Portal de Periódicos da CAPES

Human Low Density Lipoprotein Receptor Fragment

1997; Elsevier BV; Volume: 272; Issue: 41 Linguagem: Inglês

10.1074/jbc.272.41.25531

1083-351X

Trey Simmons, Yvonne M. Newhouse, K S Arnold, Thomas L. Innerarity, Karl H. Weisgraber,

Cancer, Lipids, and Metabolism

The low density lipoprotein (LDL) receptor plays a key role in cholesterol homeostasis, mediating cellular uptake of lipoprotein particles by high affinity binding to its ligands, apolipoprotein (apo) B-100 and apoE. The ligand-binding domain of the LDL receptor contains 7 cysteine-rich repeats of approximately 40 amino acids; each repeat contains 6 cysteines, which form 3 intra-repeat disulfide bonds. As a first step toward determining the structure of the LDL receptor, both free and bound to its ligands, we produced in Escherichia coli a soluble fragment containing the ligand-binding domain (residues 1–292) as a thrombin-cleavable, heat-stable thioredoxin fusion. Modest amounts (5 mg/liter) of partially purified but inactive fragment were obtained after cell lysis, heat treatment, thrombin cleavage, and gel filtration under denaturing conditions. We were able to refold the receptor fragment to an active conformation with approximately 10% efficiency. The active fragment was isolated and purified with an LDL affinity column. The refolded receptor fragment was homogeneous, as determined by sodium dodecyl sulfate or non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing. The purified fragment did not react with fluorescein-5-maleimide, indicating that all 42 cysteines were disulfide linked. In addition, the refolded fragment exhibited properties identical to those of the intact native receptor: Ca2+-dependent binding and isoform-dependent apoE binding (apoE2 binding <5% of apoE3). Furthermore, antibodies to the fragment recognized native receptors and inhibited the binding of 125I-LDL to fibroblast LDL receptors. We conclude that we have produced a properly folded and fully active receptor fragment that can be used for further structural studies. The low density lipoprotein (LDL) receptor plays a key role in cholesterol homeostasis, mediating cellular uptake of lipoprotein particles by high affinity binding to its ligands, apolipoprotein (apo) B-100 and apoE. The ligand-binding domain of the LDL receptor contains 7 cysteine-rich repeats of approximately 40 amino acids; each repeat contains 6 cysteines, which form 3 intra-repeat disulfide bonds. As a first step toward determining the structure of the LDL receptor, both free and bound to its ligands, we produced in Escherichia coli a soluble fragment containing the ligand-binding domain (residues 1–292) as a thrombin-cleavable, heat-stable thioredoxin fusion. Modest amounts (5 mg/liter) of partially purified but inactive fragment were obtained after cell lysis, heat treatment, thrombin cleavage, and gel filtration under denaturing conditions. We were able to refold the receptor fragment to an active conformation with approximately 10% efficiency. The active fragment was isolated and purified with an LDL affinity column. The refolded receptor fragment was homogeneous, as determined by sodium dodecyl sulfate or non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing. The purified fragment did not react with fluorescein-5-maleimide, indicating that all 42 cysteines were disulfide linked. In addition, the refolded fragment exhibited properties identical to those of the intact native receptor: Ca2+-dependent binding and isoform-dependent apoE binding (apoE2 binding 8.0) and the addition of a thiol exchange system. The importance of Ca2+in the folding of the LDL receptor and repeat 5 was recently pointed out (30Blacklow S.C. Kim P.S. Nat. Struct. Biol. 1996; 3: 758-762Crossref PubMed Scopus (108) Google Scholar) and may result from its ability to stabilize folding intermediates (31Creighton T.E. Trends Biochem. Sci. 1997; 22: 6-10Abstract Full Text PDF PubMed Scopus (92) Google Scholar). The cysteamine/cystamine thiol exchange system was more efficient than the more widely utilized reduced/oxidized glutathione system. This may be due to the smaller relative size of cysteamine and cystamine, which might allow better access to the interior of the relatively compact repeat structure. It is also possible that the positively charged amino group on cysteamine is more effective in the highly negatively charged environment that characterizes the receptor repeats. Another key step was the removal of both the thiol exchange system and residual guanidine by dialysis. This step proved to be absolutely essential in generating a fully folded receptor fragment. It is possible that removal of the thiol exchange system and guanidine facilitates a final oxidative refolding step in which a critical disulfide link (or links) is formed, leading directly to the fully folded state. As shown in Fig. 2, this dialysis step produces a narrow compact band above the broad heterogeneous mixture of bands, which likely contain a number of misfolded intermediates. This compact band turns out to be the fully folded and an active receptor fragment. The final step in the