Binding characteristics of a panel of monoclonal antibodies against the ligand binding domain of the human LDLr

To obtain a panel of monoclonal antibodies (MAbs) to study the folding and conformation of the low density lipoprotein receptor (LDLr), we have generated hybridomas from LDLr-deficient mice that had been immunized with the extracellular domain of the human LDLr. The 12 MAbs were specific for the ligand binding domain of the LDLr, with individual MAbs recognizing epitopes in ligand binding repeats 1, 2, 3, 5, and 7. A subset of the MAbs failed to react with the LDLr when disulfide bonds were reduced, and one MAb, specific for an epitope that spans ligand binding repeats 1 and 2, recognized two conformational forms of the LDLr with different affinities. Antibodies specific for ligand binding repeats 3, 5, and 7 completely blocked the binding of LDL particles to the LDLr on cultured human fibroblasts, whereas MAbs with epitopes in ligand binding repeats 1 and 2 partially blocked the binding of LDL to the LDLr. These anti-LDLr MAbs will serve as useful probes for further analysis of LDLr conformation and LDLr-mediated lipoprotein binding. To obtain a panel of monoclonal antibodies (MAbs) to study the folding and conformation of the low density lipoprotein receptor (LDLr), we have generated hybridomas from LDLr-deficient mice that had been immunized with the extracellular domain of the human LDLr. The 12 MAbs were specific for the ligand binding domain of the LDLr, with individual MAbs recognizing epitopes in ligand binding repeats 1, 2, 3, 5, and 7. A subset of the MAbs failed to react with the LDLr when disulfide bonds were reduced, and one MAb, specific for an epitope that spans ligand binding repeats 1 and 2, recognized two conformational forms of the LDLr with different affinities. Antibodies specific for ligand binding repeats 3, 5, and 7 completely blocked the binding of LDL particles to the LDLr on cultured human fibroblasts, whereas MAbs with epitopes in ligand binding repeats 1 and 2 partially blocked the binding of LDL to the LDLr. These anti-LDLr MAbs will serve as useful probes for further analysis of LDLr conformation and LDLr-mediated lipoprotein binding. The low density lipoprotein receptor (LDLr) is located in clathrin-coated pits on the cell surface and can bind and mediate the endocytosis of plasma lipoproteins that contain either apolipoprotein E (apoE) or apoB-100. It has an important role in regulating cholesterol homeostasis, and mutations in the LDLr gene can lead to familial hypercholesterolemia. The 839 amino acid human LDLr is organized into five structural domains (1Brown M.S. Goldstein J.L. A receptor-mediated pathway for cholesterol homeostasis.Science. 1986; 232: 34-47Crossref PubMed Scopus (4383) Google Scholar, 2Jeon H. Blacklow S.C. Structure and physiologic function of the low-density lipoprotein receptor.Annu. Rev. Biochem. 2005; 74: 535-562Crossref PubMed Scopus (234) Google Scholar). The amino terminal 292 residues constitute the ligand binding domain (LBD) that is composed of seven imperfect 40 residue repeats (R1–R7) with a short linker between R4 and R5. The LBD is followed by the 400 residue epidermal growth factor (EGF) precursor homology domain (EGFPHD) that contains three EGF-like repeats, EGF-A, EGF-B, and EGF-C, and a β-propeller subdomain inserted between EGF-B and EGF-C (3Jeon H. Meng W.Y. Takagi J. Eck M.J. Springer T.A. Blacklow S.C. Implications for familial hypercholesterolemia from the structure of the LDL receptor YWTD-EGF domain pair.Nat. Struct. Biol. 2001; 8: 499-504Crossref PubMed Scopus (185) Google Scholar, 4Rudenko G. Henry L. Henderson K. Ichtchenko K. Brown M.S. Goldstein J.L. Deisenhofer J. Structure of the LDL receptor extracellular domain at endosomal pH.Science. 2002; 298: 2353-2358Crossref PubMed Scopus (385) Google Scholar). The EGFPHD is necessary for the pH-dependent dissociation of receptor and lipoprotein in the endosome (1Brown M.S. Goldstein J.L. A receptor-mediated pathway for cholesterol homeostasis.Science. 