From the Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195
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ABSTRACT |
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Published studies demonstrate that
lipid transfer inhibitor protein (LTIP) is an important regulator of
cholesteryl ester transfer protein (CETP) activity. Although LTIP
inhibits CETP activity among different lipoprotein classes, it
preferentially suppresses transfer events involving low density
lipoprotein (LDL), whereas transfers involving high density lipoprotein
as donor are less affected. In this study, we report the purification
of LTIP and the expression of its cDNA in cultured cells.
Purification of LTIP, in contrast to other published protocols, took
advantage of the tight association of this protein with LDL.
Ultracentrifugally isolated LDL was further purified on anti-apoE and
apoA-I affinity columns. Affinity purified LDL was delipidated by
tetramethylurea, and the tetramethylurea-soluble proteins were
separated by SDS-polyacrylamide gel electrophoresis. The protein
migrating at a molecular mass of ~33 kDa was excised from the gel and
its N-terminal amino acid sequence determined. The 14-amino acid
sequence obtained showed complete homology with the sequence deduced
for apolipoprotein F (apoF) cDNA isolated from Hep G2 cells. On
Western blots, peptide-specific antibodies raised against synthetic
fragments of apoF reacted with the same 33-kDa protein in
LTIP-containing fractions purified from LDL and from
lipoprotein-deficient plasma. In contrast to that previously reported,
apoF was shown to be associated almost exclusively with LDL, identical
to the distribution of LTIP activity. The cDNA for apoF was cloned
from a human liver cDNA library, ligated into a mammalian
expression vector, and transiently transfected into COS-7 cells.
Conditioned media containing secreted apoF demonstrated CETP inhibitor
activity, whereas cells transfected with vector alone did not. This
CETP inhibitor activity was efficiently removed from the media by
nickel-Sepharose, consistent with the 6-His tag incorporated into
recombinant apoF. By Western blot, the 6-His-tagged protein had a
molecular weight slightly larger than native apoF. The CETP inhibitor
activity of recombinant apoF possessed the same LDL specificity, oleate
sensitivity, and dependence on lipoprotein concentration as previously
noted for LTIP. We conclude that LTIP and apoF are identical.
One of us (R. E. Morton), in collaboration with D. B. Zilversmit, was the first to identify and partially characterize a
novel protein in human plasma that inhibits cholesteryl ester transfer protein (CETP)1 activity
in vitro (1). Subsequent reports by Son and Zilversmit (2)
and by Nishide et al. (3) have confirmed and extended the
initial characterization of this factor as a unique ~30,000-35,000 molecular weight acidic glycoprotein. This protein, LTIP, equally inhibits the CETP-mediated transfer of TG and CE (1, 2). The mechanism
of inhibition has not been studied in detail; however, limited studies
by us and others (1, 2, 4) suggest that LTIP disrupts the association
of CETP with lipoproteins.
In contrast to the effects of other apoproteins that have been shown to
possess CETP inhibitory activity when assayed at high concentrations
in vitro (2, 5-8), LTIP shows lipoprotein specificity in
its inhibitory capacity. LTIP preferentially suppresses lipid transfers
involving LDL (1.019 < d <1.063 g/ml)(9, 10) while
having the least effect on transfers between VLDL (d <1.006
g/ml) and HDL (1.063 < d <1.21 g/ml). In mass lipid
transfer assays with whole plasma, LTIP supplementation
leads to decreased efflux of CE from LDL to VLDL and increased efflux
of CE from HDL to VLDL, resulting in HDL that are better substrates for
lecithin-cholesterol acyltransferase (10). We have proposed that this
happens because plasma TG, not CETP, is rate-limiting for net lipid
transfer in normal plasma (11, 12). Thus, in the presence of LTIP, less TG is expended by transfer to LDL, allowing transfer from VLDL to HDL
to be increased. Among control subjects, LTIP activity levels in whole
plasma correlate negatively with the rate of transfer between VLDL and
LDL and with HDL size (9). Recent studies have substantiated these
observations in a patient population deficient in LTIP activity (13).
Additionally, we have shown that CETP itself has little preference for
lipid transfer from either LDL or HDL but, rather, the preferential
transfer of CE from HDL seen in normal plasma is a function of LTIP
activity (14). High, yet physiological, concentrations of free fatty acids can inactivate LTIP (15), suggesting that LTIP activity can be
regulated by dietary status. Together, these data strongly suggest that
LTIP alters the overall pattern of lipid transfer events between
lipoproteins by functioning as a traffic policeman, impeding lipid
fluxes between some lipoproteins while allowing others to continue.
Therefore the effectiveness of lipid transfer activity in plasma is
determined by both CETP and LTIP activities.
Our efforts in the past to isolate proteins that inhibit CETP have
consistently supported the conclusion that a single protein is
responsible for most, if not all, of the inhibitory activity observed
in whole human plasma. Purification of LTIP has been hampered by its
tendency to associate with other plasma proteins, most particularly
apoD (10), during purification. Interference with CETP/LTIP assays by
low levels of many dissociating agents commonly used to disrupt
protein-protein interactions has greatly limited the methods that can
be used to dissociate LTIP-protein complexes for further purification.
