Instituto de Investigaciones Bioquímicas de La Plata-Consejo Nacional de Investigaciones Científicas y Técnicas/Universidad Nacional de La Plata, Facultad de Ciencias Médicas, Calles 60 y 120, 1900 La Plata, Argentina
Received for publication, December 21, 2000, and in revised form, February 21, 2001
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ABSTRACT |
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Previous evidence indicated that
discoidal reconstituted high density lipoproteins (rHDL) of
apolipoprotein A-I (apoA-I) can interact with lipid membranes
(Tricerri, M. A., Córsico, B., Toledo, J. D.,
Garda, H. A., and Brenner, R. R. (1998) Biochim. Biophys. Acta 1391, 67-78). With the aim of studying this
interaction, photoactivable reagents and protein cleavage with CNBr and
hydroxylamine were used. The generic hydrophobic reagent
3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine
gave information on the apoA-I regions in contact with the lipid phase
in the rHDL discs. Two protein regions loosely bound to lipids were
detected: a C-terminal domain and a central one located between
residues 87 and 112. They consist of class Y amphipathic Apolipoprotein A-I
(apoA-I)1 is the major
protein component of the high density lipoproteins (HDL), which play a
central role in "reverse cholesterol transport" (1, 2). The
interaction of lipid-free apoA-I with cell membrane binding sites
triggers free cholesterol (FC) mobilization from intracellular
membranes and from cholesteryl ester stores toward the cell
membrane as a result of signals mediated by protein kinase C (reviewed
in Ref. 3). The sequential removal of phospholipids (PL) and FC (4-7)
yields lipid-poor and discoidal HDLs of pre- A high conformational flexibility in apoA-I is needed for its existence
in different states: lipid-free, lipid-poor, and discoidal or spherical
lipoproteins of different size. Mature human apoA-I contains 243 amino
acid residues (18) with 11- and 22-mer homologous repeats (19) that are
predicted to form amphipathic -helices
that have a different distribution of the charged residues in their
polar faces by comparison with class A helices, which predominate in
the rest of the apoA-I molecule. The phospholipid analog
1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoro-methyl-3-H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, which does not undergo significant exchange between membranes and
lipoproteins, was used to identify the apoA-I domain directly involved
in the interaction of rHDL discs with membranes. By incubating either
rHDL or lipid-free apoA-I with lipid vesicles containing 125I-TID-PC, only the 87-112 apoA-I segment becomes
labeled after photoactivation. These results indicate that the central
domain formed by two type Y helices swings away from lipid contact in the discoidal lipoproteins and is able to insert into membrane bilayers, a process that may be of great importance for the mechanism of cholesterol exchange between high density lipoproteins and cell membranes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
electrophoretic migration. Discoidal pre-
HDLs, which also are secreted by liver, are then transformed into spherical
-migrating HDL by
lecithin-cholesterol acyl transferase (LCAT), which esterifies FC,
forming a hydrophobic core of cholesteryl ester (1, 2, 6). Circulating
-HDL are modified by accepting other apoproteins, such as
apolipoprotein A-II, and by lipid exchange with other serum
lipoproteins (8). This lipid and apoprotein exchange can produce
lipid-free or lipid-poor apoA-I, which can begin again the cycle of
lipid removal from cells in the interstitial space of peripheral
tissues (6, 9). Whether the interaction of apoA-I with cell membrane is
mediated by a specific receptor and the mechanism of its loading with
PL and FC is still a matter of debate (7). The recent discovery that a
defective ATP-binding cassette transporter (ABCA1) leads to Tangier
disease (10, 11) has prompted the study of the role of this protein as
a candidate apoA-I receptor (12-17). A direct molecular interaction
between ABCA1 and apoA-I is indicated by some of these studies (12,
13). However, other studies (15, 16) do not support this interaction
and suggest a role for ABCA1 in modifying the lipid distribution in the
membrane and generating the biophysical microenvironment required for
the docking of apoA-I to the cell surface.
