap between residues 118 and 132 and show a decreased immunoreactivity (Fig. 2A). Interestingly and logically, the two other epitopes (3G10 and A03) surrounding that region are unchanged. This is in agreement with what we observed previously for the same epitopes of apoA-I with another panel of reconstituted discoidal Lp2A-I, which, however, differed both in size and composition (21) . Therefore the present results prove that these changes in immunoreactivity are entirely related to a change in particle size and packing of phospholipids.
In the C-terminal half of apoA-I, we have tested three antibodies, two (A07 and A44) that react with overlapping epitopes situated between residues 149-186 and 4A12, which recognizes the sequence 173-205. Despite the significant overlap of the two first epitopes, A07 is significantly less immunoreactive with the larger LpA-I as compared with A44, which is more reactive, a phenomenon previously reported (21) . The epitope for mAb 4A12 is clearly unaffected by the size. As a consequence of this observation, we can now attribute the changes in reactivity of 4A12 with Lp2A-I particles observed in previous experiments (6, 21) to changes in lipid composition rather than to changes in particle size.
The results obtained with the competitive RIA demonstrate that increasing the size of the discoidal particles induces a major change in the conformation of the entire apoA-I molecule as specific domains distributed along most of the sequence show modifications of their immunoreactivity. However, each domain appears differently affected at different stages of particle enlargement (Fig. 2A). Starting with changes occurring between the small discs (ratios 31/1 to 94/1), the central part of apo A-I represented by the epitopes 5F6 and A11 seems modified with the initial size increments; with the larger discs (122/1 and 156/1), the changes of immunoreactivity appear predominantly in the N-terminal domain (A16, A51, A05, and 2G11), where we observe high increases in immunoreactivities, but excluding the extreme N-terminal epitope, 4H1, which exhibits a very significant decrease in immunoreactivity. The epitopes (A44 and A07) toward the C terminus show less drastic and more progressive modification of their immunoreactivity than epitopes in other domains. Furthermore, related to those changes of immunoreactivity, few RIAs have displayed significant differences in the calculated slopes, reflecting little differences in binding affinities of most mAbs for their specific epitopes with the various particles (Table 2). Only in the largest particles (ratio 156/1) do we note significant changes in antibody affinities (mAbs 4H1, 2F1, and A16).
Figure 3: Variation in competitions among pairs of mAbs binding to Lp2A-I as a function of the increase in POPC content. Each panel represents the results of the competitions with a given capture antibody: A, 2F1; B, A05; C, 3G10; D, 5F6; E, 4A12. The competing mAbs are presented on the x axis in the order in which they appear in apoA-I sequence, and for each mAb the competitions are given for each Lp2A-I preparation (see the insets). The competitions are expressed as the percentage of the maximum Lp2A-I bound in the absence of competitor; error bars represent S.D. of three separate experiments. b, c, e, f, and p < 0.01; d, p < 0.05 (comparison with POPC/A-I, 31/1).
The increasing phospholipid content causes either decreases or increases in the competition between specific mAbs, and in the absence of any major change in the affinity of these mAbs (Table 2), these changes are largely related to lipid-induced modifications of apoA-I tertiary structure that change the relative position of epitopes. However, in some cases, as we will discuss below, the binding of mAbs to Lp2A-I also appears to contribute to the conformational modification of apoA-I. There are few and rather limited structural modifications within the N-terminal domain. There is an increase in the competition of 2F1 with A05 (Fig. 3B). Further downstream, the central domain represented by the epitope for 3G10 appears to move closer to the N terminus as competitions increase between 3G10 and A05 or 2F1 (Fig. 3, A and B) and between 4H1 or A16 and 5F6 (Fig. 3D). At the same time there are increasing competitions between the mAbs reacting to the central epitope 3G10 and the C-terminal epitopes A07 (not illustrated) and 4A12 (Fig. 3E), suggesting an increasing proximity for these domains as well.
The above evidence, which indicates that
Lp2A-I size increase appears to be accompanied by a conformational
change of the molecule where the central domain appears to come closer
to both the N-terminal and the C-terminal domains, is somewhat
unexpected. However, as we will analyze these results under
``Discussion,'' we attribute these changes to the increasing
interaction of the -helix 99-121 that contains the 3G10
epitope with the lipids, thus causing a change in the orientation of
3G10 bound to its epitope.