In this report we have developed an alternative approach to LTIP
purification that takes advantage of our observations that LTIP
activity is primarily associated with LDL in plasma (10) and that a
portion of this LTIP activity is retained on LDL after its isolation by
standard ultracentrifugal techniques. We report here the isolation of
LTIP from human LDL and the cloning and expression of its cDNA.
Materials--
Cholesteryl
[1a,2a(n)-3H]oleate (50 Ci/mmol) was purchased from Amersham Pharmacia Biotech. BSA (fraction
V), diethyl p-nitrophenyl phosphate, and all reagents for
salt and buffer solutions were obtained from Sigma. Phenyl-Sepharose
CL-4B, CNBr-activated Sepharose, and dextran sulfate
(Mr = 500,000) were from Amersham Pharmacia Biotech, and CM52-cellulose was from Whatman. 1,1,3,3-Tetramethylurea, (Sigma) was repurified by distillation within 1 week of use and stored
at 4 °C in the dark. Protein G- and protein A-agarose were purchased
from Pierce.
Lipoprotein Isolation and Radiolabeling--
Fresh human plasma
from the Blood Bank of the Cleveland Clinic Foundation was the source
of VLDL, LDL, and HDL. Lipoproteins were isolated by sequential
ultracentrifugation (16), extensively dialyzed against 0.9% NaCl,
0.01% EDTA, 0.02% NaN3, pH 8.5, and stored at 4 °C.
Lipoproteins were quantitated based on their total cholesterol content.
In some instances, lipoproteins were radiolabeled with
[3H]CE by a lipid-dispersion method (1). ApoA-I was
isolated from fresh human plasma by a combination of
ultracentrifugation, HDL delipidation, and chromatofocusing of human
apoHDL (17).
CETP and LTIP Isolation from Lipoprotein-deficient
Plasma--
Partially purified CETP was isolated from
lipoprotein-deficient human plasma by hydrophobic and ion exchange
chromatography as described previously (18) and stored in 0.27 mM EDTA, pH 7.4. LTIP was partially purified from
lipoprotein-deficient plasma as described previously (1, 10).
CETP and LTIP Assays--
Assays for CETP and LTIP activity were
performed according to our previously published methods (19-21). CETP
activity was routinely measured in assays of lipid transfer between
radiolabeled LDL ([3H]CE LDL) and unlabeled HDL (10 µg
of cholesterol each). At the end of the assay, LDL and HDL were
separated by the phosphate-manganese procedure (19, 20). CETP activity
was determined from the fraction of label transferred to the acceptor
and expressed as percent kt (19).
CETP inhibitor activity was measured in the assays described above or
in assays where lipid transfer was measured between radiolabeled LDL
([3H]CE LDL) and biotinylated LDL (21). After incubation,
these lipoproteins were separated by incubation with avidin-Sepharose. Inhibitor activity is expressed as the percent inhibition of a standard
amount of CETP added to the assay.
Antibodies and Immunoadsorbants--
Goat anti-human apoA-I and
anti-apoE IgG (Calbiochem) were purified from antisera by affinity
chromatography on protein G-agarose as detailed by the manufacturer
(Pierce). Eluted IgG-containing fractions (25-28 mg) were pooled,
dialyzed versus 0.1 M NaHCO3, 0.5 M NaCl, pH 8.3, overnight, and then coupled to 10 ml of
CNBr-Sepharose (Amersham Pharmacia Biotech) following the
manufacturer's protocol. Synthetic fragments of apoF were prepared by
Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid phase chemistry
utilizing a multiple antigen peptide carrier core; antibodies to this
antigen were elicited in rabbits (Bio-Synthesis, Inc., Lewisville, TX).
Goat anti-apoF antiserum, prepared as described previously for apoF
isolated from delipidated HDL by ion exchange chromatography (22), was
the gift of Dr. Petar Alaupovic.
Purification of LTIP from LDL--
Citrated human plasma was
obtained from the Blood Bank of the Cleveland Clinic, and LDL was
isolated by sequential ultracentrifugation (50,000 rpm, 50.2 Ti rotor,
10 °C, 18 h) as the 1.019 < d < 1.063 g/ml fraction
(16). LDL was extensively dialyzed versus 0.9% NaCl, 0.02%
EDTA, pH 8.5. LDL was further purified by chromatography on two
affinity columns. Tandem columns of anti-apoA-I-Sepharose (1 × 19 cm) and anti-apoE-Sepharose (1 × 10 cm) were packed and equilibrated at room temperature with 0.9% NaCl, 0.01% EDTA, pH 7.4. LDL (5 ml, 15-18.5 mg protein) was applied to the columns and eluted
by 0.9% NaCl, 0.01% EDTA, pH 7.4, at a rate of 2 ml/h. Eluted
fractions containing LDL, as determined by cholesterol assay, were
pooled and subsequently extracted by TMU as described below. After
elution, the columns were recycled for subsequent use by extensive
washing with 0.9% NaCl, 0.01% EDTA (pH 11, adjusted with
NH4OH), followed by the same buffer at pH 7.4.
