(Received for publication, June 2, 1995; and in revised form, July 11, 1995)
From the
Two inhibitors of cynomolgus monkey cholesteryl ester transfer protein were evaluated. One, a monoclonal antibody made against purified cynomolgus monkey cholesteryl ester transfer protein, was capable of severely inhibiting triglyceride transfer, but had a variable effect on cholesteryl ester transfer. At low antibody to antigen ratios, there was what appeared to be a stoichiometric inhibition of cholesteryl ester transfer, but at high antibody to antigen ratios the inhibition of cholesteryl ester transfer was completely relieved, even though triglyceride transfer remained blocked. Fab fragments of the antibody had no effect whatsoever on cholesteryl ester transfer, but were capable of completely blocking triglyceride transfer. The other inhibitor, 6-chloromecuric cholesterol, severely inhibited cholesteryl ester transfer with minimal inhibition of triglyceride transfer. When both inhibitors were added to the assay, both cholesteryl ester and triglyceride transfer were inhibited; an indication that the inhibitors did not compete for the same binding site on cholesteryl ester transfer protein.
When the antibody was given subcutaneously to cynomolgus monkeys at a dose which inhibited triglyceride transfer in the plasma by more than 90%, there was no detectable effect on the high density lipoprotein (HDL) cholesterol level, but the HDL triglyceride levels decreased from 13 ± 2 to 1 ± 0 mol/mol of HDL (mean ± S.D.); an indication that the antibody uncoupled cholesteryl ester and triglyceride transfer in vivo. The 6-chloromecuric cholesterol could not be evaluated in vivo because it is a potent lecithin:cholesterol acyltransferase inhibitor. The fact that cholesteryl ester transfer can be inhibited without effect on triglyceride transfer and, conversely, that triglyceride transfer can be inhibited without effect on cholesteryl ester transfer indicates that these two lipids are not transferred by a single, non-discriminatory process.
Cholesteryl ester transfer protein is a plasma protein that
catalyzes the redistribution of neutral lipids, triglycerides,
cholesteryl esters, retinyl esters, and possibly other lipophilic
compounds, among lipoproteins(1) . In its absence,
neutral-lipid transfer between lipoproteins is essentially nonexistent.
The mechanism by which CETP ()catalyzes the redistribution
of neutral lipids is not entirely understood. Two models have been
proposed: the shuttle model (2) in which CETP acts as a
carrier, transporting neutral lipids back and forth between donor and
acceptor; and the ternary complex model (3) in which donor,
acceptor, and transfer protein come together in a single complex for
neutral-lipid exchange. The shuttle model has the transfer process
divided into 7 steps: association (or docking) of CETP with the donor
lipoprotein; transfer of the neutral lipid from the donor to CETP;
dissociation of CETP-lipid complex from the donor; diffusion of the
complex through the surrounding medium; docking of the CETP-lipid
complex with the acceptor; transfer of the CETP-associated lipid to the
acceptor; and finally, desorption of CETP from the acceptor. If the
donor and acceptor are identical, then there are only 4 elementary
steps: association, exchange, dissociation, and diffusion. With the
exceptions of the dissociation and diffusion components, the same
processes are required in the ternary model, although not necessarily
in the same order. Finally, it is not known whether CETP dissociates
from donors or acceptors without neutral lipid; the inability to do so
would categorize it as an ``exchange'' protein rather than a
``transfer'' protein.
CETP activity has been detected in the plasma of several species (4, 5, 6, 7, 8, 9, 10) . Of those, the cynomolgus monkey protein is the most similar in primary structure to the human protein(9) . There are just 20 amino acids out of 476 which are different between the two proteins and all but two of these substitutions are conservative. Most noteworthy, however, is the observation that the carboxyl-terminal 38 amino acids of the two proteins are identical, for that is the region of CETP thought to be essential for lipid transfer(11, 12, 13, 14, 15) . This high degree of homology between the human and cynomolgus monkey proteins implies that observations made using the monkey protein are germane to our understanding of human CETP structure and function.
An unanswered question regarding human CETP is whether it has more than one neutral lipid binding site. Early studies with both crude and purified CETP preparations indicated that some mercury-containing compounds were capable of inhibiting triglyceride transfer without inhibiting cholesteryl ester transfer(16, 17) ; an observation compatible with the premise that two neutral lipid binding sites were present. The present paper addresses that question using cynomolgus monkey CETP as the transfer protein, and shows that, in vitro, not only can triglyceride transfer can be inhibited without effect on cholesteryl ester transfer, but also that cholesteryl ester transfer can be inhibited without effect on triglyceride transfer. In addition, we present evidence that cholesteryl ester transfer can be uncoupled from triglyceride transfer in vivo.
