From the Palo Alto Medical Foundation, Palo Alto,
California 94301, ¶ Department of Medicine, College of
Physicians and Surgeons, Columbia University, New York, New York 10032,
The Scripps Research Institute, La Jolla, California 92037, and
** Stanford University School of Medicine,
Palo Alto, California 94305
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ABSTRACT |
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Hepatic lipase (HL) on the surface of hepatocytes and endothelial cells lining hepatic sinusoids, the adrenal glands, and the ovary hydrolyzes triglycerides and phospholipids of circulating lipoproteins. Its expression significantly enhances low density lipoprotein (LDL) uptake via the LDL receptor pathway. A specific interaction between LPL, a homologous molecule to HL, and apoB has been described (Choi, S. Y., Sivaram, P., Walker, D. E., Curtiss, L. K., Gretch, D. G., Sturley, S. L., Attie, A. D., Deckelbaum, R. J., and Goldberg, I. J. (1995) J. Biol. Chem. 270, 8081-8086). The present studies tested the hypothesis that HL enhances the uptake of lipoproteins by a specific interaction of HL with apoB. On a ligand blot, HL bound to apoB26, 48, and 100 but not to apoE or apoAI. HL binding to LDL in a plate assay with LDL-coated plates was significantly greater than to bovine serum albumin-coated plates. Neither heat denatured HL nor bacterial fusion protein of HL bound to LDL in the plate assays. 125I-LDL bound to HL-saturated heparin-agarose gel with a Kd of 52 nM, and somewhat surprisingly, this binding was not inhibited by excess LPL. In cell culture experiments HL enhanced the uptake of 125I-LDL at both 4 and 37 °C. The enhanced binding and uptake of LDL was significantly inhibited by monoclonal anti-apoB antibodies. In contrast to LPL, both amino- and carboxyl-terminal antibodies blocked the apoB interaction with HL to the same extent. Thus, we conclude that there is a unique interaction between HL and apoB that facilitates the uptake of apoB-containing lipoproteins by cells where HL is present.
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INTRODUCTION |
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Hepatic lipase is synthesized in hepatic parenchymal cells and functions primarily as an endothelial bound enzyme within the liver sinusoids (1-3), although some enzyme is found in adrenal glands and the ovaries (4). The enzyme hydrolyzes triglycerides and phospholipids of the circulating lipoproteins; thus, it is involved in the metabolism of high density lipoproteins (HDL)1 converting the HDL2 fraction to HDL3 and in the conversion of intermediate density lipoprotein to low density lipoprotein (LDL).
Several lines of evidence now suggest that hepatic lipase may play another role in the metabolism of apoB-containing lipoproteins. In vivo inhibition of hepatic lipase, with an antibody to the protein, delays the clearance of chylomicron remnants (5), and patients with hepatic lipase deficiency often have an accumulation of remnant-like lipoproteins in the plasma. Overexpression of hepatic lipase in transgenic rabbits results in reduced levels of plasma HDL and intermediate density lipoprotein (6). Gel filtration studies of hepatic lipase in post-heparin plasma revealed that hepatic lipase is associated with lipoproteins that overlap the elution of small LDL (7). Furthermore, it was demonstrated by this laboratory (8) that hepatic lipase significantly enhanced LDL uptake via the LDL receptor pathway in Chinese hamster ovary (CHO) cells transfected with rat hepatic lipase cDNA. The amount of the LDL receptor was virtually identical in the transfected cells as compared with the control cells; thus, the presence of hepatic lipase enhanced the affinity of the lipoprotein particle for the LDL receptor. Recently, Krapp et al. (9) reported that hepatic lipase mediates the uptake of apoE-containing lipoproteins via LDL receptor-related protein.