TMU extraction of LDL was performed by a modification of the method
Kane et al. (23). Briefly, in a disposable glass tube, affinity purified LDL (600 µg of protein) was adjusted to 1 ml with
0.9% NaCl, 10 mM EDTA, pH 9, and incubated at 37 °C for
10 min. Pre-warmed TMU (0.75 ml) was added to each 1-ml sample, mixed gently, and incubated at 37 °C in a sealed tube until the apoB-lipid pellicle floated at the top of the tube (2-3 h). After filtration through a glass wool plug, the TMU-soluble proteins were dialyzed against 0.9% NaCl, 0.01% EDTA, pH 7.4, buffer overnight and
concentrated about 10-fold (Centricon 10, Amicon, Beverly, MA).
Electrophoresis and Immunoblot Analyses--
Pre-made 7.5%
SDS-PAGE gels (Integrated Separation Systems, Natick, MA) were pre-run
for 1 h (20 mA) as recommended by the manufacturer. Samples were
combined with 5× SDS-PAGE sample buffer (0.3125 M
Tris-HCl, 25%
When detecting apoproteins on intact lipoproteins, either in serum or
in isolated form, samples were fractionated by one-dimensional agarose
gel electrophoresis (25). Lipoproteins were transferred to
nitrocellulose, which was backed with wetted filter paper, by passive
adsorption for 15 min. Blots were blocked with 5% powdered milk, 2%
calf serum, and 1% BSA in 0.9% NaCl, 0.1% EDTA, pH 7.6. Buffers for
the blocking step and all subsequent steps contained 1% BSA in 0.9%
NaCl, 0.1% EDTA, pH 7.6, the latter being important to prevent lipid
oxidation in the nitrocellulose-bound lipoproteins. Immune complexes
were visualized by reaction with ECL as indicated above. Alternatively,
gels were dried and lipoproteins visualized by staining with Fat Red 7B
(25).
Cloning of ApoF cDNA--
Based on a 14-amino acid
N-terminal sequence obtained for the 33-kDa protein from LDL, a
26-nucleotide primer (5'-GAGGACTGTGAGAATGAGAAGGAGCA-3') was synthesized
(Genosys, The Woodlands, TX) and used to screen a human liver cDNA
library by 3'-rapid amplification of cDNA ends PCR with
Marathon-Ready cDNA (CLONTECH, Palo Alto, CA).
The single, 1.2-kilobase pair product was ligated into a pCR 3.1-Bi
plasmid by TA cloning (Eukaryotic TA Cloning Kit, Invitrogen, Carlsbad, CA) and transformed into Escherichia coli (TOP10F')
according to the methods described by the manufacturer (Invitrogen).
DNA sequencing was performed with a BigDye Terminator Cycle Sequencing Kit using the ABI PRISM 377XL DNA sequencer (26). Based on homology of
sequenced clones with apoF (27), additional gene-specific primers (see
"Results") were prepared to permit PCR cloning of the cDNA
fragment of apoF encoding for amino acids 147-308, which corresponds
to the complete sequence of the secreted protein. A 492-base pair
HindIII/XbaI restriction fragment was subcloned into a mammalian expression vector, pSecTag (Invitrogen), to permit the
synthesis and secretion of apoF with six C-terminal histidine residues.
Express of ApoF cDNA in COS-7 Cells--
COS-7 cells were
transfected by the LipofectAMINE method (Life Technologies, Inc.).
Briefly, in six-well culture plates, 1 × 105 cells
were seeded per well in 2 ml of the appropriate growth medium
supplemented with 5% fetal bovine serum and incubated at 37 °C in a
CO2 environment until the cells were 50% confluent. Each
well received DNA (1 µg) and 5 µl of LipofectAMINE in a final volume of 1 ml of serum-free medium. After incubating with DNA complexes for 24 h, cells were washed and incubated in serum-free media for 48 h. The media were collected, briefly centrifuged, concentrated on Centricon 10 membranes (Amicon), and CETP inhibitory activity in the conditioned media determined.
Other Analytical Methods--
Protein was quantitated by the
method of Lowry et al. (28) as modified by Peterson (29),
with BSA as standard. Total cholesterol of lipoproteins was assayed by
a colorimetric, enzymatic method using Cholesterol 100 reagent kit
(Sigma). For amino acid analyses, proteins were transferred to
polyvinylidene difluoride membranes by electrophoresis in transfer
buffer (10 mM CAPS, 10% methanol, pH 11.0) at 10 °C for
2 h (30). Transferred proteins were visualized by brief staining
with 0.1% Coomassie Blue (Sigma) in 50% methanol, and bands of
interest were excised. N-terminal protein sequence analysis was carried
out with an Applied Biosystems Procise, model 492 protein sequencer
fitted with a 140c microgradient system, 785A programmable absorbance
detector, and a 610A (version 2.1) data analysis system (31, 32).