Recombinant cynomolgus monkey CETP was purified from the culture media of Chinese hamster ovary cells that had been transfected with cynomolgus monkey CETP cDNA exactly as described previously(18) . SDS-polyacrylamide gel electrophoresis and amino-terminal sequence analysis indicated that the CETP isolated by this method was >97% pure.
Monoclonal antibodies were prepared using the purified, recombinant CETP essentially as described previously(11) . These antibodies were designated CMTP-1 and CMTP-2. CMTP-1 had no effect on either cholesteryl ester or triglyceride transfer when evaluated in the radioactivity-based assay described here or a fluorescence-based assay described previously(19) . At certain concentrations (see ``Results'') CMTP-2 inhibited both cholesteryl ester and triglyceride transfer in both assays.
CMTP-2 antigen binding fragments were prepared and purified exactly as described by Margulies(20) , except that the IgG was purified from the ascites using a commercially available kit (E-Z-SEP for ascites, Pharmacia Biotech, Piscataway, NJ). Analysis of the Fab preparation by SDS-polyacrylamide gel electrophoresis showed a major band of approximately 50 kDa and no detectable IgG.
The compound 6-chloromecuric cholesterol (U-617) was synthesized and purified by chemists at the Upjohn Co.
In the absence of transfer protein, redistribution of radioactivity was essentially zero. In the presence of CETP, the distribution of the radioactive lipids between the LDL and HDL approached equal specific activity. This occurred regardless of whether the radioactive lipid was initially in LDL or HDL. The redistribution kinetics were those of a simple, two-pool, closed system, in which there was no appreciable net mass transfer.
Fig. 1shows a typical radioactivity redistribution pattern when LDL was the donor and HDL the acceptor. The HDL radioactivity is expressed as a fraction of the expected equilibrium value, where equilibrium is defined as the point at which the specific activity of a given lipid is the same in the donor and acceptor. Note that both the triglyceride and the cholesteryl ester radioactivity content of HDL were approaching a plateau by 8 h (Fig. 1), even though their mass ratios were different (Table 1). That was taken as indication that the CETP-mediated redistribution of the radioactivity was behaving as predicted.
Figure 1:
Effect of cynomolgus monkey
CETP on the redistribution of neutral lipids between LDL and HDL. Human
LDL (11 µg of LDL protein) containing
[4-C]cholesteryl oleate (20,000 dpm) and
[9,10-
H]triolein (46,000 dpm) was incubated with
cynomolgus monkey HDL (39 µg of HDL protein) in the presence (squares) and absence (circles) of cynomolgus monkey
LPDP for the times indicated. The reaction was stopped by precipitation
of the LDL and the radioactivity in the supernatant (HDL) measured. The
chemical composition of the LDL and HDL used in this assay is contained
in Table 1. Equilibrium was defined as the point at which the
distribution of a given radioactive lipid between LDL and HDL was the
same as that lipids mass. The data were analyzed by a non-linear least
squares method using a simple first order reaction model. The good
agreement of the theoretical curves with the experimental points was
taken as support for the model.
,
, cholesteryl ester
radioactivity;
,
, triglyceride
radioactivity.
To evaluate CETP inhibition, that
inhibitor was added at the indicated concentration and the assay
mixture incubated for 3 h. The CMTP-2 and the CMTP-2 Fab fragments were
contained in saline. The U-617 was contained in MeSO. The
final Me
SO concentration in the reaction mixture was 1%
(v/v); a concentration that had no detectable effect on the assay.
The plasma cholesterol and triglyceride concentrations and the HDL composition and Stokes' radius were determined as described previously (22) .
Fig. 2A shows the effect of increasing CMTP-2
concentration on the redistribution of radioactivity between LDL and
HDL, as measured with cynomolgus monkey LPDP as the source of CETP.
Triglyceride transfer was progressively inhibited with increasing
amounts of the antibody, whereas cholesteryl ester transfer was
progressively inhibited only at antibody concentrations 16
nM. Beyond this point, the inhibition decreased with
increasing antibody concentration until at concentrations near 100
nM, cholesteryl ester transfer was almost normal. Subsequent
studies (not shown) demonstrated that at CMTP-2 concentrations above 1
µM, cholesteryl ester transfer was not inhibited
whatsoever.