As the phospholipid to apoA-I ratio increases, enhancement of binding of one mAb (i.e. the capture antibody) by another mAb has also been observed. In the case of 3G10 as the capture mAb, most mAbs reacting in the N-terminal region enhance its binding of Lp2A-I and in the largest particles this reaches significance for A16, A05, and 2G11 (Fig. 3C). These enhancements reflect conformational changes of apoA-I induced by the binding of the competing mAb, and these have been shown to be associated with modifications of LpA-I interactions with lecithin:cholesterol acyltransferase (22) or cholesterol transfers (32) .
Previous immunochemical studies from this laboratory on the effect of lipid composition on apoA-I structure in native HDL (6) and in discoidal reconstituted LpA-I (21) have demonstrated the significant effects of phospholipids and cholesterol in distinct domains. In discoidal LpA-I, the variation in phospholipid/apoA-I ratio causes an increase in epitope expression in two large domains of the N-terminal and C-terminal regions (spanning residues 14-90 and 148-209, respectively). Between these domains, the epitopes of a central region (residues 99-143) decreased in immunoreactivity as the particle size increased(21) . These observations first suggested to us (20) that these regions might constitute the two lipid-binding domains separating a hinge domain postulated by others to explain the existence of discrete size populations in HDL particles(16) . Here the progressive changes in the concentrations of either phospholipids or cholesterol in defined Lp2A-I provide a precise analysis of their respective effect on apoA-I conformation and confirm the notion of its three domain structure.
Figure 4:
Location of apoA-I domains with
differently immunoreactive epitopes depending on POPC or cholesterol
content of discoidal Lp2A-I. On a planar representation of apoA-I
supersecondary structure, the predicted elements of secondary structure
are drawn on a line proportional to their position in the primary
structure. Amphipathic -helices,
-turns, and random coils are
respectively indicated with boxes, curvilinear
sections, and curves; the numbers along the line and the boxes refer to residues and are intended as
approximate reference points for the elements of secondary structure.
The location of epitopes is represented by a shaded area underlayed below the elements of secondary structure. The epitopes
sharing the same change in immunoreactivity in response to
phospholipids or cholesterol are given the same shading. The N-terminal
sequence containing the 4H1 epitope has been positioned close to the
central domain containing the epitopes for A11 and 5F6 to reflect the
close structural (immunochemical) and functional properties of these
domains as indicated in the text.
In the N-terminal third of apoA-I, for which we
earlier proposed a complex and condensed structure based on its high
content of multiple overlapping discontinuous epitopes(20) ,
all epitopes display very significant increases in immunoreactivity as
the size of Lp2A-I increases. This effect could be mediated by the
formation of short helices to accommodate the increasing disc size.
Several studies have documented the increase in -helix content
with the size increase (33) , which can be related to increase
in the number of helical repeats (34) or to the formation of
new short segments(23) . The changes in lysine
pK
reported by Sparks et al.(23) also implicated major changes in the N-terminal lysines
that reflect the structural rearrangements of this domain with the
increase in particle size.
The linkage of the epitope at the N terminus, 4H1, with those in the central domain, A11 and 5F6, based on the similarity of their response to cholesterol is now well documented as a result of this and previous studies(21, 42) . The specificity of the cholesterol effect on the expression of the three epitopes is clear from the present study (Fig. 4). We have also shown previously that mAbs reacting with these three epitopes exert a similar enhancement on the reaction of lecithin:cholesterol acyltransferase with small Lp2A-I(22) , a property that we tentatively attributed to conformational modifications of apoA-I that affect either its interaction with lecithin:cholesterol acyltransferase or the accessibility of cholesterol to the enzyme. In another study, we have shown that only 4H1, 3G10, and 5F6 (A11 was not included in the study) were found to have no effect on the passive transfer of cholesterol between LpA-I and low density lipoprotein, whereas all other mAbs tested (A05, A16, A51, 2G11, A03, A44 and 4A12) enhanced the transfer of cholesterol relative to that of phospholipids from Lp2A-I to low density lipoprotein(32) . These results have been interpreted as evidence that the N terminus containing 4H1 shares a common structural association with the central domain containing 5F6. We postulate that the folding of the N-terminal region brings the N-terminal sequence in close proximity with the central domain located between residues 99 and 121, as shown on the apoA-I model in Fig. 5and Fig. 6. The effects of cholesterol may be explained either by its heterogeneous distribution within the lipid phase and in relation to apoA-I chain or by changes in lipid phase fluidity or by a combination of both.