isolation was affinity chromatography. The refolded mixture was applied to an LDL-Sepharose column, and as shown in Fig. 2, only the thin compact band was bound. This refolded receptor fragment could be eluted with either a salt or a pH gradient. At this point, the receptor fragment is approximately 99% pure. We estimate that approximately 10% of the starting material is successfully refolded. Our overall yield is approximately 0.5 mg of purified and refolded receptor fragment per liter of cells. At various stages of purification, the receptor binding activity of the receptor fragment was monitored with an LDL plate assay. Little, if any, activity is present in the 80 °C supernatant or the thrombin-digested mixture (Fig. 3). A low level of activity is present following the gel filtration step, indicating that some refolding and reoxidation occurs in 4 m guanidine and 0.1% β-mercaptoethanol or occurs after dialysis. It is possible that this activity represents the presence of a significant fraction of partially folded and hence weakly active species, rather than a small amount of fully active receptor fragment. Western blot experiments with monoclonal antibody C7 (32Beisiegel U. Schneider W.J. Goldstein J.L. Anderson R.G.W. Brown M.S. J. Biol. Chem. 1981; 256: 11923-11931Abstract Full Text PDF PubMed Google Scholar, 33van Driel I.R. Goldstein J.L. Südhof T.C. Brown M.S. J. Biol. Chem. 1987; 262: 17443-17449Abstract Full Text PDF PubMed Google Scholar), which recognizes only correctly folded repeat 1, demonstrated that the majority of the receptor was partially folded even at this early stage, whereas no species migrating in the position of the fully folded receptor fragment could be detected on SDS gels (data not shown). Refolding led to a 10-fold increase in total binding activity, followed by another 10-fold increase in activity after the affinity step (Fig. 3). Taking the differences in molecular mass into account, the 292 receptor fragment has approximately the same level of activity as the full-length bovine receptor, as assessed in the LDL plate assay, suggesting the fragment has full binding activity. However, this comparison must be viewed as a qualitative rather than a quantitative argument given the potential for differences in both intrinsic activity and antibody recognition between the two species. To assure that a homogeneous, correctly folded, and fully active fragment was produced, the fragment was subjected to a variety of analyses based on the known properties of the intact native receptor, the results of which are presented below. Although the refolded receptor fragment appeared to be homogeneous on non-reducing SDS gels, it was important to establish that it does, in fact, represent a single molecular species. As shown in Fig.4, the refolded fragment migrated as a single species on a non-denaturing polyacrylamide gel, as well as on an isoelectric focusing gel, providing strong evidence that it comprises a structurally homogeneous population. As expected from the amino acid composition, the pI of the receptor fragment is acidic (pI ∼4.0). The disulfide connectivities of the 6 cysteine residues in the independently folded repeats 1, 2, and 5 are the same in each repeat: Cys-1–Cys-3, Cys-2–Cys-5, and Cys-4–Cys-6 (16Daly N.L. Scanlon M.J. Djordjevic J.T. Kroon P.A. Smith R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6334-6338Crossref PubMed Scopus (162) Google Scholar, 17Daly N.L. Djordjevic J.T. Kroon P.A. Smith R. Biochemistry. 1995; 34: 14474-14481Crossref PubMed Scopus (84) Google Scholar, 30Blacklow S.C. Kim P.S. Nat. Struct. Biol. 1996; 3: 758-762Crossref PubMed Scopus (108) Google Scholar). Thus, it is reasonable to expect that the same disulfide connectivity would occur in all 7 repeats in the intact native receptor and that all 42 cysteine residues would be disulfide bonded with each repeat containing 3 intra-repeat disulfide bonds. Therefore, if the receptor fragment is properly folded, it should contain no free cysteine residues. To determine if this was the case, we used fluorescein-5-maleimide, a fluorescent cysteine-specific reagent that reacts with free –SH groups. As shown in Fig. 5, the refolded receptor fragment when modified under denaturing conditions did not fluoresce, indicating that it contains no detectable free –SH groups. For comparison, apoE3, which contains a single cysteine residue, was easily detected when loaded in a equimolar amount, whereas apoE4, which contains no cysteines, did not fluoresce, as expected. This experiment provides strong evidence that all of the cysteines in the refolded receptor fragment are disulfide bonded. Another characteristic of the native LDL receptor is that it exhibits a Ca2+ requirement for high affinity binding to LDL (33van Driel I.R. Goldstein J.L. Südhof T.C. Brown M.S. J. Biol. Chem. 1987; 262: 17443-17449Abstract Full Text PDF PubMed Google Scholar). To test whether the receptor fragment also exhibited Ca2+-dependent binding, the binding activity of the receptor fragment in the presence and absence of Ca2+was examined. The standard binding buffer for the plate assay contains 2 mm CaCl2, and the LDL binding of the fragment in this buffer was defined as 100%. As shown in Fig.6, binding was significantly reduced if Ca2+ was not included in the binding buffer. Although no Ca2+ was added, some is likely to be receptor-bound because Ca2+ is present at a relatively high concentration in the refolding and affinity isolation procedures. This residual binding was virtually eliminated by adding EGTA to the incubation mixture. Hence, like the native receptor, the receptor fragment shows highly Ca2+-dependent binding activity. In addition to apoB-100 on LDL, apoE-containing lipoproteins are the other major ligand for the LDL receptor (34Innerarity T.L. Mahley R.W. Biochemistry. 