Figure 2:
Effect of CMTP-2 and CMTP-2 Fab on
CETP-mediated neutral lipid transfer. The indicated amount of antibody (A) or Fab (B) was added to the standard CETP assay
ingredients (see ``Materials and Methods'') and those
mixtures incubated at 37 °C for 3 h, after which the reaction was
stopped and the amount of radioactivity present in the HDL quantified.
Cynomolgus monkey LPDP (100 µl) was the source of CETP. Control
tubes (containing no antibody or Fab) were run in parallel and the HDL
radioactivity in a given assay tube expressed as a percentage of the
HDL radioactivity in the control tube. The latter values were:
[4-C]cholesteryl ester (filled
squares), 9,917 dpm; [9,10-
H]triglyceride (open squares), 18,921 dpm. The icons (a-d) in the
lower portion of the graphs represent: a, antibody with both
antigen binding sites occupied with CETP; b, antibody with
only one of the antigen binding sites occupied with CETP; c and d, Fab fragments occupied with
CETP.
The icons in the lower portion of the graphs are intended to show the theoretical relationship between antigen (depicted as the gray globular structure) and antibody (depicted as the black y-shaped structure) at various antibody concentrations. If the affinity of the antibody for CETP is high, then at antibody concentrations below stoichiometry the antibody should be saturated with CETP, i.e. both antigen binding sites should be occupied. This form is depicted by the icon (a) in the lower left of Fig. 2A. As the antibody concentration increases, the degree of saturation decreases, such that at antibody concentrations well above stoichiometry only one of the antigen binding sites should be occupied. That form of the antibody-CETP complex is depicted by the icon (b) in the lower right portion of Fig. 2A.
We hypothesized that only the saturated form of the antibody (Fig. 2A, icon a) is capable of inhibiting cholesteryl ester transfer, and the loss of inhibition evident at high antibody concentrations (>16 nM) coincides with the accumulation of the hemisaturated form of the antibody (Fig. 2A, icon b). To test this premise, we produced antibody binding (Fab) fragments of CMTP-2 and evaluated the effects of those Fab fragments on cholesteryl ester and triglyceride transfer. The results of those experiments are shown in Fig. 2B. The CMTP-2 Fab fragments, like the intact antibody, were capable of completely inhibiting triglyceride transfer, but no inhibition of cholesteryl ester transfer was detectable, even at Fab concentrations near 1 µM. These experiments not only support the hypothesis that both antigen binding sites on CMTP-2 must be occupied for cholesteryl ester transfer to be inhibited, but also demonstrate that CETP-mediated cholesteryl ester transfer can be uncoupled from triglyceride transfer.
To eliminate the possibility that a distinct protein was responsible for each type of neutral lipid transfer, the Fab studies were repeated using recombinant cynomolgus monkey CETP that had been purified from the media of Chinese hamster ovary cells expressing the protein. Fig. 3shows the results of those experiments. Note that, as with LPDP, there was a selective inhibition of triglyceride transfer by the Fab fragments and no inhibition of cholesteryl ester transfer whatsoever. Thus, triglyceride transfer could be selectively inhibited regardless of whether LPDP, or purified, recombinant CETP was used as the source of transfer activity.
Figure 3:
Effect
of CMTP-2 Fab on CETP-mediated neutral lipid transfer. The assay was
the same as that described in the legend to Fig. 2, except that
recombinant cynomolgus monkey CETP, rather than LPDP, was the source of
CETP. The black bars show the amount of
[4-C]cholesteryl ester radioactivity recovered
in the HDL; the gray bars show the amount of
[9,10-
H]triglyceride radioactivity recovered in
the HDL. The absolute amount of HDL-associated
C or
H radioactivity recovered in the control tube (dose
= 0) is the same as indicated in the legend to Fig. 2.
The question then arose as to whether the converse condition could
be created, i.e. whether cholesteryl ester transfer could be
inhibited without affecting triglyceride transfer. Fig. 4shows
the effects of the compound 6-chloromecuric cholesterol (U-617) on
neutral lipid transfer mediated by cynomolgus monkey LPDP. Those data
show that selective inhibition of cholesteryl ester transfer is indeed
possible. Separate studies of U-617 inhibition of cholesteryl ester
transfer using purified, recombinant cynomolgus monkey CETP and
synthetic donors and acceptors(19) , indicated that U-617 was a
competitive inhibitor of cholesteryl ester transfer that had no
detectable effect on triglyceride transfer. ()
Figure 4:
Effects of 6-chloromecuric cholesterol
(U-617) on CETP-mediated neutral lipid transfer. The assay is the same
as that described in the legend to Fig. 2, except that the
compound U-617 was present at the concentrations indicated. U-617 was
dissolved in MeSO for addition to the assay mixture. The
final Me
SO concentration never exceeded 1% (v/v); a
concentration that has no detectable effect on CETP-mediated neutral
lipid transfer.