The mechanisms whereby hepatic lipase facilitates the metabolism of plasma lipoproteins have not been elucidated. One study (10) suggested that hydrolysis of the lipid core by the enzyme facilitates the uptake of lipoproteins. However, a number of recent reports (9) suggest that nonenzymatic functions of hepatic lipase facilitate the uptake of plasma lipoproteins through an interaction between cell surface heparan sulfate proteoglycans and the apoB of lipoprotein particles. An association of lipoprotein lipase (LPL) with apoB-containing lipoproteins by a specific protein-protein interaction between LPL and apoB was recently reported by Choi et al. (11). Analysis of amino acid sequences of hepatic lipase and LPL has shown that there is an extensive amino acid homology between these lipases (12). Additionally, both LPL and hepatic lipase bind to proteoglycans (13). Thus, the present studies were designed to test the hypothesis that a nonenzymatic function of hepatic lipase may facilitate the uptake of apoB-containing lipoproteins.
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MATERIALS AND METHODS |
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Preparation of Lipoproteins-- Human LDL (d = 1.019-1.063 g/ml) and HDL (d = 1.063-1.21 g/ml) were isolated from EDTA-containing plasma by ultracentrifugation (14). Chylomicrons were prepared by the methods previously described (15), and chylomicron remnants were prepared by the modification of the method of Redgrave and Martin (16) previously described (15). Apolipoprotein patterns of all the lipoprotein preparations were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). Total protein concentration of lipoproteins was determined by the method of Lowry et al. (17) with bovine serum albumin (BSA) as a standard.
Preparation of Rat Hepatic Lipase and Anti-hepatic Lipase Antibodies-- CHO cells transfected with rat hepatic lipase cDNA were grown in flasks in Dulbecco's modified Eagle's medium-Coon's F-12 (1:1) plus 10% fetal calf serum until just subconfluent, and the medium was replaced with induction medium containing 30 µM ZnSO4 as described previously (8). The enzyme was then purified on octyl-Sepharose (Amersham Pharmacia Biotech) followed by heparan-Sepharose columns (18, 23). Fractions containing lipase activity were concentrated using Centricon 30 filters and stored in liquid nitrogen. The purified enzyme was catalytically active, and its molecular mass was approximately 50 kDa (data not shown).
A bacterial fusion protein of recombinant rat hepatic lipase was prepared as described previously (18). The fusion protein migrated as a single protein band of about 50 kDa on 8% SDS-PAGE as previously characterized (18). Since this enzyme was produced in Escherichia coli, it was not glycosylated and was presumably not folded correctly. Additionally, it was not catalytically active in a triolein emulsion assay (data not shown). An antibody to rat hepatic lipase was prepared against recombinant rat protein and has previously been characterized (18). The antibody was recently used to assess the role of hepatic lipase in vivo (5) and does not cross-react with lipoprotein lipase or apoB fragments when analyzed by Western blotting (Fig. 1).SDS-PAGE and Ligand Blotting-- Samples were analyzed using 6% polyacrylamide gels containing 0.5% SDS. Proteins were transferred to a nitrocellulose membrane (Bio-Rad) followed by incubation in phosphate-buffered saline containing 3% BSA. Immunoblotting was performed as described previously (19) using rabbit polyclonal antibodies against rat hepatic lipase.
Microtiter Plate Assays-- Solid phase plate assays were performed as described by Williams et al. (20). For binding studies, LDL, HDL, apoAI, or BSA diluted in Tris-buffered saline (TBS) (50 mM Tris, 150 mM NaCl, pH 7.4) containing 5 mM Ca2+ were added to 96-well plates and incubated at 4 °C overnight. The unbound lipoproteins were removed, and the plates were washed three times with 0.3% BSA in TBS followed by incubation with 3% BSA in TBS, 5 mM Ca2+ for 1 h at room temperature to block nonspecific binding sites. After washing with 0.3% BSA in TBS, hepatic lipase in TBS containing 5 mM Ca2+ and 3% BSA was added and incubated at 4 °C for 24 h. The plates were washed and incubated for another 2 h at room temperature with polyclonal rabbit anti-hepatic lipase antibodies. Unbound antibodies were removed, and horseradish peroxidase-conjugated goat anti-rabbit IgG (Oreganon Teknika Corp.) at 1:500 dilution was added. One h later, 100 µl of substrate solution containing o-phenylenediamine dihydrochloride (Sigma) and 5 µl of 50% H2O2 in 0.1 M citric acid (pH 5) was added to each well. After 20 min at room temperature, absorbance at 490 nm was measured. The protein molecular masses of 550 kDa for apoB100 and 47 kDa for hepatic lipase monomer (18) were used to calculate the molar concentrations of LDL and hepatic lipase, respectively.