-helices (20, 21) that interact with
lipids through their hydrophobic face. The helices are linked by short
and flexible
-turns usually containing a proline residue. Lipid-free
apoA-I is thought to be a bundle of helices (22) in a molten
globular-like state (23), and sedimentation velocity experiments
indicate significant conformational heterogeneity (24), supporting a
flexible structure. In the spherical
-HDL, it has been proposed (25)
that the amphipathic helices are oriented parallel to the surface of
the phospholipid monolayer with the hydrophobic faces embedded into the
hydrocarbon region and with the hydrophilic faces interacting with the
phospholipid polar groups and the aqueous phase. It is generally
accepted that discoidal lipoproteins are composed of a lipid bilayer
disc with the apoA-I at their edge, but the orientation of the helices
with respect to the acyl chains is controversial (see Ref. 26 for a
review; Fig. 1) The "picket fence"
model (27-32) proposed eight antiparallel amphipathic
-helices
connected by
-turns oriented parallel to the phospholipid acyl
chains. However, recent experimental evidence supports the "belt"
model (33-37), which proposes that apoA-I wraps around the edge of the
disc with the axis of its
-helices oriented perpendicular to the
acyl chains. The size of discoidal complexes is primarily determined by
the number of apoA-I molecules per particle (38), but discs with the
same number of apoA-I molecules and different amounts of lipids do also
exist (39-41). A hinge domain, which may be excluded from the
interaction with lipids in small discoidal complexes (39, 40, 42), has
been proposed to be present in the central region of apoA-I (28,
43-45) to explain the ability of apoA-I to form different sized
discoidal complexes.
View larger version (59K):
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Fig. 1.
Proposed models for apoA-I structure in
spherical and discoidal HDL complexes.
The conformational flexibility of apoA-I can also play a role in the
interaction and lipid exchange of HDL with membranes. It was
hypothesized (46, 47) that the interaction of HDL discs with membranes
may involve helices of apoA-I that can swing away from lipid contact on
the disc to insert into the phospholipid bilayer, thus facilitating
cholesterol exchange. In spherical HDL, this apoA-I domain would be
interacting with the particle lipids and would not be able to interact
with membranes (47). According to this hypothesis the morphologic
change from disc to sphere or the enrichment in cholesteryl ester would
reduce the affinity of HDL particles for membranes, allowing the
liberation of spherical -HDLs to the circulation. The major problem
of this hypothesis is that the morphologic change from disc to sphere requires LCAT whose activity is very low in lymph and interstitial fluid (48) but high only in plasma (6). In previous reports (49, 50) we
suggested, as a possible alternative to this mechanism, that the
increase in size or in the cholesterol content in the discs could
cause, independently of LCAT activity, a conformational change in
apoA-I that decreases the affinity for membranes. Then large
cholesterol-rich discs would be liberated into circulation and
transformed into
-HDL by LCAT in plasma. This alternative hypothesis
is supported by the fact that the increase of size and cholesterol
content in the discs modifies apoA-I conformation (45, 49) and
decreases the interaction (49) and cholesterol exchange rate (50) with
lipid vesicles. The interaction of discoidal complexes of apoA-I with
lipid vesicles produces a transient leakage of the internal aqueous
contents, which is faster compared with the leakage produced by
lipid-free apoA-I (49), and suggests the penetration of an apoA-I
domain into the bilayer of the lipid vesicles. It was also shown that
apoA-I discs are able to interact with lipid monolayers by
increasing their lateral pressure (70). To gain further insight into
the mechanism of this interaction, we have used two photoactivable
reagents to obtain information on the lipid-bound regions of apoA-I in
discoidal complexes and to identify the apoA-I region involved in the
interaction of discoidal complexes with membranes.
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EXPERIMENTAL PROCEDURES |
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Photoactivable Reagents--
The reagents used were:
3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine
(125I-TID) and
1-O-hexadecanoyl-2-O-(9-[[(2-[125I]iodo-4-(trifluoromethyl-3-H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine (125I-TID-PC) (Fig. 2). Upon
photolysis, the trifluoromethyl diazirine group of these reagents is
rapidly converted to carbene, which is capable of reacting with the
full range of functional groups occurring in biomolecules, including
paraffinic carbon-hydrogen bonds (51). In membranes or
lipoproteins, the environment of carbene consists largely of acyl
chains and protein segments inserted into or in contact with the
hydrophobic region of the phospholipid. Membrane inserted domains of
several proteins (52-56) were identified by using these reagents
followed by chemical or enzymatic cleavage. These photoreagents exhibit