Figure 5: Planar model of two apoA-I as they may appear on the side of an Lp2A-I disc. Summary of the intra- and intermolecular competitions among pairs of mAbs binding to an Lp2A-I of median composition (POPC/apoA-I, 80/1). Each apoA-I model is adapted from Fig. 4, and the N-terminal sequence including the 4H1 epitope has been positioned close to the central domain to reflect their common properties. Well defined epitopes are represented by different shaded areas underlayed below the elements of secondary structure where they are located. Several overlapping epitopes are grouped in the N- and C-terminal regions. The arrows linking epitopes identify a mAb competing significantly with another mAb, and the double-headed arrows indicate reciprocal competitions (data derived from Fig. 3, A-E). The intramolecular competitions are compatible with the position of the epitopes involved that are adjacent on the primary sequence. The existence of intermolecular competitions between mAbs reacting at N- and C-terminal epitopes supports the model proposed for the N- to C-terminal arrangement of the two apoA-I around the disc.
Figure 6: Effects of increasing Lp2A-I POPC content on the observed competitions among pairs of mAbs. The planar model of apoA-I in an Lp2A-I particle is the same as that in Fig. 5. However, the arrows linking the epitopes recognized by competing mAbs indicate not only the existence of a competition or an enhancement of binding but also either a significant increase in the competitions (A) or a significant increase in enhancement of binding (B) as a function of the increase in POPC content and/or in the size of the particle. These changes, which are most evident in the central domain (residues 99-121) containing the epitope for 3G10, are interpreted as evidence that as the particle enlarges, the orientation of this domain changes to become more closely associated with lipids.
For the interpretation of these competitions, we can consider that the observed competitions are either exclusively intramolecular or both intramolecular and intermolecular depending on the position of the epitopes and the relative position of the two apoA-I at the surface of the particle (Fig. 5). Considering first the competitions observed in a constant composition mode with the median size Lp2A-I, the results are consistent with the known position of epitopes in the primary sequence and suggest a rather compact structure with competitions within the N-terminal region, competitions between epitopes in the central domain and epitopes in the N- or C-terminal regions, and competitions within the C-terminal region. Most of these competitions can be logically interpreted as intramolecular competitions as presented in the model of Lp2A-I (Fig. 5). However, the competitions between mAbs A16 and A05, which react in the N-terminal domain, and mAb 4A12, which reacts with the farthest epitope in the C-terminal domain, are not compatible with an intramolecular competition in the apoA-I model of Fig. 5. However, in this Lp2A-I model where we only assume that the apoA-I follow one another in an N- to C-terminal arrangement around the discoidal particle, we can easily propose that the competitions between these mAbs are intermolecular. This model does not require a particular orientation of the central domain and logically accommodates all competitions based on the positions of epitopes on the primary sequence. Therefore, these results are compatible with the model and provide the first indirect evidence documenting that the relative arrangement of apoA-I molecules in a discoidal Lp2A-I should be in an N- to C-terminal belt model around the disc.
As the ratio of
phospholipids to apoA-I increases in Lp2A-I, the changes occurring in
the competitions among pairs of mAbs are summarized in Fig. 6.