Coprecipitation of LDL with Hepatic Lipase-- Interaction between 125I-LDL and hepatic lipase was assessed in solution using heparin-agarose (Bio-Rad) saturated with hepatic lipase. One ml of heparin-agarose was incubated with 3 ml (approximately 600 µg of protein/ml) of purified rat hepatic lipase overnight at 4 °C on a rocker. The unbound hepatic lipase was then removed, and the gel was washed three times with TBS.
Radiolabeled LDL at 0-900 nM in 50 µl of TBS containing 3% BSA was incubated with 50 µl of hepatic lipase-saturated heparin-agarose gel for 3 h at 4 °C on a rocker. The unbound LDL was then removed, and the gel was washed three times with TBS. Fifty-fold excess unlabeled LDL was added in some experiments to correct for the nonspecific or background binding. After incubation, the gel was precipitated by centrifugation for 1 min at 6000 rpm, and the pellet was washed 5 times with TBS. All the experimental procedures were carried out at 4 °C. The 125I-LDL precipitated with hepatic lipase was then determined by measuring 125I radioactivity in a gamma counter.Preparation of Monoclonal Antibodies against Human ApoB-- Anti-human apoB monoclonal antibodies were purified from ascites using mono Q column chromatography and the fast protein liquid chromatography system (Amersham) or protein G (Pierce). The monoclonal antibodies mAb3 and mAb47 have determinants in the amino-terminal region (21) and the LDL receptor binding site of apoB close to the carboxyl terminus (22), respectively.
Degradation and Binding Assays of LDL by CHO Cells-- Preparation of the expression vector for hepatic lipase and transfection into CHO cells has been described previously (8). Additionally, the preparation of CHO cells expressing hepatic lipase anchored to the cell surface by a glycophosphatidylinositol anchor was recently described (23). Stably transfected and wild-type CHO cells were cultured in Dulbecco's modified Eagle's medium/Coon's 1:1 supplemented with 10% fetal calf serum at 37 °C in a 5% CO2 atmosphere until just subconfluent. The medium was then replaced with the same medium containing 30 µM ZnSO4 and 10% fetal calf serum and incubated overnight to induce recombinant hepatic lipase production. Specific degradation and binding of LDL were carried out as described previously (8) in the presence or absence of monoclonal anti-apoB antibodies.
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RESULTS |
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Hepatic Lipase Binding to Apolipoproteins as Determined by Ligand Blotting-- The specificity of the anti-hepatic lipase antibodies was first characterized for cross-reactivity with lipoprotein lipase or apoB fragments. In these experiments, hepatic lipase, bovine lipoprotein lipase, or thrombin-digested human LDL were applied to 6% SDS-PAGE and transferred to nitrocellulose paper followed by immunoblotting using the anti-hepatic lipase antibody. The antibody does not cross-react with lipoprotein lipase or apoB fragments in the thrombin-digested LDL (Fig. 1).