1978; 17: 1440-1447Crossref PubMed Scopus (250) Google Scholar). To bind with high affinity to the LDL receptor, apoE must be complexed with lipid (35Innerarity T.L. Pitas R.E. Mahley R.W. J. Biol. Chem. 1979; 254: 4186-4190Abstract Full Text PDF PubMed Google Scholar). This is typically done by reconstituting apoE into vesicles with dimyristoyl phosphatidylcholine (DMPC) to mimic its natural environment (35Innerarity T.L. Pitas R.E. Mahley R.W. J. Biol. Chem. 1979; 254: 4186-4190Abstract Full Text PDF PubMed Google Scholar). Using apoE·DMPC complexes in the plate assay, we demonstrated the same Ca2+-dependent binding as was observed for LDL (data not shown). Interestingly, lipid-free apoE bound to the microtiter wells also bound with high affinity to the LDL receptor fragment. Thus, it appears that apoE binding to a plastic surface mimics binding to lipid with respect to the high affinity receptor interaction. Another key characteristic of the native LDL receptor is its differential binding to the three common human apoE isoforms, apoE2, apoE3, and apoE4. These isoforms are distinguished by cysteine and arginine differences at positions 112 and 158; apoE3 contains cysteine and arginine at these positions, respectively, whereas apoE2 contains cysteine and apoE4 contains arginine at both positions (36Weisgraber K.H. J. Lipid Res. 1990; 31: 1503-1511Abstract Full Text PDF PubMed Google Scholar, 37Rall Jr., S.C. Weisgraber K.H. Mahley R.W. J. Biol. Chem. 1982; 257: 4171-4178Abstract Full Text PDF PubMed Google Scholar). Whereas apoE3 and apoE4 bind to the LDL receptor with essentially the same affinity, apoE2 displays defective binding (<1% of that of apoE3 or apoE4) (38Weisgraber K.H. Innerarity T.L. Mahley R.W. J. Biol. Chem. 1982; 257: 2518-2521Abstract Full Text PDF PubMed Google Scholar). The defective binding of apoE2 is an underlying cause of type III hyperlipoproteinemia, a lipoprotein disorder associated with elevated plasma lipid levels and premature heart disease (39Rall Jr., S.C. Weisgraber K.H. Innerarity T.L. Mahley R.W. de Gennes J.L. Polonovski J. Paoletti R. Latent Dyslipoproteinemias and Atherosclerosis. Raven Press, Ltd., New York1984: 157-163Google Scholar). If the receptor fragment is correctly folded and fully active, it should exhibit differential binding to the apoE isoforms. This expectation was tested with either apoE·DMPC complexes or lipid-free apoE in a plate assay. The results with both forms of apoE were essentially identical; the data for the lipid-free apoE isoforms are presented in Fig. 7. As shown, the amounts of receptor fragment bound to apoE3 and apoE4 were identical, whereas binding to apoE2 was less than 5% of that of apoE3 and apoE4 (comparable amounts of each isoform were bound to the wells). These results clearly demonstrate that the isoform specificities of the intact LDL receptor are mirrored by the receptor fragment. As a further test of the authenticity of the receptor fragment, we reasoned that an antibody raised to the refolded receptor fragment should recognize the native receptor. As determined by Western blot analysis, an anti-receptor fragment antibody bound to LDL receptors from rat liver and bovine adrenal membranes, whereas it did not bind to misfolded or non-folded receptor fragments, i.e. fusion protein or S-300 fraction (data not shown). In addition, the antibodies completely inhibited the binding of human LDL to LDL receptors on human fibroblasts (Fig. 8), demonstrating that the antibodies recognized the native human receptor. In summary, the evidence presented above strongly supports the conclusion that the binding activity of the LDL receptor fragment is biologically relevant and that the receptor fragment has been successfully refolded to its native conformation. With this correctly folded receptor fragment available, we can now begin to address structural questions, such as the overall topology of the 7 cysteine-rich repeats, the role of calcium in ligand interaction, the number of repeats involved in apoE interaction, and how the Arg-150 conformational change in apoE2 reduces receptor binding activity. In addition, with the methods developed here, production and refolding of the ligand-binding domains of other members of the LDL receptor superfamily are potentially feasible. The authors thank K. Humphrey for manuscript preparation, J. Carroll, B. Clark, S. Gonzales, and A. Corder for graphic arts, and G. Howard and S. Ordway for editorial assistance.