LTIP Purification from LDL--
To facilitate the purification of
LTIP by an approach that minimizes the potential for apoD contamination
(10), we devised a protocol to purify this protein from LDL, which
contains most all LTIP activity in native plasma (10). Based on the
recovery of LTIP activity in the d >1.21 g/ml fraction
after ultracentrifugation, compared with that in lipoprotein-deficient
plasma prepared by dextran sulfate-manganese precipitation (which
dissociates all LTIP from lipoproteins (9)), we have estimated that
~30% of plasma LTIP activity is retained in this lipoprotein
fraction after isolation. Although LDL only retains a small portion of its original LTIP, this approach provides a means of avoiding apoD
contamination since apoD resides on apoA-I-containing lipoproteins (33).
Beginning with isolated LDL, the purification scheme involved affinity
adsorption of minor contaminating proteins, apoE and A-I, with solid
phase antibodies to these apoproteins, followed by TMU extraction (23)
of the affinity purified LDL. The lipid-free, TMU-soluble proteins from
LDL exhibited inhibitory activity. This purification scheme typically
resulted in ~530-fold purification of CETP inhibitor activity with
~8% recovery (Table I). The CETP inhibitor activity in the TMU-soluble LDL fraction manifested the same
preference for lipid transfers involving only LDL and suppression by
sodium oleate as we have previously observed for LTIP in the
CM-cellulose fraction of LTIP isolated from lipoprotein-deficient plasma (Table II). In contrast to the
inhibitory activity in the CM-cellulose and LDL-TMU fraction,
apolipoprotein A-I, which suppresses CETP activity under some
conditions (2, 15), was unaffected by 10 µM oleate (data
not shown). The TMU-soluble fraction from LDL was further resolved by
SDS-polyacrylamide gel electrophoresis (Fig.
1, 3rd lane). A
slightly diffuse staining band near 33 kDa was observed in the
LDL-derived proteins, but neither VLDL nor HDL contained detectable
protein of similar molecular weight, consistent with the near-exclusive
association of LTIP with LDL. This molecular mass compares well with
that determined for LTIP (30-35 kDa) based on the elution of LTIP
activity on gel filtration under dissociating conditions (1). Although
other small molecular weight proteins (<10,000 molecular weight,
presumably apoCs) were present in the LDL-derived fraction, these
proteins were not found in LTIP prepared from lipoprotein-deficient
plasma (Fig. 1, 2nd lane), suggesting that these
contaminants do not contribute to the LTIP activity measured in the LDL
protein fraction. Assuming the inhibitor activity in the TMU-soluble
LDL fraction is due to the 33-kDa component, 35 pmol of inhibitor
protein was sufficient to suppress CETP activity by 50% in the
standard LDL to LDL transfer assay, which contains 33 pmol of
lipoprotein. This equates to an IC50 of ~0.05
µM.
Following transfer to polyvinylidene difluoride membranes, the 33-kDa
band was excised and its N-terminal amino acid sequence determined.
N-terminal sequence analysis yielded a single sequence consisting of
SLPTEDXENEKEQA. A search of sequence data bases revealed
complete homology of this 14-amino acid sequence with amino acids
147-160 of human apoF (27). Amino acid 147 corresponds to the
N-terminal amino acid of secreted apoF, which is derived from the
cleavage of a larger precursor protein. No other proteins of similar
sequence were identified. ApoF has a calculated isoelectric point of
4.11, which is near the acidic pI we and others (1, 2) previously
reported for partially purified LTIP under dissociating conditions.
Anti-ApoF Antibodies--
Peptides fragments of apoF, selected
based on surface probabilities and lack of homology with other proteins
in published data bases, were synthesized and injected into rabbits to
raise antibodies against selected regions of apolipoprotein F. Antibodies to fragment 1 (anti-apoF Ab1, amino acids 1-14 of mature,
secreted apoF, i.e. SLPTEDCENEKEQA) and fragment 2 (anti-apoF Ab2, amino acids 94-107, i.e. QYYQDQKDANISQP)
reacted with the same 33-kDa band on SDS-PAGE/immunoblots of two
distinct preparations of CETP inhibitor activity (CM-cellulose fraction
and the LDL-TMU fraction, Fig. 2).
Antibodies to fragments of apoF could also be used to detect this
apoprotein in serum resolved by one-dimensional agarose electrophoresis
and subsequently blotted onto nitrocellulose (Fig. 3, data shown for anti-apoF Ab2 only).
ApoF was detected almost exclusively on LDL in whole serum (Fig. 3).