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Hepatic Lipase Binding to ApoB-containing Lipoproteins in a Solid Phase Assay-- To obtain more precise and quantitative information, hepatic lipase binding to lipoproteins in a solid phase assay system was assessed. Ninety-six-well microtiter plates were coated with lipoproteins at 50 µg/ml, a concentration that was above the amount required to saturate the binding sites of the plate (11). Hepatic lipase binding to BSA-coated wells was measured to estimate nonspecific binding. Hepatic lipase binding was quantitated by the amount of anti-hepatic lipase antibody that bound to the plates. At all concentrations of hepatic lipase in the range of 0-50 nM, hepatic lipase binding to LDL or chylomicron remnants was significantly greater than to BSA-coated plates (Fig. 3). For instance, at 25 nM hepatic lipase, approximately 4-fold more hepatic lipase bound to the LDL-coated plates than to BSA-coated plates. A concentration curve revealed that the binding was saturable. Binding of hepatic lipase to chylomicron remnant-coated plates was also severalfold more than to BSA-coated plates.
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Structural Requirement of Hepatic Lipase Interaction with ApoB-- To assess whether the secondary or tertiary structure of hepatic lipase was required for the interaction of hepatic lipase with apoB, the enzyme was heat-denatured by boiling for 2 min. When analyzed by SDS-PAGE, the heat-treated protein migrated as a single band (data now shown). The interaction of hepatic lipase with LDL was virtually eliminated by heat denaturation of hepatic lipase (Fig. 4A), indicating that the secondary and/or tertiary structure of hepatic lipase is required for its interaction with apoB. To confirm this, a solid phase assay using a bacterial fusion protein rather than hepatic lipase derived from a mammalian system was performed. Hepatic lipase expressed in E. coli did not interact with LDL (Fig. 4B). Additionally, the fusion protein did not bind to LDL on a ligand blot (data not shown). Since this enzyme was produced in E. coli, it was not glycosylated and was presumably not folded correctly. To support this, the fusion protein migrated with a slightly faster electrophoretic mobility on SDS-PAGE than did native rat hepatic lipase as described previously (18). Thus, proper protein folding or processing or both is required for binding of hepatic lipase to apoB.
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Inhibition of Hepatic Lipase-ApoB Interaction by Monoclonal Anti-apoB Antibodies-- To assess the functional significance of the protein-protein interaction between apoB and hepatic lipase, monoclonal antibodies against apoB were used in solid phase experiments and in cell culture experiments. 125I-Labeled LDL (2.5 µg/ml) was added to the media of wild-type or hepatic lipase-secreting CHO cells. Specific cell association and degradation of LDL were determined. As compared with the wild-type cells, hepatic lipase-secreting cells had significantly greater LDL degradation than those that had not (Fig. 5A). These data are consistent with previous reports from this laboratory (8). mAb3 reduced the degradation of LDL by those cells that secreted hepatic lipase to a level comparable with that of the cells that did not secrete hepatic lipase. The addition of mAb47 in contrast inhibited virtually all of the specific LDL degradation. Specific cell association of LDL was also enhanced by more than 2-fold in hepatic lipase-transfected cells (Fig. 5B). Again, the addition of mAb3 (250 µg/ml) inhibited the portion of cell association that could be attributed to hepatic lipase stimulation, and mAb47 (250 µg/ml) eliminated virtually all of the specific LDL cell association.
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Kinetic Analysis of 125I-LDL Interaction with Hepatic
Lipase--
To carry out kinetic measurements, a precipitation assay
for the interaction was devised. Agarose beads were saturated with hepatic lipase. They were incubated with radiolabeled LDL and then
precipitated by centrifugation. This assay allows kinetic analysis in
terms of LDL concentration. In the absence of hepatic lipase, only a
trace amount of 125I-LDL (<3%) of the
125I-LDL bound to the gel. In the presence of hepatic
lipase, however, a significant amount of 125I-LDL was
precipitated with the gel (Fig. 6).
Kinetic analysis by the method of Scatchard (24), shown in the
inset of Fig. 6, indicated that LDL bound to hepatic lipase
with a Kd of 52 nM and Bmax
of 24 × 103 nmol/ml gel.