very different monomer solubility in water, with one of the factors
determining intermembrane exchange/transfer rates and the spontaneous
incorporation in preformed membranes or lipoprotein structures (51,
52). 125I-TID is easily incorporated into the lipid phase
of preformed structures such as membranes or lipoproteins with a
partition coefficient highly favorable to the lipid phase (39,000 bound ligands × µl external solution/free ligand × mg of
lipids) (57). It is supposed to be uniformly distributed across the
hydrophobic region of the phospholipid bilayer, being a good candidate
for the analysis of the apoA-I regions directly in contact with the acyl chains in discoidal complexes. On the other hand, phospholipid vesicles containing the phospholipid analog 125I-TID-PC,
which does not undergo significant intermembrane transfer (51) and
should locate the active diazirine group very deep into the lipid
bilayer, were used to identify the apoA-I region able to penetrate into
the vesicle bilayer:
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125I-TID (10 µCi/nmol) was purchased from Amersham
Pharmacia Biotech. 125I-TID-PC was prepared by
radioiodination of its nonradioactive tin-containing precursor
1-O-hexadecanoyl-2-O-
(9-[[[2-(tributylstannyl)-4-(trifluoromethyl-3-H-diazirin-3-yl)benzyl]oxy]carbonyl] nonanoyl]-sn-glycero-3-phospho-choline according to Weber
and Brunner (51). The tin precursor was generously donated by Prof. J. Brunner from the Swiss Federal Institute of Technology (Zurich, Switzerland). The dried tin-containing precursor (~20 nmol) was dissolved in 10 µl of acetic acid in a 1-ml Reacti-Vial (Pierce). [125I]NaI (1 mCi) was added, and the iodination was
started by the addition of peracetic acid (2 µl of a 32% solution in
acetic acid). After 2 min at room temperature, the reaction was
quenched with 50 µl of 10%
Na2S2O5. Then 40 µl of
CHCl3/methanol (2:1) was added and vortexed. The organic
phase was collected and concentrated using a charcoal filter to adsorb
volatile radioactivity. The residue was dissolved in 20 µl of
methanol/CHCl3/H2O (9:1:1) and subjected to
reverse-phase high pressure liquid chromatography using the same
solvent and a 208HS54 C8 column (Vydac) in a Merck-Hitachi instrument
with UV detection at 254 nm. The flow rate was 1 ml/min, and fractions
of 0.5 ml were collected. 125I-TID-PC eluted at ~20 min,
whereas the excess of tin-containing precursor eluted at ~40 min. An
aliquot (5 µl) of each fraction in the elution region of
125I-TID-PC was analyzed by TLC on silica gel plates (LK6D,
60 Å; Whatman, Clifton, NJ) and subjected to autoradiography.
Fractions containing radioactivity were pooled and concentrated by
co-evaporation with toluene/ethanol (1:1). 125I-TID-PC was
stored at 20 °C and dissolved in ethanol/toluene (1:1) at ~1
mCi/ml.
Other
Materials--
1-Palmitoyl-2-oleoyl-phosphatidylcholine (POPC) was
purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Cholesterol and sodium cholate were from Sigma.
L--Dipalmitoyl-[2-palmitoyl-9,10-3H(N)]-phosphatidylcholine
([3H]PC), [4-14C]cholesterol
([14C]FC), and [125I]NaI were from
PerkinElmer Life Sciences.
ApoA-I Purification and Preparation of Discoidal Reconstituted High Density Lipoproteins (rHDL)-- ApoA-I was purified from the HDL fraction of human serum (kindly donated by the Banco de Sangre del Instituto de Hemoterapia de la Provincia de Buenos Aires, La Plata, Argentina), according to the procedure previously described (58) with some modifications (49, 50). The cholate dialysis method of Jonas et al. (39) was used to prepare the apoA-I rHDL complexes using initial mixtures of cholate/POPC/apoA-I in the molar ratio 250/100/1 or cholate/POPC/FC/apoA-I in the molar ratio 250/100/25/1. [3H]PC and [14C]FC were included to allow PL and FC quantification. Differently sized rHDL were separated by gel filtration fast protein liquid chromatography (49, 50) using two columns in tandem (Superose 6 and Superose 12 HR 10/30 from Amersham Pharmacia Biotech). The purity and size of the isolated rHDL were determined by nondenaturating gradient gel electrophoresis (41), and their composition was obtained by measuring the protein content by Lowry's method (59). PL and FC contents were measured by scintillation counting.
Preparation of Lipid Vesicles Containing 125I-TID or 125I-TID-PC-- Large unilamellar vesicles (LUV) of POPC or POPC/FC (4/1 mol/mol) were prepared (0.5 mM in POPC) by extrusion through polycarbonate membranes with pore diameters of 100 nm (Avestin Inc., Otawa, Canada). Lipids in CHCl3 were mixed, dried under a stream of N2, and resuspended in 10 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM N3Na (buffer A) by vortexing. Then lipid suspensions were passed 11 times through the polycarbonate filters by using a Liposofast-extruder system (Avestin). To incorporate 125I-TID into the LUV, 10 µCi of the photoactivable reagent in ethanol were added to 0.2 ml of the LUV suspension and incubated with stirring for at least 2 h at room temperature until use. On the other hand, to prepare the LUV containing 125I-TID-PC (200 µCi/µmol of POPC), the photoreagent was mixed with the lipids in CHCl3 previous to the LUV preparation.