Minor immunoreactivity was noted with VLDL, whereas HDL in whole serum
(Fig. 3) or in isolated form (data not shown) did not react with
anti-apoF Ab2. Notably, the distribution of apoF in serum is the same
as the distribution of LTIP activity in plasma determined by gel
filtration (10). Although the high sample concentrations used in these blots permitted us to assess whether low levels of apoF were associated with other lipoproteins, they do not reflect the relative apoF content of these fractions due to the nonlinear response in this dose
range. Based on the immunoreactivities of serially diluted serum and
isolated LDL fractions, we determined that isolated LDL contains
~25% of the apoF originally on this lipoprotein in serum, with the
reminder lost to the d >1.21 g/ml fraction during centrifugation (data not shown). This closely mirrors the recovery of
LTIP activity in the d >1.21 g/ml fraction of
plasma compared with the LTIP activity in lipoprotein-deficient plasma
prepared by divalent cation precipitation (see above); divalent cations effectively dissociate all LTIP from lipoproteins (9, 34).
This lipoprotein distribution for apoF is contradictory to that
previously reported by Alaupovic and co-workers (22). With a goat
antiserum against apo"F" provided to us by Dr. Alaupovic, the major
antigen for this antibody was recovered in the lipoprotein-free fraction (d >1.21 g/ml) of plasma, with minor reactivity
detected in HDL and LDL (data not shown). This antigen distribution is distinct from that obtained with antibodies prepared against pure apoF
fragments (Fig. 3), clearly showing that these two antisera are
primarily directed against different proteins. Data from Day et
al. (27) also suggest that this goat antibody reacts with a number
of plasma proteins. We interpret these data to mean that the original
antisera against apoF (that prepared by Alaupovic et al.) is
not specific for apoF, although it does detect apoF under certain
conditions. Based on our data with antibodies directed against
synthetic fragments of apoF, we conclude that apoF is primarily an
LDL-associated apoprotein although minor amounts may be recovered in
other lipoproteins after ultracentrifugation.
It is important to note that although anti-apoF Ab2 antibodies could
react with apoF on intact LDL when the lipoprotein was adsorbed onto
nitrocellulose, they did not react with apoF on LDL in
solution. That is anti-apoF Ab2 antibodies (nor anti-apoF Ab1
antibodies) were not able to mediate the immunoadsorption of
LDL-associated apoF even when added in apparent excess (data not
shown). Consistent with this, anti-apoF peptide antibodies were not
able to block LTIP activity nor could they mediate immunoadsorption of
inhibitory activity to a significant degree. This indicates that the
antigenic sites for these antibodies are not accessible on native apoF.
ApoF Cloning and Expression--
Based on the 14-amino acid
sequence obtained for the 33-kDa band shown above, we prepared a
26-nucleotide primer (see "Experimental Procedures") and screened a
human liver cDNA library by 3'-rapid amplification of cDNA ends
PCR. The single, 1.2-kilobase pair product was ligated into the pCR3.1
vector and transformed into E. coli. The nucleotide sequence
of isolated clones was homologous with the published sequence for the
apoF cDNA cloned from Hep G2 cells (27). The nucleotide sequence of
liver apoF was 95.6% homologous to Hep G2 cDNA; the bulk of this
non-identity was due to the absence of one of the two 32-nucleotide
repeats present in the 3'-untranslated region of the Hep G2 sequence
(base 1545-1608). Subsequently, additional primers were prepared to
permit PCR cloning of a cDNA fragment of apoF coding for amino
acids 147-308, corresponding to the complete sequence of secreted apoF
(Fig. 4). This fragment was subcloned
into a mammalian expression vector, pSecTag, to permit the synthesis
and secretion of apoF with six C-terminal histidine (6-His)
residues.
After transfection into COS-7 cells and secretion, media from cells
transiently transfected with vector containing apoF cDNA expressed
inhibitor activity that suppressed CETP in a dose-dependent manner, whereas cells transfected with the vector (Fig.
5A) or LipofectAMINE alone
(data not shown) did not markedly affect CETP activity. Treatment of
conditioned media with nickel-agarose to bind the 6-His-tagged
recombinant apoF removed >93% of the inhibitor activity. The proteins
eluted from the nickel-Sepharose matrix also demonstrated CETP
inhibitor activity (Fig. 5B). The eluted protein fraction
was >95% pure recombinant apoF as determine by SDS-PAGE and
immunoblot analysis (Fig. 5C). The apparent molecular weight
of recombinant apoF was slightly higher than that of native apoF
(~35,000 versus ~33,000), consistent with the additional amino acids incorporated into the expressed protein. Recombinant apoF
showed the same specificity for lipid transfer assays involving only
LDL and inactivation by oleate as previously noted for LTIP (10, 15)
(Fig. 6 A and B).