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Competition between Hepatic Lipase and LPL for 125I-LDL Binding-- To further establish that the binding site for hepatic lipase on apoB is different from its binding site for LPL, hepatic lipase-gel precipitation experiments as described under "Materials and Methods" were carried out in the presence of an excess amount of LPL. 125I-LDL (5 µg/ml) was precipitated with hepatic lipase-saturated heparin-agarose gel in the presence or absence of excess LPL. The presence of LPL did not reduce LDL binding to hepatic lipase-saturated gel (Fig. 7A). The same was true in the converse experiments in which excess hepatic lipase was added to compete for the LDL binding to LPL-saturated heparin gel (Fig. 7B). In other experiments, hepatic lipase binding to LDL-coated plates was measured in the presence of excess LPL in the solid phase assay described above, and no competition between hepatic lipase and LPL was observed (data not shown). These data suggest, surprisingly, that LPL and hepatic lipase do not share the same binding site(s) on the apoB molecule.
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DISCUSSION |
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The present experiments provide strong support for the hypothesis that an interaction between hepatic lipase and apoB facilitates the uptake of apoB-containing lipoproteins. There had been considerable evidence suggesting that hepatic lipase plays a role in the removal of apoB-containing lipoproteins (5, 25-28). This could be due to the lipolytic activity of the enzyme causing remodeling of the particles allowing enhanced uptake, as has been suggested by Aviram et al. (10); alternatively, however, the recent evidence that lipoprotein lipase facilitates LDL uptake by binding LDL led us to explore this as a possible mechanism of hepatic lipase action.
Evidence for a direct interaction of apoB with hepatic lipase was provided by ligand blots, solid phase and solution assays, and cell culture experiments. On a ligand blot, hepatic lipase bound to several forms of apoBs including B48 and B100 and the kallikrein cleavage products of apoB100, B26, and B74 (29) present on lipoproteins but not to other apolipoproteins including apoE and apoAI. The interaction between apoB and hepatic lipase was further studied by solid phase and solution assays. In a solution assay, where hepatic lipase was in excess, LDL binding to hepatic lipase clearly shows saturation kinetics with a Kd of 52.86 nM. In a solid phase assay with apoB in excess, the requirement for the hepatic lipase secondary and/or tertiary structure was demonstrated. The functional significance of the interaction was demonstrated in cell culture where monoclonal antibodies against apoB on both the amino- and carboxyl-terminal regions inhibited the hepatic lipase-mediated LDL uptake. Lastly, LDL binding to hepatic lipase-saturated agarose gel was not inhibited by the presence of excess LPL, suggesting that these proteins bind to distinct regions of apoB.
ApoB100 is a large glycoprotein with a molecular mass of 550 kDa and is virtually the only protein component of LDL particles. The carboxyl-terminal region of apoB is involved in binding to LDL receptors, and this region also contains five of seven apoB heparin binding sites (30). Under physiologic ionic conditions, however, LDL binds weakly to heparin (31). The amino-terminal region of apoB is hydrophilic and thought to be a globular structure that extends away from the lipid core of lipoproteins (32). Possible functions of the amino-terminal region of apoB have recently been elucidated (33-35). Using monoclonal antibodies against amino and carboxyl regions of apoB we recently demonstrated that the amino-terminal, but not the carboxyl-terminal, region of apoB interacts with LPL (36). In contrast to LPL, the present experiments suggest that both the amino and carboxyl regions of apoB have binding domains for hepatic lipase. Evidence supporting this was provided primarily by solid phase assays in which the binding of LDL to hepatic lipase-expressing CHO cells or purified hepatic lipase, respectively, was inhibited by both carboxyl- and amino-terminal anti-apoB antibodies (i.e. mAb47 and mAb3, respectively). Ligand blots using thrombin-digested apoB also showed that hepatic lipase bound to both amino- and carboxyl-terminal fragments of apoB (data not shown).