Photolabeling and Delipidation of ApoA-I-- The photoreagent-containing LUV (25 nmol of POPC) were incubated with the rHDL complexes or with lipid-free apoA-I (1 nmol of apoA-I) in buffer A containing 50 mM glutathione at room temperature. After the indicated times, mixtures (0.3 ml) in glass cuvettes were irradiated for 30 s with a Xenon lamp (450 Watts) at a distance of 25 cm. As a control, the photoreagent-containing LUV were also irradiated before mixing with apoA-I. 125I-TID was directly incorporated into the rHDL complexes by adding the reagent (1.5 µCi) in ethanolic solution to the rHDL suspension (1 nmol of apoA-I) in buffer A with 50 mM glutathione and incubating for at least 4 h with stirring before the irradiation. After irradiation, 3 volumes of CHCl3/methanol (2:1) were added and vortexed, and the organic phase was discarded. ApoA-I was precipitated with 10% trichloroacetic acid and redissolved as indicated below for chemical cleavage or in 25 µl of sample buffer for direct analysis by SDS-polyacrylamide gel electrophoresis (PAGE).
Chemical Cleavage of ApoA-I with CNBr and with Hydroxylamine-- For cleavage with NH2OH, samples were dissolved and incubated at 45 °C for 4 h in 50 µl of 2 M NH2OH, 2 M urea, 0.2 M K2CO3, pH 9.0. For cleavage with CNBr, samples were dissolved in 50 µl of 70% formic acid, and CNBr was added in a 30:1 ratio (CNBr: protein w/w) and incubated at 25 °C under a N2 atmosphere for 24 h. Then the samples were dried in a Speed Vac system and redissolved in 20 µl of sample buffer for Tricine SDS-PAGE analysis.
Analysis by SDS-PAGE and Autoradiography--
SDS-PAGE was
carried out according to Schägger and von Jagow (60) in 16.5%
acrylamide Tris-Tricine. After Coomassie Blue staining, gels were dried
and exposed to X-Omat film (Kodak) at 80 °C for different times
depending on the amount of radioactivity.
N-terminal Sequencing of ApoA-I Fragments--
Peptide fragments
were transferred from SDS-polyacrylamide gels to polyvinylidene
difluoride membranes using a Trans-Blot semi-dry transfer cell
(Bio-Rad). N-terminal sequencing was performed on the bands in
polyvinylidene difluoride membranes by the Laboratorio Nacional de
Investigación y Sevicio en Péptidos y Proteínas (Universidad de Buenos Aires-Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina) and by
the Protein Structure Core Facility, University of Nebraska Medical
Center (Omaha, NE).
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RESULTS |
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Characterization of ApoA-I Discoidal rHDL Particles-- Four kinds of discoidal lipoprotein complexes of apoA-I were used in this study. Two cholesterol-free (Lp2 and Lp4) and two cholesterol-containing (Lp2C and Lp4C) rHDL complexes were purified by gel filtration fast protein liquid chromatography after dialysis of the cholate-containing mixtures. They were pure as judged by nondenaturating gradient gel electrophoresis. Their size corresponded to that previously determined (49) for rHDL containing two (85 Å Stokes diameter for Lp2 and Lp2C) or four (150 Å for Lp4 and Lp4C) apoA-I molecules/rHDL particle. The determined molar compositions were as follows: 57/1 and 108/1 (POPC/apoA-I) for Lp2 and Lp4 and 59/4/1 and 101/18/1 (POPC/FC/apoA-I) for Lp2C and Lp4C, respectively.
Photolabeling of ApoA-I in the Lipid-free State or as Discoidal
Complexes by Incubation with Lipid Vesicles Containing
125I-TID-PC--
The incubation of POPC LUV containing
125I-TID-PC with apoA-I discoidal complexes or lipid-free
apoA-I resulted in the labeling of the protein after photoactivation.
The protein cleavage with CNBr and analysis by SDS-PAGE and
autoradiography yielded the results shown in Fig.
3. ApoA-I contains three methionine
residues at which cleavage with CNBr occurs: residues
Met86, Met112, and Met148 (see Fig.
7), and only four polypeptides should be obtained by complete cleavage.
However, because of incomplete cleavage, a more complex pattern, which
can be observed in Fig. 3A, is obtained. All of the peptide
bands can be assigned as indicated in Fig. 3A by taking into
account their molecular masses and the indicated N-terminal sequence.