1986; 232: 34-47Crossref PubMed Scopus (4383) Google Scholar). The third domain (O-linked sugar domain) is rich in threonine and serine residues that become O-glycosylated during the intracellular maturation of the receptor. A short transmembrane domain is followed by a 50 residue cytoplasmic tail that is required to localize the LDLr in clathrin-coated pits on the cell surface and for the endocytosis of ligands. Each of the LBD repeats contains a site for the coordination of a calcium ion and six cysteine residues that form three intrarepeat disulfide bonds (5Blacklow S.C. Kim P.S. Protein folding and calcium binding defects arising from familial hypercholesterolemia mutations of the LDL receptor.Nat. Struct. Biol. 1996; 3: 758-762Crossref PubMed Scopus (108) Google Scholar, 6Bieri S. Djordjevic J.T. Daly N.L. Smith R. Kroon P.A. Disulfide bridges of a cysteine-rich repeat of the LDL receptor ligand-binding domain.Biochemistry. 1995; 34: 13059-13065Crossref PubMed Scopus (51) Google Scholar, 7Fass D. Blacklow S.C. Kim P.S. Berger J.M. Molecular basis of familial hypercholesterolaemia from structure of LDL receptor module.Nature. 1997; 388: 691-693Crossref PubMed Scopus (308) Google Scholar, 8Kurniawan N.D. Atkins A.R. Bieri S. Brown C.J. Brereton I.M. Kroon P.A. Smith R. NMR structure of a concatemer of the first and second ligand-binding modules of the human low-density lipoprotein receptor.Protein Sci. 2000; 9: 1282-1293Crossref PubMed Scopus (38) Google Scholar). Folding of newly synthesized LDLr occurs posttranslationally and is nonvectorial with the formation of transient, nonnative, long-range disulfide bonds that are subsequently isomerized into the native intrarepeat disulfide bonds that characterize the LDLr LBD (9Jansens A. van Duijn E. Braakman I. Coordinated nonvectorial folding in a newly synthesized multidomain protein.Science. 2002; 298: 2401-2403Crossref PubMed Scopus (139) Google Scholar). Binding of lipoproteins to the LDLr appears to be mediated by an interaction between acidic residues in the LDLr LBD and basic residues of apoE and apoB-100. By systematic deletion of individual ligand binding repeats of the LDLr, it has been shown that the repeats contribute differently to apoB-100- and apoE-mediated lipoprotein binding to the LDLr (10Esser V. Limbird L.E. Brown M.S. Goldstein J.L. Russell D.W. Mutational analysis of the ligand binding domain of the low density lipoprotein receptor.J. Biol. Chem. 1988; 263: 13282-13290Abstract Full Text PDF PubMed Google Scholar, 11Russell D.W. Brown M.S. Goldstein J.L. Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins.J. Biol. Chem. 1989; 264: 21682-21688Abstract Full Text PDF PubMed Google Scholar). Deletion of individual repeats R3–R7 results in a loss of LDL binding (apoB-100-mediated), whereas β-VLDL binding (apoE-mediated) is impaired only when R5 is deleted. An LDLr fragment consisting of R4 and R5 is sufficient to bind to apoE-phospholipid vesicles (12Fisher C. Abdul-Aziz D. Blacklow S.C. A two-module region of the low-density lipoprotein receptor sufficient for formation of complexes with apolipoprotein E ligands.Biochemistry. 2004; 43: 1037-1044Crossref PubMed Scopus (57) Google Scholar). The crystal structure of an LDLr segment composed of the LBD and EGFPHD has been solved at pH 5.3 (4Rudenko G. Henry L. Henderson K. Ichtchenko K. Brown M.S. Goldstein J.L. Deisenhofer J. Structure of the LDL receptor extracellular domain at endosomal pH.Science. 