, cholesteryl ester radioactivity;
,
triglyceride radioactivity.
To determine whether the CMTP-2 Fab binding site on cynomolgus monkey CETP overlapped the U-617 binding site, monkey LPDP was preincubated with high concentrations of the Fab fragments after which the effects of U-617 on lipid transfer were measured. As expected, triglyceride transfer was inhibited greater than 95%; the effects on cholesteryl ester transfer are shown in Fig. 5. Note that the Fab fragments did not block the ability of the compound to inhibit cholesteryl ester transfer and, in fact, appeared to increase the potency of U-617 somewhat. That indicates that the site on CETP at which U-617 binds is different from that which binds the Fab.
Figure 5:
Effects of combining 6-chloromecuric
cholesterol and CMTP-2 Fab on CETP-mediated cholesteryl ester transfer.
The assay is the same as that described in the legend to Fig. 4,
except that only the effects on cholesteryl ester transfer are shown.
, effects of U-617 alone;
, effects of U-617 + Fab.
The CETP source (100 µl of cynomolgus monkey LPDP), Fab fragments
(1 µM final concentration), and assay buffer were mixed
and incubated at 37 °C for 2 h, after which the radiolabeled LDL,
HDL, and the U-617 were added. The latter mixture was then incubated
for an additional 3 h at 37 °C.
Given the observations that the transfer of cholesteryl ester and triglyceride could be selectively inhibited in vitro, the question arose whether the same would occur in vivo. Previous studies (25, 26, 27) indicated that inhibition of cholesteryl ester transfer resulted in an increase in HDL-cholesterol levels, but it has not been firmly established what effect selective inhibition of triglyceride transfer would have on plasma lipoprotein levels. To see whether triglyceride transfer could be inhibited in vivo to a significantly greater extent than cholesteryl ester transfer, 30 mg of purified CMTP-2 were administered to each of four cynomolgus monkeys subcutaneously. Analysis of plasma samples taken from those monkeys at various times after antibody administration indicated that triglyceride transfer was inhibited approximately 90% from 24 to 96 h and that it was inhibited to a much larger extent than cholesteryl ester transfer (Fig. 6).
Figure 6:
Evidence for disproportionate inhibition
of triglyceride transfer by CMTP-2 in vivo. Thirty mg of the
indicated antibody was administered subcutaneously to each of four
cynomolgus monkeys and blood samples were taken at the indicated times.
Plasma (100 µl) from those blood samples was then used as the
source of CETP in the standard assay and the ratio of the triglyceride
and cholesteryl ester transfer activities determined in each plasma
sample. Those ratios were normalized (i.e. divided by the
ratio in the t = 0 plasma sample) to make visual comparison
easier. , plasma samples from monkeys that received CMTP-1;
, plasma samples from monkeys that received
CMTP-2.
Table 2shows the effects of antibody administration on the plasma cholesterol and triglyceride concentrations in those monkeys. Note that there was not a significant effect of the antibody on either total or HDL cholesterol levels; however, there was a clear decrease in total plasma triglyceride concentrations as a result of antibody treatment, and that decrease appeared to be due largely to the decrease in the HDL fraction. In separate studies run in parallel with these, four other monkeys were injected each with 30 mg of CMTP-1, a monoclonal antibody to cynomolgus monkey CETP which had no detectable effect on either triglyceride or cholesteryl ester transfer in vitro, and in these studies no effect on total or HDL triglyceride or cholesterol concentrations was detected. That indicates that the change in HDL triglycerides evident in Table 2was due to inhibition of CETP-mediated triglyceride transfer by CMTP-2.
To evaluate more carefully the effects of CMTP-2 administration on the structure of the HDL, three different monkeys were given 30 mg each of the antibody subcutaneously, and a blood sample taken 48 h later for HDL composition analysis. The results of those analyses are contained in Table 3and show that treatment with the antibody for 48 h resulted in an HDL that was virtually devoid of triglyceride, but was otherwise unaffected. That was taken as evidence that triglyceride transfer was inhibited well out of proportion to cholesteryl ester transfer, and therefore, that the antibody also uncoupled cholesteryl ester and triglyceride transfer in vivo. It also indicated that essentially all of the triglyceride in the HDL of the cynomolgus monkey was transferred there from other triglyceride-rich lipoproteins (e.g. very low density lipoprotein and chylomicrons) by CETP.