It must be noted that because of the lower Coomassie staining
sensitivity to smaller peptides, the real cleavage extension is larger
than the apparent one. Autoradiography analysis (Fig. 3B)
shows that the radioactivity is located almost exclusively (more than
90%) in the smallest peptide band of a molecular mass similar
to glucagon or the insulin -chain (about 3 kDa). The N-terminal
sequence (Ser-Lys-Asp-Leu) indicates that this band corresponds to
fragment 87-112. Fig. 3B (lane 8) shows that no labeling was found when the 125I-TID-PC-containing POPC LUV
were photoactivated previously to the incubation with lipid-free
apoA-I. The same apoA-I fragment becomes almost exclusively labeled by
the incubation of these 125I-TID-PC-containing vesicles
with the different discoidal complexes (Lp2, Lp2C, Lp4, or Lp4C) or
even with lipid-free apoA-I, although a different level of labeling can
be observed, which is possibly due to differences in affinity or the
ability to interact as previously reported (49). The same apoA-I
fragment is also labeled by photoactivation of mixtures of apoA-I
discoidal complexes with POPC/FC (4/1 mol/mol) vesicles containing the
phospholipidic reagent 125I-TID-PC (data not shown). By
increasing the time of gel exposure to the autoradiographic films (not
shown), it was possible to observe that the larger fragments that
contain the 87-112 region (amino acids 1-243, 1-148, 1-112, and
87-148) become also labeled, although some unspecific label (less than
5%) is also observed in the band corresponding to fragments 1-87 and
149-243. These results strongly indicate that an apoA-I domain located
between residues 87 and 112 is able to penetrate the bilayer of POPC or POPC/FC vesicles when either lipid-free apoA-I or discoidal complexes are used.
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Photolabeling of ApoA-I in Discoidal Complexes by Direct Incubation
with 125I-TID or by Incubation with Lipid Vesicles
Containing 125I-TID--
Fig.
4 (lanes 1 and 2)
shows that the photoactivation of mixtures of Lp2 complexes with
125I-TID-containing POPC/FC (4/1 mol/mol) vesicles also
results in the labeling of apoA-I. No appreciable difference in the
level of labeling is found when Lp2 complexes are preincubated
for 0.5 or 5 min with the vesicles before the photoactivation. If
lipid-free apoA-I is incubated with these vesicles, the protein becomes
also labeled but at a level that is significantly lower than that found for the Lp2 complexes (lane 3). ApoA-I is also labeled if
125I-TID is directly added to the Lp2 complexes (lane
5); in this case we added only 0.5 µCi of 125I-TID
to the rHDL sample to keep the same 125I-TID/POPC ratio as
in the incubation mixtures containing Lp2 and vesicles. On the other
hand, no labeling of apoA-I is found when the
125I-TID-containing LUV are photoactivated before their
incubation with lipid-free apoA-I (lane 6).
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Samples photoactivated as in Fig. 4 were cleaved with CNBr, and the
corresponding SDS-PAGE and autoradiography analyses are shown in Fig.
5. Lane 5 shows the labeling
pattern obtained by photoactivation after the direct addition of
125I-TID to Lp2 discs. With this experimental approach, the
apoA-I regions that are in contact with the lipid bilayer of the discs should be labeled, although it cannot be totally discarded that some
hydrophobic domains not in contact with the bilayer could also be
labeled. It is remarkable that the 87-112 fragment (which was almost
exclusively labeled by 125I-TID-PC-containing vesicles)
does not become labeled by the direct addition of 125I-TID
to Lp2 discs. This indicates that the 87-112 region, although is able
to penetrate into the lipid bilayer of vesicles, is loosely bound to
the disc lipid bilayer.
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The incubation of Lp2 discs with 125I-TID-containing vesicles during a short period of time (30 s) before the photoactivation, results in a detectable labeling of the 87-112 fragment (lane 2). However, if the preincubation time is extended up to 5 min (lane 3), the label in the 87-112 fragment is no longer detected, and a similar labeling pattern to that obtained by the direct addition of the reagent to the discs is obtained. These results indicate that 125I-TID, as expected from its higher monomer solubility in water and its intermembrane exchange/transfer rate as compared with 125I-TID-PC (51), diffuses rapidly from the lipid bilayer of the vesicles toward the bilayer of the discs, where it should label the apoA-I regions in contact with the disc lipids. Only at a short time after mixing the 125I-TID-containing vesicles with the rHDL discs should the reagent concentration in the vesicles be high enough to produce a detectable labeling of the 87-112 apoA-I region. With the time course after mixing, the reagent concentration decreases in the vesicles and increases in the discs until the equilibrium is reached. By assuming a similar partition of 125I-TID between both vesicle and disc lipid bilayer, it can be calculated that in the conditions used here, the reagent concentration in the vesicles would decrease at the equilibrium more than 3- or 5-fold when incubated with Lp2 or Lp4, respectively. Moreover, it must be also considered that at any time, only a small proportion of the discs (depending on the membrane affinity of each kind of rHDL disc) are interacting with vesicles through the 87-112 apoA-I region, whereas most of the discs would be free and thus with the apoA-I 87-112 region unaccessible to be labeled by the reagent located in the vesicles. On the contrary, 100% of the apoA-I molecules are interacting with the disc lipid bilayer and thus are accessible to be labeled in their contact regions by the reagent located there. This fact makes difficult the detection of label in the membrane inserted domain when a significant amount of reagent is transferred from the vesicles toward the disc bilayer.