The cell culture studies provide strong support for the concept that the interaction between apoB and hepatic lipase is of physiologic significance. In cell culture both antibodies also decreased LDL binding to cells that secreted hepatic lipase. Interpretation of the result with mAb3 is straight forward; its ability to eliminate the increment of binding due to the secretion of hepatic lipase supports the hypothesis that the hepatic lipase effect is due to an interaction with apoB. mAb47, however, binds to an epitope near the LDL receptor binding domain of apoB. Accordingly, its effect could be due to direct interference with the receptor-ligand interaction. Consistent with this was its ability to decrease cell association and degradation in control cells as well as in hepatic lipase-transfected cells. Interestingly, in the binding studies the antibody did not completely inhibit binding in either cell line but reduced binding in HL secretors to that of nonsecretors. Thus, if its main effect on LDL processing is on internalization, the effect on binding to the secretor could be due to elimination of the apoB-HL interaction. This requires further exploration. Taking the cell culture and solid phase assay data together, however, it appears that there are hepatic lipase binding sites on both the amino-terminal region of apoB and in the region near the LDL receptor binding domain.
Earlier studies have clearly demonstrated the role of hepatic lipase in remodeling larger lipoproteins such as intermediate density lipoprotein and HDL2 into the smaller particles, LDL and HDL3, respectively. The lipolytic activity, however, may not be required for the facilitation of lipoprotein uptake by tissues. First, a previous in vivo study showed that, in mice, injection of anti-rat hepatic lipase antibody resulted in a small but significant reduction in the rate of chylomicron remnant removal from plasma (5); however, the antibodies did not inhibit lipolysis by mouse hepatic lipase, suggesting that lipolysis was not the mechanism by which hepatic lipase enhanced remnant uptake. Second, in the present study the binding experiments were performed at 4 °C (Fig. 4B) to minimize lipid hydrolysis by hepatic lipase, and again, LDL binding was significantly enhanced in hepatic lipase-expressing cell lines, suggesting that a nonenzymatic function of hepatic lipase played a role in lipoprotein uptake. Further, the presence of phenylmethylsulfonyl fluoride (100 µM), an inhibitor of lipolytic activity, did not reduce the binding of hepatic lipase to LDL in solid phase assays. This is consistent with an expanding body of evidence that LPL, a closely related enzyme, does not require its catalytic activity for its interaction with LDL. Future experiments with active-site mutant proteins should help to elucidate this definitively.
Possible nonenzymatic mechanisms of action of hepatic lipase include bridging of lipoproteins and heparan sulfate proteoglycans. Previously, it was demonstrated that hepatic lipase enhanced the uptake of LDL in CHO cells transfected with rat hepatic lipase cDNA and that anti-LDL receptor antibodies virtually eliminated the hepatic lipase-mediated LDL uptake (8). The amount of LDL receptor and of LDL receptor-related protein in the transfected cells was identical to that in wild-type cells on both Western and Northern blots. Furthermore, kinetic studies indicated that the increased LDL binding in the hepatic lipase-secreting CHO cells was the result of a higher affinity of the particle for the LDL receptor. Together, these data suggest that hepatic lipase enhanced the uptake of LDL via the LDL receptor-mediated pathway by increasing its affinity for the receptor.
A number of previous studies have suggested that lipoprotein lipase can anchor lipoproteins to cell surface and matrix proteoglycans (37, 38), and this molecular bridge has been postulated to increase lipoprotein retention by subendothelial cell matrix and increase cellular lipoprotein uptake. Similarly it has been suggested (39) that hepatic lipase binding to lipoproteins requires heparan sulfate proteoglycans, and it was recently reported (9) that hepatic lipase mediates the uptake of apoE-containing lipoproteins via the LDL receptor-related protein and that this effect was absent in proteoglycan deficient cells.
Based on the present study we propose that hepatic lipase enhances the uptake of apoB-containing lipoproteins by binding both to the cell surface, presumably to heparan sulfate proteoglycans, and to apoB. Thus, hepatic lipase functions as a bridge. This could both facilitate hydrolysis of lipoproteins and increase their uptake via the LDL receptor by providing high affinity multifooted binding. Kinetic analysis suggests this is a plausible explanation.