The studies reported here demonstrate that the CETP-mediated transfer of cholesteryl ester can be inhibited without effect on triglyceride transfer and, conversely, triglyceride transfer can be inhibited without effect on cholesteryl ester transfer. From this we conclude that the two lipids are not transferred by a single, non-discriminatory process. Given that the same protein is responsible for the transfer of both types of neutral lipid, the question becomes, how is that discrimination possible? We would propose, based on currently accepted models of the transfer process(2, 3) , that two specific, nonoverlapping binding sites are involved in the transfer of the two classes of lipid.
An underlying assumption here is that both of the inhibitors
presented in this paper block transfer by interacting directly with
CETP, and not by altering the donor or acceptor. In the case of U-617,
we base that deduction on previous studies which showed
that U-617 is a competitive inhibitor which probably reacts with an
unpaired cysteine in CETP. In the case of CMTP-2, we know that the
antibody recognizes an epitope on CETP, since it was obtained by
screening hybridomas using a direct binding assay. Finally, if either
or both of the current models of the transfer process (2, 3) are correct, then the inhibitors must be
interacting directly with the protein, for it would not be possible to
selectively inhibit transfer of one or the other lipid by blocking the
docking, dissociation, or diffusion steps.
To confirm that the selective inhibition detected using the in vitro assay was, in fact, real, and not an artifact of the assay, we evaluated the effect of the inhibitor of triglyceride transfer in vivo. The reasoning was that if selective inhibition is occurring, it should manifest itself as a selective change in the lipoprotein composition. For example, previous studies(25, 26, 27, 28, 29, 30) have shown that inhibition of cholesteryl ester transfer causes an increase in the plasma HDL-cholesterol levels. If, as indicated by the in vitro assay, CMTP-2 at high concentrations inhibits triglyceride transfer but not cholesteryl ester transfer, in vivo administration of the antibody should change the HDL triglyceride content with no effect on the HDL cholesterol levels. That is precisely what occurred: the triglyceride content of HDL was reduced more than 10-fold by antibody treatment while the cholesterol content of HDL remained essentially unchanged. Thus, CETP-mediated cholesteryl ester and triglyceride transfer can be uncoupled in vivo, in full agreement with the two-site hypothesis.
Others (16, 17, 31) have reported that CETP-mediated
triglyceride transfer could be selectively inhibited, but it has been
argued (32) that, because triglycerides have a larger molecular
volume (1600 Å) than cholesteryl esters (1090
Å
), a given inhibitor could partially obstruct access
to a single binding site such that the cholesteryl esters could
exchange but not the triglycerides. However, in these and previous
studies,
U-617 was found to inhibit transfer of the smaller
molecule but not that of the larger. Therefore, that explanation is not
adequate in this instance. On the other hand, by postulating the
existence of two neutral lipid binding sites, one can invoke the same
principle to explain the selectivity of U-617 for cholesteryl esters; i.e. that a second, but smaller, neutral lipid binding site
exists on CETP which is accessible to cholesteryl esters, but not
triglycerides, and with which U-617 interacts specifically.
If discrete binding sites exist, and each of the inhibitors used here is, in fact, selective for a distinct site, then the binding of one inhibitor should not reduce the effectiveness of the other. Incubation of CETP with CMTP-2 Fab fragments completely blocks triglyceride transfer, but does not interfere with the ability of U-617 to inhibit cholesteryl ester transfer. Thus, the binding sites of the two inhibitors do not overlap; a conclusion in full agreement with the two binding site hypothesis.
Finally, the two-binding site hypothesis is entirely compatible with the observation that CMTP-2 inhibition of cholesteryl ester transfer was reversible beyond a certain antibody concentration, but triglyceride transfer was not. We would propose that the site principally responsible for triglyceride transfer is the epitope recognized by the antibody (i.e. blocked by attachment of the antigen binding region of the antibody) and that the cholesteryl ester binding site is a separate site which does not react with the antibody, but is located such that access to it is hindered by a second CETP molecule bound to the same antibody (Fig. 2A, icon a). Thus, at low antibody concentration (below 16 nM; Fig. 2A), both antigen binding sites are occupied and cholesteryl ester transfer is inhibited; however, as the antibody concentration increases beyond 16 nM and the unsaturated form of the antibody (Fig. 2A, icon b) becomes the predominant species, access to the cholesteryl ester transfer site is restored and inhibition of cholesteryl ester transfer is relieved. The fact that CMTP-2 Fab fragments had no observable effect on cholesteryl ester transfer is taken as support for this explanation of the reversible transfer, and therefore, for the premise that topologically distinct, neutral lipid binding sites exist on cynomolgus monkey CETP.