Even by using a short incubation time before the photoactivation, no detectable labeling of the 87-112 fragment was observed when 125I-TID-containing vesicles were incubated with large (Lp4) or FC-containing (Lp2C or Lp4C) rHDL discs (Fig. 5, lanes 7-9). Because the 87-112 fragment was labeled when these discs were incubated with vesicles containing the nondiffusible reagent 125I-TID-PC (Fig. 3), it must be concluded that these large and FC-containing discs interact with vesicles in a way similar to that of Lp2 through the membrane insertion of a central domain in the 87-112 region. The lack of labeling of this apoA-I region by 125I-TID-containing vesicles in Lp2C, Lp4, and Lp4C is very likely to be due to their lower ability to interact with lipid membranes in comparison with Lp2, as it was previously shown (49).
Although 125I-TID was not useful in detecting the membrane
inserted domain, because of its higher intermembrane transfer rate, it
was very helpful to detect the apoA-I regions loosely bound to the disc
lipid bilayer. Incomplete CNBr cleavage of apoA-I allows us to conclude
that, in addition to the central 87-112 region, a C-terminal apoA-I
domain is also not in contact with the lipid bilayer of discoidal
complexes. As already mentioned the complete cleavage should produce
four peptides: 1-86, 87-112, 113-148, and 149-243. In contrast to
the 87-112 fragment, the 113-148 peptide becomes labeled by the
125I-TID, and it should be in contact with the lipid
bilayer of the discs (Fig. 5). The N- and C-terminal fragments 1-86
and 149-243 co-migrate on SDS-PAGE, giving a unique band that contains
a high level of radioactivity. Co-migration of these peptides does not allow us to conclude whether both or only one of them becomes labeled.
However, the radioactivity distribution in peptides 1-112 and
113-243, which are produced by incomplete cleavage, indicates that the
C-terminal region becomes labeled at a very low level. Fragment 1-112
contains a higher level of radioactivity than the 113-243 fragment,
despite its lower mass as indicated by the intensity of Coomassie
staining. Moreover, fragment 1-112 contains the 87-112 region, which
is not labeled, and fragment 113-243 contains the 113-148 region,
which is labeled. For all of these reasons we conclude that the
C-terminal region of apoA-I is poorly labeled and that it should be
loosely bound to the lipid bilayer of discs. To confirm this, a second
cleavage strategy was used. Hydroxylamine cleaves apoA-I at its unique
Asn184-Gly185 bond (Fig.
6), giving a large N-terminal polypeptide
(1) and a shorter C-terminal fragment (185) as can be
observed in Fig. 6A. Autoradiography shows that only the
large 1-184 fragment becomes labeled by photoactivation of mixtures of
Lp2 or lipid-free apoA-I with vesicles containing 125I-TID
(Fig. 6B). No appreciable radioactivity was found in the shorter C-terminal 185-243 fragment, indicating that this apoA-I region has a low contact with the lipid bilayer of the discs.
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DISCUSSION |
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The results obtained with the 125I-TID reagent
indicate that two apoA-I regions have little if any contact with the
lipid bilayer of the discoidal lipoprotein complexes: a C-terminal
region and a central one located between the residues 87-112. Fig.