2002; 298: 2353-2358Crossref PubMed Scopus (385) Google Scholar). In this structure, R4 and R5 are docked onto the β-propeller with an interface that includes histidine 192 (His192) in R5 and His562 and His586 in the β-propeller. It has been proposed that, on the cell surface, the extracellular domain of the LDLr would adopt an elongated structure, as has been visualized by cryoelectron microscopy (13Jeon H. Shipley G.G. Vesicle-reconstituted low density lipoprotein receptor. Visualization by cryoelectron microscopy.J. Biol. Chem. 2000; 275: 30458-30464Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) with R4 and R5 accessible for binding to ligand. When the LDLr-ligand complex is internalized and exposed to the acidic environment of the endosome (∼pH 5.3), the LDLr would undergo a conformational change so that the extracellular domain would fold back on itself and the β-propeller could displace bound lipoprotein (14Innerarity T.L. LDL receptor’s β-propeller displaces LDL.Science. 2002; 298: 2337-2339Crossref PubMed Scopus (27) Google Scholar, 15Rudenko G. Deisenhofer J. The low density lipoprotein receptor: ligands, debates and lore.Curr. Opin. Struct. Biol. 2003; 13: 683-689Crossref PubMed Scopus (54) Google Scholar). In support of this model, it has been demonstrated that mutation of His192, His562, and His586 leads to an LDLr variant that binds LDL with high affinity at neutral pH but fails to release the ligand at pH 5.3 (16Beglova N. Jeon H. Fisher C. Blacklow S.C. Cooperation between fixed and low pH-inducible interfaces controls lipoprotein release by the LDL receptor.Mol. Cell. 2004; 16: 281-292Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). To obtain a panel of monoclonal antibody (MAb) probes to study the folding of the newly synthesized LDLr and the conformation of the mature receptor, we have generated hybridomas from LDLr-deficient (Ldlr−/−) mice that had been immunized with the extracellular domain of the human LDLr. Here, we describe the binding characteristics of these antibodies. The mammalian expression plasmid, pCMV5-LDLr1–692, encoding residues 1–692 of the LDLr (LDLr1–692) was a gift from Dr. David Russell (University of Texas Southwestern Medical Center). Chinese hamster ovary K1 cells were cotransfected with pCMV5-LDLr1–692 (15 μg of DNA) and pSV2neo (1.5 μg) by calcium phosphate precipitation (17Chen C. Okayama H. High efficiency transformation of mammalian cells by plasmid DNA.Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4824) Google Scholar). Stably transfected cell clones were selected using 700 μM G418 and were subsequently maintained with 500 μM G418. For the purification of LDLr1–692 from cell supernatants, a high-expressing, LDLr1–692-transfected clone was adapted for growth in CHO S-SFM II medium (Invitrogen, Burlington, Ontario, Canada) that was supplemented with 1% fetal bovine serum and cultured in a Spinner Basket 1 Cell Culture Bioreactor (New Brunswick Scientific, Edison, NJ) according to the manufacturer’s recommendation. Cells were maintained for up to 1 month, and 400 ml of medium was harvested and replaced every 2 days. The anti-human LDLr MAb, C7 (18Beisiegel U. Schneider W.J. Goldstein J.L. Anderson R.G.W. Brown M.S. Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia.J. Biol. Chem. 1981; 256: 11923-11931Abstract Full Text PDF PubMed Google Scholar), was immobilized on cyanogen bromide-activated Sepharose 4B beads (Amersham, Baie D’Urfeé, Queébec, Canada) according to the manufacturer’s instructions. The beads were washed with TBS containing 20 mM CaCl2 (TBS-CaCl2). Culture medium (400 ml) was passed over the beads, the column was washed with TBS-CaCl2, and bound LDLr1–692 was eluted with 0.1 M glycine, pH 3. The fractions were dialyzed against TBS-CaCl2 and stored in liquid nitrogen. Characterization of LDLr1–692 will be described in detail elsewhere. Ldlr−/− mice (19Ishibashi S. Brown M.S. Goldstein J.L. Gerard R.D. Hammer R.E. Herz J. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery.J. Clin. Invest. 