Although the ratio of inhibitor activities in the LDL/LDL and LDL/HDL
assays tended be lower for recombinant apoF (5.8 ± 1.8 (n = 3) versus 7.4 ± 1.8 (n = 4) for apoF and CM-LTIP, respectively) this
difference was not significant (p = 0.47). Based on its
protein concentration, determined by comparison of
nickel-Sepharose-purified recombinant apoF with known proteins on
protein-stained SDS-PAGE gels, we estimate a specific activity of 14%
inhibition/µg of protein, resulting in an IC50 for
recombinant apoF of <0.15 µM. This is comparable
to the 0.05 µM IC50 value determined for
purified, native apoF (LDL-TMU fraction). Like CM-LTIP, the inhibitory
activity of recombinant apoF was strongly and inversely related to the
assay content of lipoproteins (Fig. 6C). Recombinant apoF
equally suppressed the capacity of CETP to mediate TG and CE transfers
(ratio of TG/CE inhibition = 0.95 ± 0.06, mean ± S.D.,
n = 5), as was the case for CM-LTIP (0.99 ± 0.10, mean ± S.D.), indicating that inhibition is achieved by blocking
all CETP function, not by modifying the capacity of CETP to interact
with specific lipid substrates.
Our current understanding of LTIP derives from studies mostly
performed on the CM-cellulose fraction of partially purified LTIP
isolated from lipoprotein-deficient plasma. Based on measurements of
LTIP activity, we and others (1-3, 10) have shown that LTIP is an
acidic glycoprotein (pI The data presented in this report strongly support the identity of LTIP
and apolipoprotein F. Similarities in molecular weight, isoelectric
point, lipoprotein distribution in plasma, and co-recovery in the
d >1.21 fraction after ultracentrifugation provide strong evidence for such a conclusion. This conclusion is further strengthened by our finding that two preparations of LTIP activity isolated by
independent protocols each contain apoF. And finally, recombinant apoF
manifests CETP inhibitory activity that has specificity for inhibiting
transfer reactions involving LDL and is suppressed by fatty acids as
has been reported for LTIP (9, 10, 15, 21). Based on these several
lines of evidence, we conclude that LTIP is apoF. In assigning
this identity, we follow the nomenclature of Day et al. (27)
where apoF is defined as the C-terminal 162 amino acids derived from
the cleavage of a 308-amino acid precursor. Given the location of the
putative signal peptide, it appears likely that this cleavage occurs
extracellularly, although this has not been determined.
The identification of LTIP as apoF finally assigns a function to this
little understood apoprotein. We suggest that this function of apoF is
unique among apoproteins since the reported CETP inhibitory activities
of better studied apolipoproteins have not been borne out by further
studies in more physiological settings. For example, although purified
apoA-II was shown in several studies to inhibit CETP activity in
in vitro assays (5-7), CETP activity is not decreased in
transgenic mice co-expressing apoA-II and CETP (35), and HDL particles
containing apoA-I only or both apoA-I and apoA-II are equal in their
CETP substrate capacity (36). In other studies, pure apoA-I, -C-I, and
-C-III inhibited CETP activity with IC50 values of 0.5 to
0.8 µM (2, 7). However, co-expression of apoC-III in CETP
transgenic mice does not decrease plasma CETP activity in these mice
(35), and co-expression of human apoA-I in CETP transgenic mice leads
to increased, not decreased, CETP activity (37). Consistent with this
finding, in other studies isolated apoA-I, apoA-II, and phospholipid
transfer protein have been shown to stimulate CETP activity under some
assay conditions (38-41). The baboon plasma CETP inhibitor (N-terminal
38 amino acids of apoC-I) reported by Kushwaha et al. (8)
also suppresses CETP activity in vitro, but with an
IC50 value of ~100 µM (7) it seems unlikely
that it has significant in vivo function in human plasma
where apoC-I concentrations are In contrast to that previously reported, we demonstrate here that apoF
is an LDL-associated protein and that little, if any, is associated
with HDL in serum. The observation that apoF can be isolated from HDL
suggests that some of this apoprotein may redistribute to this
lipoprotein fraction with ultracentrifugation. Since the majority of
LDL-associated apoF is lost during ultracentrifugation, the recovery of
apoF in HDL under some centrifugation conditions seems probable. The
near-exclusive association of LTIP/apoF with LDL and the observation
that LTIP preferentially suppresses transfer reactions with LDL
suggests that lipoprotein association is important to its function.
This is consistent with limited kinetic analysis which suggests that
LTIP binds to lipoproteins and inhibits CETP by preventing its
association with the lipoprotein surface (4), and with our current data
and with published data (1, 2, 4) showing that inhibitor activity is
inversely related to assay lipoprotein concentration. With native
LTIP/apoF (LDL-TMU fraction), we observed here that a 1:1 mole ratio of
inhibitor to lipoprotein is sufficient to suppress CETP activity by
50%. This clearly suggests that there are a limited number of sites where LTIP/apoF and CETP compete for interaction on the lipoprotein surface. The plasma concentration of apoF has been reported to be 27 µg/ml (0.82 µM) (43), which is similar to a 70 µg/ml
(~2 µM) value that can be estimated from our
purification data. Thus, at a typical plasma LDL concentration of 1.87 µM (4), apoF is sufficiently abundant to reside on most
LDL particles. This suggests that most LDL particles in plasma may
support CETP activity at a reduced level due to the competition of apoF
and CETP for residence on the lipoprotein surface.