7 shows the amino acid sequence of apoA-I
and the amphipathic -helices, which are predicted to be present in
this protein (21, 25, 26). Segrest and colleagues (21) and others
(15) proposed that the
-helices of apolipoproteins are not
all equivalent in their affinities toward lipids. The differences in
affinity do not appear to be only related to their hydrophobic moment
but also to the distribution of charged residues along the axis of the
helix. Amphipathic helices are classified into seven major and distinct
classes (A, H, L, G, K, C, and M) based upon a detailed analysis of
their physico-chemical and structural properties (21). The most
frequent helix class found in exchangeable apolipoproteins is class A,
which is characterized by a unique clustering of positively charged
amino acid residues at the polar-nonpolar interface and negatively
charged residues at the center of the polar face. As shown in Fig. 7,
six class A helices have been identified in apoA-I (25, 26): helices
44-65, 66-87, 121-142, 143-164, 166-186, and 187-208. In
addition, two other helix types were also identified: class G* (helix
8-33) and class Y (helices 88-98, 99-120, 209-219, and 220-241).
Class G* helices are distinguished by a random radial arrangement of
positive and negative residues, which is similar but not identical to
that of the class G helices found in globular proteins. The basic
features of the class Y motif are two negative residue clusters on the
polar face separating the two arms and the base of the Y motif formed
by three positive residue clusters (25). Interestingly, the two apoA-I
regions not labeled with 125I-TID, indicating little
contact with the lipid bilayer of discoidal complexes, are rich in the
class Y motif. In both regions, a short helix of 10-11 residues is
followed by a long one of 21 residues. This fact would agree with the
hypothesis that lipid affinity correlates with the extent to which a
helix domain fits to class A motif (25, 61). However, a synthetic
peptide containing the 220-241 apoA-I has been shown to have the
highest affinity toward lipids among the other apoA-I helices (62).
These discrepancies could be due to different affinity toward the edge
of a lipid bilayer disc compared with the affinity toward the surface
monolayer of vesicles or spherical particles and due to the fact that
other factors such as interhelix interactions are playing an important role.
|
The central region 87-112, although loosely bound to the lipids of the discoidal complexes, is able to penetrate the bilayer of lipid vesicles as indicated by the results obtained with the 125I-TID-PC reagent. It is remarkable that the same 87-112 segment of apoA-I becomes labeled when 125I-TID-PC-containing vesicles are mixed either with discoidal complexes or with lipid-free apoA-I. This fact suggests that apoA-I could be liberated from the discs and that it then could interact with the vesicle bilayer as lipid-free apoA-I. This possibility, however, is not likely because it was shown (49) that discoidal complexes induce leakage of the vesicle internal aqueous space much faster than lipid-free apoA-I. Also, the lateral pressure increase in lipid monolayers occurs more rapidly with discoidal complexes than with lipid-free apoA-I (70).
The interaction between apoA-I discs and lipid vesicles results also in resonance energy transfer from tryptophan fluorescence to a nonexchangeable fluorescent phospholipid (diphenylhexatrienyl-phosphatidylcholine) in the vesicles (70). However, tryptophan fluorescence is not quenched when the vesicles contain phospholipid analogs (doxyl-phosphatidylcholines) having a collisional quencher group in different positions.2 These observations indicate that no tryptophan residue of apoA-I penetrates the lipid bilayer of the vesicles so that it could be quenched by collision with doxyl-phosphatidylcholines, although at least one tryptophan residue would lie at a distance short enough to produce an energy transfer to the fluorescent phospholipid. The 87-112 apoA-I fragment that becomes labeled with 125I-TID-PC contains a tryptophan residue at the 108 position. This residue would not be inserted into the bilayer of the lipid vesicles but would be located very near the interface, so that it would be responsible for the resonance energy transfer to the fluorescent phospholipid.
Fig. 8 shows the Edmundson wheel
representation of the type Y amphipathic -helices of this apoA-I
segment. It contains the short 88-98 helix and the first half
(99) of the long 99-120 helix. Because the hydrophobic faces of
these helices are narrower than the hydrophilic ones, an interaction
between both helices through their hydrophobic faces would expose only
the hydrophilic residues, so that this domain could avoid contact with
the lipid bilayer in the discoidal complexes, independently of which
model is considered. To penetrate the membrane bilayer, rotation of the
helices should occur to expose the nonpolar residues to the hydrophobic
lipid phase environment. There are at least two possibilities for the membrane penetration by amphipathic helices (Fig.