If CETP does, in fact, contain a distinct binding site for each of
the two substrates, then there would be the possibility that genetic
drift could alter the protein such that the ratio of the specificities
toward the two substrates would be different for different species.
Such species to species differences should be less evident in the case
of a single, non-discriminatory binding site. Comparing CETP activity
from three species and using identical assay conditions for measuring
those activities, we do, indeed, find substantial species-dependent
differences in the substrate specificities. As shown in Table 4,
the ratio of transfer rates, V/V
, ranges from
0.76 for hamster CETP to 7.5 for the human CETP. Thus, substrate
specificity is an intrinsic property of the protein and that
specificity can vary considerably among species, suggesting divergent
adaptation of the two sites.
There is abundant precedent for the existence of multiple lipid binding sites on a single protein. Probably the best example of this is plasma albumin, which has two major binding domains (designated subdomains IIA and IIIA(33) ), and possibly multiple lipid binding sites within a subdomain(34) . The experiments described here suggest that two or more neutral lipid binding sites may also exist on cynomolgus monkey CETP, but offer no information as to whether these represent distinct structural domains, or separate binding sites within a single domain. They do, however, suggest that the binding sites are dissimilar, since one appears to have a higher affinity for triglycerides and the other a higher affinity for cholesteryl esters.
Morton and Zilversmit (16) have argued against the two-site hypothesis, based largely on the observation that increasing the cholesteryl ester concentration in the donor particle decreased the rate of transfer of triglycerides ((16) , Table 2) which they interpreted as indication that the two lipids were competing for a single site. However, since increasing the fraction of cholesteryl esters in the lipid core automatically decreased that of the triglycerides, these observations may simply reflect the fact that the transfer rate of each substrate depends on the concentration of that substrate in the donor, i.e. the triglyceride transfer rate may have decreased, not because of competition for the binding site, but simply because the triglyceride concentration in the donor was reduced. Thus, these data do not prove that these two lipids compete with each other for a single binding site. Nonetheless, we would agree with Morton and Zilversmit (16) that the two lipids are probably mutually exclusive (binding at one site precludes binding at the other), for otherwise it is difficult to explain the ``net mass transfer'' phenomenon. Mutual exclusivity could occur as a result of conformational changes induced in one site when the other is occupied; or, because of their spatial relationship, CETP may simply be unable to present both sites to the lipid donor simultaneously. In the latter case, one would have to hypothesize that, once either site becomes occupied, CETP dissociates, so that only 1 mol of a given lipid would be transferred at a time, otherwise net mass transfer could not occur.
The single
most compelling evidence that two sites exist is the observation that
exchange of either lipid can continue, even though transfer of the
other is blocked. Several
studies(16, 17, 24, 31, 37) have demonstrated that that can occur. Ko et
al.(24) , for example, produced a monoclonal antibody
against rabbit CETP that had an inhibitory pattern which was very
similar to that observed with CMTP-2, i.e. at high antibody
concentrations triglyceride transfer was completely blocked, but
cholesteryl esters continued to exchange. Despite these results, those
authors proposed that there was but one lipid binding site, and invoked
the steric hindrance hypothesis (discussed above) to explain their
observations. However, this seems unlikely given the observations that
compounds such as U-617 and PD 140195 (37) can selectively
inhibit transfer of the smaller of the two lipids. Ko et al.(24) also observed that, even though cholesteryl ester exchange
continued after triglyceride exchange was blocked, net mass transfer of
cholesteryl ester appeared to have been blocked. Thus, by blocking one
site, one apparently renders CETP incapable of net mass transfer.
Although no direct evidence exists for the one or two lipid binding sites on CETP, many of the observations reported to date are difficult to reconcile with a single binding site, whereas all observations are consistent with the two-site hypothesis. The question of one or two sites is more than academic and leads to the physiologically important question of whether cholesteryl ester and triglyceride binding and transfer are mutually exclusive, or can a ternary CETP-CE-triglyceride complex be formed? We feel that the mutual exculsion of the two lipids from CETP is probably real but is due to the fact that CETP is unable to present both binding sites simultaneously to the lipid donor.