The major changes involve antibody 3G10, whose competition with all
other mAbs reacting at adjacent epitopes increases. There are also
significant changes in the N-terminal domain where the competitions of
4H1 with 5F6 and 2F1 with A16 increase (Fig. 6A). The
increase in phospholipid to apoA-I ratio is also accompanied by the
enhancement of 3G10 binding by most mAbs reacting in the N-terminal
domain (4H1, A16, A05, and 2G11) and by A03 reacting at an epitope
downstream. In the absence of any significant modification of the
affinity of the antibodies, these variations in immunoreactivity that
appear centered on mAb 3G10 can be interpreted as follows. As the
Lp2A-I particle enlarges, it is commonly assumed that more amphipathic
-helices become associated with the lipid phase (7, 14) by a process probably involving a hinged
domain(16, 21) . Our results suggest that the epitope
for 3G10, which spans the
-helix between residues 99 and 121,
either represents or is contained in the hinge domain possibly
constituted by the two central
-helices between residues 99 and
143. In small Lp2A-I the hinged domain is repulsed away from the lipid
phase for lack of stabilizing interactions with adjacent helices,
possibly also because of charge repulsions involving the proximity of
glutamic and aspartic acid residues on the adjacent faces of the
helices between residues 68 and 88 and between residues 100 and
119(17) . The particular position of these glutamic acid
residues has also been proposed (43) to be important for
lecithin:cholesterol acyltransferase activation. We also propose that
the presence of a class Y amphipathic
-helix with an intermediate
mean hydrophobic moment and an intermediate nonpolar face
hydrophobicity (17) facilitates the repulsion of the hinged
domain away from the lipid phase. The increase in POPC content of
Lp2A-I facilitates the interaction of helix 99-121 with the lipid
phase, which in turn increases the competition of mAb 3G10 binding at
this site with all mAbs reacting at adjacent epitopes. Simultaneously
because of the decreased constraints in the center of the molecule
nearly all mAbs reacting with adjacent epitopes upon binding to Lp2A-I
can contribute to increase the interaction of helices with the lipids,
including that of helix 99-121, which increases both helix
stability and binding of 3G10 to its epitope. We have earlier provided
evidence for the increase in
-helical content of Lp2A-I upon
binding of most mAbs(32) , which supports this interpretation.
The mobility of the domain containing the 3G10 epitope is also
supported by its sensitivity to proteolytic cleavage (44) and
by its high antigenicity(20) . During this process of particle
enlargement, other conformational modifications occur as indicated by
the increase in the competitions between A03 and 2F1 and between A07
and 2F1.
This study implies a great sensitivity of apoA-I
conformation to the lipid composition of reconstituted LpA-I. The
modifications of these different domains, described here by an
immunochemical approach, probably reflect the difference in
hydrophobicity of the pairs of amphipathic -helices distributed
along the apoA-I sequence (17) and their ability to form stable
structure with the adjacent helices. In small Lp2A-I with a limited
amount of POPC, the central domain may be excluded from direct lipid
interactions because of its low lipid affinity. As the amount of POPC
increases, the interaction of that pair of helices with lipids is
enhanced and a larger disc is formed also causing a reorganization of
the N-terminal region. In contrast with increasing cholesterol, the
-helix content decreases (37) and the central domain is
probably unstable, giving a higher expression of the central epitopes
(more evident with 5F6 than A11, Fig. 2B).
Other
evidence for the conformational sensitivity of the -helix
99-121 containing the epitope for 3G10 is the effect of
increasing cholesterol content in Lp2A-I, which decreases the normal
enhancement of 3G10 binding by mAbs binding to the N-terminal domain.
Indeed, cholesterol would be expected to increase the acyl chain order
and thus decrease the mobility of the lipid-associated protein
domains(11, 44) .
In conclusion, this study confirms the notion that we previously introduced of a three domain structure for apoA-I, an N-terminal domain extending up to residue 99, a central domain centered between residues 99 and 121 but possibly extending to residue 143, and a C-terminal domain. We have demonstrated that variations in the lipid components of discoidal reconstituted Lp2A-I can have significant effects on the conformation of the entire apoA-I, particularly in its central domain. Cholesterol and phospholipid contents have specific and opposite effects on the conformation of this region of apoA-I, which has been suggested to be important in lecithin:cholesterol acyltransferase activation (22, 41) and may also be implicated in apoA-I-mediated efflux of cellular cholesterol(8) . Thus the lipid composition of LpA-I can modulate its metabolism through changes of apoA-I conformation. This appears to be mediated not only by the amount of cholesterol but also by the size of the disc. We also confirm here the existence of a link (structural, functional, or both) between the extreme N terminus, represented by the epitope for 4H1, and the central domain. Both appear significantly involved or affected by cholesterol. Finally, the domain for which we observed the least modification of conformation in this study is located in the C-terminal half of the protein, a region involved in lipid binding and possibly also in interactions with cellular binding sites. Thus, apoA-I conformation appears to be crucial in HDL metabolism, with different domains having specific functions.