9): (a) a vertical penetration
with the helix axis parallel to the hydrocarbon phospholipid chains,
which would require two or more helices interacting through their
hydrophilic faces to expose the hydrophobic faces to the lipid
environment, and b) a horizontal interaction with the helix axis
parallel to the membrane surface, which requires the insertion of the
nonpolar face into the hydrophobic interior of the external phospholipid monolayer; the hydrophilic face remains exposed to the
aqueous phase and/or interacts with the phospholipid polar groups.
|
|
The vertical membrane penetration of only these two helices interacting
by their polar faces seems to be very unlikely. Although the charged
residue distribution at both sides of proline 99 would make the
interaction possible through salt bridges between both helices, the
hydrophilic faces are too wide, a fact that would result in the
exposure of polar residues to the lipid environment. Moreover, this
would require a large conformational change because a 180° rotation
of each helix is needed to switch from the lipid-unbound state in the
discoidal complexes to the membrane inserted state. Another possibility
is suggested by the analogy found with the three-dimensional PSSM
program (63) of this apoA-I central region with ectatomin. This is a
toxic peptide of 7.9 kDa that inserts into cellular and artificial
membranes to form ionic channels (64, 65) and whose spatial structure
in aqueous solution was resolved by 1H NMR (66). Ectotamin
contains two homologous polypeptide chains (37 and 34 residues) of
similar conformation linked by disulfide bridges. Each chain consists
of two -helices and a hinge region of four residues giving an
overall structure of a four-helix bundle (66). On insertion into
artificial membrane bilayers, two ectatomin molecules rearrange with
considerable movement of their helical parts (64) to form an ion pore.
Thus, it should be possible for the two helices of the 87-112 region
of apoA-I to interact with the same region of another apoA-I molecule
to vertically penetrate the vesicle bilayer in a way similar to
ectatomin. As recently determined (37), positions 132 of each
apoA-I monomer in Lp2 discs are in close proximity, suggesting the
possibility that the 87-112 regions are also near each other.
Moreover, it is also possible that central domains of apoA-I molecules
belonging to different discoidal particles can interact in a way
similar to that of ectatomin to be inserted into membranes. Other
possibility to be considered is that helix content could change during
the membrane insertion of the central domain. Although the crystal structure of an apoA-I fragment lacking the 43 N-terminal amino acids
confirmed that the 87-112 region has a helical conformation (33), the
helical content has shown to change among the lipid-free and different
lipid-bound states (23, 39, 40, 67)
Although more experimental evidence would be required to determine how this central apoA-I domain is inserted into the bilayer of lipid vesicles, the horizontal insertion parallel to the membrane surface seems to be more likely. This type of interaction was postulated for apolipoprotein insertion into the surface phospholipid monolayer of spherical lipoproteins (25, 61), and it is known as the "snorkel" model.
From the reports of Segrest et al. (25) and Palgunachari
et al. (62), a relatively low affinity for the membrane
bilayer of the 87-112 region of apoA-I could be expected. Cholesterol exchange between HDL and membranes or other lipoproteins can occur by
free diffusion through the aqueous medium (68, 69), but there is also
evidence (47, 50) that it can be facilitated by the binding of HDL to
membranes. The low affinity of the central apoA-I domain in the 87-112
region could be important for the functional need of a reversible and
transient anchoring of HDL discs to cell membranes.
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ACKNOWLEDGEMENTS |
---|
We acknowledge Banco de Sangre del Instituto de Hemoterapia de la Provincia de Buenos Aires (Argentina) for the provision of human serum, Prof. J. Brunner from the Swiss Technological Institute of Zurich (Switzerland) for the provision of the tin-containing precursor of 125I-TID-PC, Prof. Ana Jonas from the Department of Biochemistry of the University of Illinois at Urbana-Champaign for revision of the manuscript and for very important suggestions, Dr. Autino (Departamento de Química Orgánica, Facultad de Ciencias Exactas, Universidad Nacional de La Plata) and Dr. Burton (Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires) for technical suggestions about the preparation of peracetic acid, and Laura Hernandez for expert technical assistance.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Member of the Carrera del Investigador Científico,
Consejo Nacional de Investigaciones Científicas y
Técnicas, Argentina. To whom correspondence should be addressed:
INIBIOLP, Facultad de Ciencias Médicas, Calles 60 y 120, 1900 La Plata, Argentina. Tel.: 54-221-4834833; Fax:
54-221-4258988; E-mail: hgarda@ atlas.med.unlp.edu.ar.
Published, JBC Papers in Press, March 2, 2001, DOI 10.1074/jbc.M011533200
2 J. D. Toledo and H. A. Garda, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: apoA-I, apolipoprotein A-I; HDL, high density lipoproteins; FC, free cholesterol; PL, phospholipids; LCAT, lecithin-cholesterol acyl transferase; 125I-TID, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine; 125I-TID-PC, 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3-H-diazirin-3-yl)benzyl]oxy] carbonyl] nonanoyl]-sn-glycero-3-phosphocholine; POPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine; PC, phosphatidylcholine; LUV, large unilamellar vesicles; rHDL, reconstituted high density lipoproteins; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis- (hydroxymethyl)ethyl]glycine.
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