The coupling of lipoprotein association and inhibitory function
suggests that LTIP activity could be altered if its binding to
lipoproteins is prevented or altered, perhaps by changes in lipoprotein
composition. In preliminary studies, we have observed that a portion of
LDL-associated LTIP/apoF is lost to the VLDL fraction when plasma is
incubated at 37 °C overnight in the presence of lecithin-cholesterol
acyltransferase inhibitors.2
The elucidation of lipoprotein properties that facilitate LTIP binding
and the metabolic and pathologic conditions that alter this binding are
under investigation.
In summary, we present data demonstrating that apoF possess the same
biochemical and functional properties as LTIP. The weight of these data
leads to the conclusion that apoF is LTIP. This the first report of a
function for this minor apoprotein. Based on our understanding of LTIP
from in vitro studies of its functional properties (1-4, 9,
10, 13-15), we suggest that apoF is an important regulator of
cholesterol transport between plasma lipoproteins which directly
affects the sterol content of individual lipoprotein fractions. The
advances presented in this report will permit a more direct testing of
this conclusion than previously possible.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
-mercaptoethanol, 50% glycerol, 0.5 mg/ml bromophenyl blue, pH 8.3) and heated for 5 min at 100 °C. Samples were applied and gels developed at 20 mA for 1.5 h. Proteins were visualized by silver staining (Dai-ichi silver stain kit, Integrated Separation Systems) or Coomassie Blue (Sigma). For Western blot analyses, proteins in SDS-PAGE gels were electrotransferred to nitrocellulose at 4 °C overnight as described by Towbin et
al. (24). Membranes were blocked with 5% dry milk in
phosphate-buffered saline, reacted with the indicated antibody followed
by reaction with peroxidase-conjugated secondary antibody. Reaction
products were visualized by reaction with ECL (Amersham Pharmacia
Biotech) and exposure to x-ray film.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
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Purification of LTIP activity from LDL
Characterization of CETP inhibitory activity
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Fig. 1.
SDS-polyacrylamide gel of LTIP preparations
and reference lipoproteins. SDS-treated, reduced proteins were
separated by SDS-PAGE as described under "Experimental Procedures."
1st lane, molecular weight standards;
2nd lane, CM-cellulose fraction of LTIP;
3rd lane, TMU-soluble proteins from LDL;
4th lane, TMU-soluble proteins from VLDL;
5th lane, TMU-soluble proteins from HDL.
Fractions were loaded at similar protein levels (0.1-0.3 µg).
Proteins were visualized by silver staining. Note: the major band in
the CM-cellulose fraction of LTIP is apoD as determined by N-terminal
amino acid sequencing. Thus, the novel protein derived from LDL is
slightly smaller than apoE and slight larger than apoD.
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Fig. 2.
Western blot of inhibitor fractions with
anti-apoF peptide antibodies. CETP inhibitor-containing
preparations isolated from LDL or from lipoprotein-deficient plasma by
phenyl-Sepharose and CM-cellulose chromatography (CM,
i.e. standard LTIP preparation) were electrophoresed on
SDS-PAGE gels and blotted onto nitrocellulose. Samples were incubated
with the indicated anti-apoF peptide antiserum or preimmune serum
(1:100 dilution). Immune complexes were reacted with anti-rabbit IgG
peroxidase-conjugated IgG (1:1000 dilution) and visualized by enhanced
chemiluminescence. Similar amounts of CETP inhibitor activity were
applied for CM and LDL inhibitor fractions (42 and 72% inhibition,
respectively).
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Fig. 3.
Immunoreactivity of sera and lipoprotein
fractions. The indicated samples were electrophoresed on agarose
gels, blotted onto nitrocellulose, and incubated with isolated
anti-apoF peptide IgGs (apoF Ab1 + Ab2, 125 µl of serum
equivalent each) or IgG from non-immune sera (250 µl of serum
equivalent). Immune complexes were reacted with anti-rabbit IgG
peroxidase-conjugated IgG (1:5000 dilution) and visualized by ECL. LDL
and d <1.21 samples were concentrated 2.4- and 1.9-fold
relative to starting plasma, respectively. Org,
origin.
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Fig. 4.
Nucleotide sequence of Hep G2 apoF cDNA
and a clone isolated from a human liver cDNA library. The
nucleotide sequence of apoF cDNA fragment amplified by PCR from a
human cDNA library is compared with the published sequence of a
portion of the apoF cDNA from Hep G2 cells (27). PCR primers are
indicated by the underlined sequences. Nucleotides indicated
in bold are unique to the clone and were integrated into
primers to aid subcloning. Numbers in parentheses
note the amino acid numbers for secreted apoF, which correspond to
amino acids 147 and 308 of the full-length protein. The stop codon,
noted by * in the native sequence, was deleted when cloned into
pSecTag.