1993; 92: 883-893Crossref PubMed Scopus (1280) Google Scholar) (a gift from Dr. Stewart Whitman, University of Ottawa Heart Institute) were immunized by subcutaneous injection of LDLr1–692 (50 μg) emulsified in complete Freund’s adjuvant. The mice received two additional boosts with 50 μg of LDLr1–692 in incomplete Freund’s adjuvant (Sigma-Aldrich, Oakville, Ontario, Canada) at 3 week intervals. Serum antibody titers were monitored 1 week after each boost by a solid-phase RIA with LDLr1–692 as the immobilized antigen (20Milne R.W. Weech P.K. Marcel Y.L. Immunological methods for studying and quantifying lipoproteins and apolipoproteins.in: Converse C. Skinner E. Lipoprotein Analysis: A Practical Approach. Oxford University Press, Oxford1992: 61-84Google Scholar). Four days the and at 3 after the a boost (50 μg of LDLr1–692 in of was by tail The for the of from immunized mice with cells has been described (20Milne R.W. Weech P.K. Marcel Y.L. Immunological methods for studying and quantifying lipoproteins and apolipoproteins.in: Converse C. Skinner E. Lipoprotein Analysis: A Practical Approach. Oxford University Press, Oxford1992: 61-84Google Scholar). to days after the were for antibodies by solid-phase (20Milne R.W. Weech P.K. Marcel Y.L. Immunological methods for studying and quantifying lipoproteins and apolipoproteins.in: Converse C. Skinner E. Lipoprotein Analysis: A Practical Approach. Oxford University Press, Oxford1992: 61-84Google Scholar) with LDLr1–692 as the immobilized Cells in were on at a density of one cell was in mice by injection of The of MAbs was using a Monoclonal was from or from by Sepharose Protein affinity according to the manufacturer’s of the were by and by Sepharose Protein A as described R.W. R. Marcel Y.L. Characterization of monoclonal antibodies against human low density 3: PubMed Google Scholar). The and (10Esser V. Limbird L.E. Brown M.S. Goldstein J.L. Russell D.W. Mutational analysis of the ligand binding domain of the low density lipoprotein receptor.J. Biol. Chem. 1988; 263: 13282-13290Abstract Full Text PDF PubMed Google Scholar, 11Russell D.W. Brown M.S. Goldstein J.L. Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins.J. Biol. Chem. 1989; 264: 21682-21688Abstract Full Text PDF PubMed Google Scholar) were a gift from Dr. David Russell (University of Texas Southwestern Medical (10Esser V. Limbird L.E. Brown M.S. Goldstein J.L. Russell D.W. Mutational analysis of the ligand binding domain of the low density lipoprotein receptor.J. Biol. Chem. 1988; 263: 13282-13290Abstract Full Text PDF PubMed Google Scholar, 11Russell D.W. Brown M.S. Goldstein J.L. Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins.J. Biol. Chem. 1989; 264: 21682-21688Abstract Full Text PDF PubMed Google Scholar). The and were generated from with the using the and cells were transfected with 2 μg of the plasmid to be using (Invitrogen, Burlington, Ontario, Cells were cultured in the of fetal bovine serum to the expression of the LDLr, and after the cells were harvested and for Cell were and by as described for the C7 anti-human LDLr monoclonal antibody U. Schneider W.J. Brown M.S. Goldstein J.L. analysis of low density lipoprotein in from with familial hypercholesterolemia.J. Biol. Chem. Full Text PDF PubMed Google Scholar). In cell were to The of binding of the anti-LDLr and to LDLr1–692 were by surface using a LDLr1–692 (15 in mM pH was to a using the by the to surface of were as were to analysis to Binding of the to the immobilized LDLr1–692 was using a of mM pH containing mM mM and at a of 40 For MAbs and 20 mM pH containing mM mM and was were with 50 mM for 3 were using plasma from was supplemented with 1 mM mM and LDL was at by between of and The and of lipoproteins in human Clin. Invest. 