In our previous experiments (8) the affinity Kd of
LDL for the cell surface in wild-type CHO cells was 6.6 nM and about 10-fold higher, 0.6 nM (6 × 1010 M), to cells secreting hepatic lipase.
The present studies provide support for the hypothesis that the
increase in affinity is due to the polyvalent or multifooted binding
created by the ability of apoB to bind to both the LDL receptor and
hepatic lipase on the cell surface. Comparisons of polyvalent with
monovalent binding have been carried out in studies of immunoglobulin
(40). It was found that IgG anti-dinitrophenyl antibodies bound to
aggregated dinitrophenyl with 103 to 105
greater affinity than to monomeric dinitrophenyl. This is because when
multiple sites are present, the actual affinity may be much higher than
the intrinsic affinity, due to the cooperation between sites. Thus even
though the affinity of apoB for hepatic lipase, 52 nM, was
about one-thirtieth that of LDL for the LDL receptor, which was
determined to be 1.6 nM in a plate assay (41), it is highly
plausible to suggest that the affinity of apoB for the multiple binding
sites generated by both HL and LDL receptor was 10-fold enhanced
compared with that for its binding to the LDL receptors alone. In fact
the increase of 1 order of magnitude may be less than might have been
expected from the immunoglobulin studies. This could be due to failure
of each particle to undergo multifooted binding. It is, however, of
comparable magnitude to the increase in the affinity for the LDL
receptor of liposomes containing four molecules of apoE compared with
those containing one molecule of apoE. We recently reported that the
Kd of LDL for LPL was 3.76 nM in
solution assays using LPL-saturated heparin-agarose beads (42).
Thus, hepatic lipase, which has a Kd of 52 nM for LDL, does not have as high an affinity as LPL.
Nonetheless, these considerations illustrate how the presence of a low
affinity site in the proximity of a higher affinity site, by allowing
multifooted binding, may account for a considerable enhancement of LDL
uptake in the organ that expresses hepatic lipase bound to its
surfaces.
The previous report (11) suggested that the amino-terminal region of apoB is involved in its protein-protein interaction with LPL. In contrast, hepatic lipase binds to both the amino- and carboxyl-terminal region of apoB on ligand blot. Thus, hepatic lipase binding to apoB is not identical to LPL binding to apoB, although both enzymes appear to interact with apoB by a specific protein-protein interaction. This was further supported by the somewhat surprising lack of competition between hepatic lipase and lipoprotein lipase for apoB binding. This suggests that apoB, which has numerous potential sites for protein-protein interaction in regions where it is not interacting with the lipid surface can undergo interaction with a number of molecules, and these interactions may help to define its fate.
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ACKNOWLEDGEMENTS |
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We thank Dr. Joseph Obunike (Columbia University) for providing bovine LPL and Dr. Loren Fong (Palo Alto Medical Foundation Research Institute) for providing human LDL and valuable scientific discussion. We also thank Rick Cuevas (Palo Alto Medical Foundation Research Institute) for manuscript preparation and Kathie Richards (The Scripps Research Institute) for technical assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK38318 (to A. D. C.), HL45095 (to I. J. G.), and HL58034 (to S. Y. C.) and pilot/feasibility studies from Digestive Disease Center, School of Medicine, Stanford University (to S. Y. C.).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: Research Institute, Palo Alto Medical Foundation, 860 Bryant St., Palo Alto, CA 94301. Tel.:650-326-8120; Fax: 650-329-9114.
The abbreviations used are: HDL, high density lipoprotein; LDL, low density lipoprotein; LPL, lipoprotein lipase; apo, apolipoprotein; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; TBS, Tris-buffered saline; mAb, monoclonal antibody; HL, hepatic lipase.
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REFERENCES |
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