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Fig. 5.
CETP inhibitor activity in conditioned
media. COS-7 cells were transfected with the indicated vector by
the LipofectAMINE method. After 24 h, cells were washed and
incubated in serum-free media for 48 h. CETP inhibitory activity
in the conditioned media was assayed as described under "Experimental
Procedures." A, dose response of CETP inhibitor activity
in conditioned media from cells expressing vector + apoF (closed
squares) or vector alone (closed circles). Media were
concentrated 10-fold for assay of inhibitor activity. Values are
mean ± S.D. B, nickel-Sepharose-retained proteins (100 µl) were eluted and assayed for CETP inhibitor activity. Condition
media (45 ml) were adjusted to 500 mM NaCl and treated with
5 ml of nickel-Sepharose. Following a 150 mM imidazole
wash, proteins were eluted with 500 mM imidazole and
concentrated 30-fold relative to the starting media. Values are
mean ± S.D. C, nickel-Sepharose purified proteins were
applied to SDS-PAGE gels and stained for protein or electrotransferred
and immunoblotted with anti-apoF Ab2 antisera. Lanes 1 and
3, nickel-Sepharose-purified proteins from apoF-transfected
cells; lanes 2 and 4, nickel-Sepharose-purified
proteins from cells transfected with vector alone.
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Fig. 6.
Properties of CETP inhibitory activity in
COS-7 conditioned media. A, the CETP inhibitor activity
expressed in assays measuring transfer from LDL to biotin-LDL (LDL/LDL)
and LDL to HDL (LDL/HDL) was determined. Samples are as follows: LTIP
(CM-cellulose fraction) and recombinant apoF (rApo F,
conditioned media from COS-7 cells transiently expressing apoF). For
clarity, activity values are expressed as that measured in 50 µl (15 µg protein) of LTIP or that in 1 ml of 10× concentrated media for
recombinant apoF. LDL specificity is calculated as the ratio of these
two activities (LDL/LDL/LDL/HDL). These results are representative of 4 and 3 determinations for LTIP and recombinant ApoF, respectively.
B, inactivation of inhibitor activity by sodium oleate. LTIP
(CM-cellulose fraction) and recombinant ApoF inhibitor activities were
assayed by the LDL to biotin-LDL assay in the presence of the indicated
amount of oleate. Data are the mean ± S.D. for five
(LTIP) or three (rApoF) determinations.
C, dependence of inhibitor activity on the assay
concentration of lipoproteins. LTIP (CM-cellulose fraction) and
recombinant ApoF inhibitor activities were determined in assays
measuring lipid transfer from LDL to HDL. The assay content of
lipoproteins was varied as indicated while maintaining an LDL to HDL
ratio of 1. Data are the mean ± S.E. for 2-4
determinations.
DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References
4) of 30,000-35,000 molecular weight
that is associated almost exclusively with LDL. This LDL-specific association is consistent with the observation that LTIP has greater suppressive activity toward CETP activity involving LDL compared with
other lipoprotein substrates (9, 10). These properties of LTIP, based
on its activity, are important criteria for comparing LTIP with CETP
inhibitory activity isolated from plasma by other approaches.
15 µM (42). Overall, the capacity of various known apoproteins to stimulate and/or inhibit
CETP activity has been highly assay-dependent and not supported by in vivo studies. Additionally, effects of these
isolated apoproteins have not been demonstrated in more physiologic
assays that approximate the conditions of whole plasma. By comparison, the inhibitory activity of LTIP/apoF has been shown in a wide variety
of assays using isolated lipoproteins, synthetic liposomes, and
unfractionated plasma as substrate. The estimated IC50
value for LTIP/apoF is 0.05-0.15 µM, much lower than
that reported for other apoproteins.
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ACKNOWLEDGEMENTS |
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The Molecular Biotechnology Core of the Lerner Research Institute is acknowledged for performing N-terminal amino acid sequence analyses and DNA sequencing. We thank Dr. Satya P. Yadav for help in protein sequence analyses.
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FOOTNOTES |
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* This research was supported by Grant HL29582 from the NHLBI, National Institutes of Health.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.
To whom correspondence should be addressed: Cell Biology, NC10,
Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-5850; Fax: 216-444-9404; E-mail:
mortonr{at}cesmtp.ccf.org.
The abbreviations used are: CETP, cholesteryl ester transfer protein; LTIP, lipid transfer inhibitor protein; CE, cholesteryl ester; TG, triglyceride; VLDL, very low density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein; apoF, apolipoprotein F; TMU, 1,1,3,3-tetramethylurea; BSA, bovine serum albumin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; CAPS, 3-(cyclohexylamino)propanesulfonic acid.
2 R. E. Morton, unpublished observations.
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