34: PubMed Scopus Google Scholar). LDL was dialyzed against containing 1 mM and by and stored at for up to 3 LDL was with as described by and D.W. S. The of low density lipoprotein PubMed Scopus Google Scholar). The for between the anti-LDLr MAbs and for binding to the LDLr on the surface of cultured human was adapted from that described for the of MAbs to the binding of to the LDLr R. R. R. Weech P.K. E. J. Marcel Y.L. The of monoclonal antibodies to localize the low density lipoprotein domain of apolipoprotein Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar). MAbs (50 μg were in of the a we 12 hybridomas that MAbs specific for the LDLr1–692 To localize the epitopes recognized by the MAbs the LDLr structure, we in a of LDLr that had of individual LBD repeats, of the linker between R4 and of individual or of EGF-like repeats, or of the Cell containing the variant were and for with the MAbs by The of of the MAbs is shown in 1, and a of the of of the MAbs is in MAbs with the receptor. MAbs and react with an LDLr variant that and a variant was recognized by MAbs and Deletion of either or the binding of MAbs and The epitopes for MAbs and to be in and whereas the epitope recognized by appears to be in Deletion of the EGF-like repeats or the complete EGFPHD the binding of of the antibodies. An epitope on these results is shown in of disulfide bridges in the LDLr or of from the medium has been to the binding of LDL and the anti-LDLr MAb, to the LDLr U. Schneider W.J. Brown M.S. Goldstein J.L. analysis of low density lipoprotein in from with familial hypercholesterolemia.J. Biol. Chem. Full Text PDF PubMed Google Scholar, Goldstein J.L. Brown M.S. cysteine-rich repeat in ligand-binding domain of low density lipoprotein receptor binds and monoclonal but Biol. Chem. 1987; Full Text PDF PubMed Google Scholar). we the panel of MAbs for with LDLr1–692 that had been to and Antibodies and only with the LDLr1–692 by in whereas the MAbs and that C7 LDLr1–692 after MAbs recognized the LDLr in the of when by either or an in the binding of C7 to immobilized LDLr1–692 to be of the LDLr epitopes recognized by the panel of MAbs as by Binding epidermal growth LDLr, low density lipoprotein MAb, monoclonal in a epitope of The epitopes recognized by the panel of anti-LDLr MAbs were to the structure of LDLr1–692 on the results shown in with are only for additional epidermal growth LDLr, low density lipoprotein MAb, monoclonal antibodies were selected to each of the for further antibodies can be by the The of binding of to immobilized LDLr1–692 were with a The for MAbs and to a binding model, for these antibodies to be The for a ligand in the antibody would two of the immobilized LDL receptor with different the were by the for a have after purification or on the LDLr In with an we had that bound to LDLr1–692 with affinity at pH 5.3 at pH was the for the at pH the binding at pH 5.3 a ligand model, with the in the affinity to an in and a in dissociation In of the structural of the LDLr (12Fisher C. Abdul-Aziz D. Blacklow S.C. A two-module region of the low-density lipoprotein receptor sufficient for formation of complexes with apolipoprotein E ligands.Biochemistry. 2004; 43: 1037-1044Crossref PubMed Scopus (57) Google Scholar), we the binding at pH 5.3 of the fragment that is specific for R5. In to and dissociation at pH 5.3 at pH on the surface the epitope to be accessible at pH and pH 5.3 and affinity for the interaction of the MAbs with immobilized LDLr as by to interaction the for binding of from the against and could be the for binding of from the against and could be in a The antibodies were for to the binding of LDL to the LDLr on the surface of cultured human MAbs specific for epitopes in R5 and the binding of LDL to the LDLr, whereas MAbs specific for epitopes in and blocked and of the binding of LDL to the LDLr. had been (18Beisiegel U. Schneider W.J. Goldstein J.L. Anderson R.G.W. Brown M.S. Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia.J. Biol. Chem. 1981; 256: 11923-11931Abstract Full Text PDF PubMed Google Scholar), is specific for an epitope in Goldstein J.L. Brown M.S. cysteine-rich repeat in ligand-binding domain of low density lipoprotein receptor binds and monoclonal but Biol. Chem. 1987; Full Text PDF PubMed Google Scholar), blocked of whereas a specific for human Y.L. H. E. E. The epitopes of apolipoprotein structural domains a Biol. Chem. Full Text PDF PubMed Google Scholar), LDL binding to the LDLr. a panel of MAbs from Ldlr−/− mice that had been immunized with a fragment of the human LDLr composed of the LBD and Ldlr−/− mice for the be by to the LDLr, as would be the with we that a would be in Ldlr−/− mice after with LDLr1–692 that would antibodies to as on the For the this to be the were that MAbs specific for epitopes in five of the seven ligand binding repeats as as an that appears to the In we failed to antibodies that epitopes in the to in on the structure A for PubMed Scopus Google Scholar, J. A for the of a Biol. PubMed Scopus Google Scholar) that the LDLr EGFPHD be at as as the It is that to of of the LDLr gene the to the human LDLr EGFPHD in Ldlr−/− mice as a the is against the a panel of 20 MAbs to human that are specific for at of are located in the of R. A. E. R. of human analysis using monoclonal 1996; Full Text PDF PubMed Google Scholar, V. V. V. S. E. R. that and a Sci. 8: PubMed Scopus Google Scholar). in the it is a of the a as mice have a gene A. for the of a gene in mice and Biochem. Biochem. Biol. 2003; PubMed Scopus Google Scholar). was to obtain anti-LDLr MAbs that could be to study LDLr conformation and of the MAbs have specific conformational for binding to the LDLr. MAbs only the LDLr, epitopes native disulfide these MAbs are specific for epitopes in and will be useful probes to the folding of the LBD of newly synthesized LDLr. The binding of to immobilized LDLr1–692 monitored by surface a ligand that two conformational forms of LDLr1–692 with different and dissociation at pH 5.3 at pH LDLr that either or appears to an epitope that spans and The NMR structure of a concatemer of and that are and that the residue linker between modules N.D. Atkins A.R. Bieri S. Brown C.J. Brereton I.M. Kroon P.A. Smith R. NMR structure of a concatemer of the first and second ligand-binding modules of the human low-density lipoprotein receptor.Protein Sci. 2000; 9: 1282-1293Crossref PubMed Scopus (38) Google Scholar). The antibody a conformational of the LDLr that is in low at neutral pH but is at pH at the the of residues in LDLr1–692 and in The between these two on the structural of the extracellular domain of the LDLr at pH 5.3 (3Jeon H. Meng W.Y. Takagi J. Eck M.J. Springer T.A. Blacklow S.C. Implications for familial hypercholesterolemia from the structure of the LDL receptor YWTD-EGF domain pair.Nat. Struct. Biol. 2001; 8: 499-504Crossref PubMed Scopus (185) Google Scholar), one that the epitope in R5 would be accessible at pH was the the epitope accessible when R5 is docked with the β-propeller at pH when immobilized on the LDLr1–692 its Antibodies specific for and the binding of LDL to the LDLr on cultured human fibroblasts, whereas is with MAbs to or the results for LDL binding to LDLr in individual LBD repeats were LDLr and could bind whereas deletion of LDL binding and deletion of had (10Esser V. Limbird L.E. Brown M.S. Goldstein J.L. Russell D.W. Mutational analysis of the ligand binding domain of the low density lipoprotein receptor.J. Biol. Chem. 1988; 263: 13282-13290Abstract Full Text PDF PubMed Google Scholar, 11Russell D.W. Brown M.S. Goldstein J.L. Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins.J. Biol. Chem. 1989; 264: 21682-21688Abstract Full Text PDF PubMed Google Scholar). To this is the first of MAbs that are of LDL binding to the LDLr. The of of LDL binding to the LDLr by its low affinity at pH it is binding would be in the of the In we describe the and of a panel of MAbs to the LBD of the LDLr that be useful probes to study the folding and conformation of the LDLr. as of the antibodies can the binding of LDL to the LDLr, be for the of the LDLr to lipoprotein binding. this study is Kim E. of monoclonal antibodies specific for apolipoprotein in apolipoprotein Full Text Full Text PDF PubMed Google Scholar, V. J. J. J. T.L. R.W. that human when in human 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) of the of mice for the of monoclonal antibodies. was by from the of The Dr. David Russell for gift of and Dr. for of

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