Global Defects in the Expression and Function of the Low Density Lipoprotein Receptor (LDLR) Associated with Two Familial Hypercholesterolemia Mutations Resulting in Misfolding of the LDLR Epidermal Growth Factor-AB Pair

Abstract

The low density lipoprotein (LDL) receptor is a modular protein involved in the endocytosis of cholesterol-rich lipoproteins from the circulation. Mutations to the receptor result in familial hypercholesterolemia, and over 60 of these occur in the calcium-binding epidermal growth factor-like domain pair. Two selected mutations in this region (G322S and R329P) were introduced into the domain pair and analyzed by in vitro refolding. Both exhibited differing levels of protein misfolding with R329P being the most pronounced. Solution NMR studies of the mutant domain pairs after purification established that a fraction of protein maintains a native-like fold and that this fraction contains two intact calcium-binding sites. An in vivo analysis of intact receptors containing these binding sites showed significantly reduced cell-surface expression compared with the native LDL receptor levels, again with R329P showing the most severe decrease. The sum of these results suggests that either local changes in structure or domain misfolding may be associated with the mutations. There is also the possibility that the misfolding of the calcium-binding epidermal growth factor-like pair region is propagated to other regions of the intact receptor, resulting in more global defects. Surprisingly, for both mutants, those full-length receptors that fold and reach the cell surface retain the ability to bind LDL and release the ligand upon exposure to low pH. This analysis provides significant insight into the protein defect resulting from each of the two mutations and allows their classification to be 2B (partially transport-defective). The results also highlight a range of misfolding defects that may be associated with familial hypercholesterolemia and may enable the prediction of the consequences of homologous disease-causing mutations to other proteins. The low density lipoprotein (LDL) receptor is a modular protein involved in the endocytosis of cholesterol-rich lipoproteins from the circulation. Mutations to the receptor result in familial hypercholesterolemia, and over 60 of these occur in the calcium-binding epidermal growth factor-like domain pair. Two selected mutations in this region (G322S and R329P) were introduced into the domain pair and analyzed by in vitro refolding. Both exhibited differing levels of protein misfolding with R329P being the most pronounced. Solution NMR studies of the mutant domain pairs after purification established that a fraction of protein maintains a native-like fold and that this fraction contains two intact calcium-binding sites. An in vivo analysis of intact receptors containing these binding sites showed significantly reduced cell-surface expression compared with the native LDL receptor levels, again with R329P showing the most severe decrease. The sum of these results suggests that either local changes in structure or domain misfolding may be associated with the mutations. There is also the possibility that the misfolding of the calcium-binding epidermal growth factor-like pair region is propagated to other regions of the intact receptor, resulting in more global defects. Surprisingly, for both mutants, those full-length receptors that fold and reach the cell surface retain the ability to bind LDL and release the ligand upon exposure to low pH. This analysis provides significant insight into the protein defect resulting from each of the two mutations and allows their classification to be 2B (partially transport-defective). The results also highlight a range of misfolding defects that may be associated with familial hypercholesterolemia and may enable the prediction of the consequences of homologous disease-causing mutations to other proteins. The low density lipoprotein (LDL) 1The abbreviations used are: LDL, low density lipoprotein; AIEX, anion-exchange; EGF, epidermal growth factor; EGF-A, first EGF domain of the LDLR; EGF-B, second EGF domain of the LDLR; EGF-AB, EGF domain pair from LDLR; FH, familial hypercholesterolemia; HPLC, high pressure liquid chromatography; HSQC, heteronuclear single-quantum correlation; LB medium, Luria-Bertani medium; LDLR, low-density lipoprotein receptor; RP, reverse-phase; WT, wild type.1The abbreviations used are: LDL, low density lipoprotein; AIEX, anion-exchange; EGF, epidermal growth factor; EGF-A, first EGF domain of the LDLR; EGF-B, second EGF domain of the LDLR; EGF-AB, EGF domain pair from LDLR; FH, familial hypercholesterolemia; HPLC, high pressure liquid chromatography; HSQC, heteronuclear single-quantum correlation; LB medium, Luria-Bertani medium; LDLR, low-density lipoprotein receptor; RP, reverse-phase; WT, wild type. receptor mediates the cellular uptake of cholesterol-rich lipoproteins from the circulation by binding to two ligand molecules, apolipoprotein B and apolipoprotein E, of LDL and β-very LDL particles. Receptor-ligand complexes enter the cell through endocytosis at clathrin-coated pits. The bound lipoprotein is released subsequently in the low pH environment of the endosome, and the receptors are recycled back to the cell surface (1Brown M.S. Goldstein J.L. Science. 1986; 232: 34-47Crossref PubMed Scopus (4288) Google Scholar). Mutations to the LDL receptor (LDLR) are associated with familial hypercholesterolemia (FH), an autosomal-dominant inherited disease affecting ∼1 in 500 in most populations. FH is characterized clinically by a lifelong elevation of LDL-bound cholesterol in blood and is associated with an increased risk of coronary artery disease and cardiovascular disease (2Goldstein J.L. Hobbs H.H. Brown M.S. Scriver C.R. Sly W.S. Childs B. Beaudet A.L. Valle D. Kinzler K.W. Vogelstein B. 8th Ed. The Metabolic and Molecular Bases of Inherited Disease. II. McGraw-Hill, New York2001: 2863-2913Google Scholar). Almost 900 LDLR mutations have been associated with FH (3Villeger L. Abifadel M. Allard D. Rabes J.P. Thiart R. Kotze M.J. Beroud C. Junien C. Boileau C. Varret M. Hum. Mutat. 2002; 20: 81-87Crossref PubMed Scopus (90) Google Scholar), collated in two online databases, 2Available at www.ucl.ac.uk/fh and www.umd.necker.fr/ldlr/Home_Page.html.2Available at www.ucl.ac.uk/fh and www.umd.necker.fr/ldlr/Home_Page.html. and many have been classified based on their effect on the synthesis (class 1), transport (class 2), ligand binding activity (class 3), internalization (class 4), or recycling (class 5) of the LDLR (4Hobbs H.H. Brown M.S. Goldstein J.L. Hum. Mutat. 1992; 1: 445-466Crossref PubMed Scopus (920) Google Scholar). Despite the wealth of biochemical data available for the LDL receptor and atomic resolution structural data for the extracellular region of the protein (5Fass D. Blacklow S. Kim P.S. Berger J.M. Nature. 1997; 388: 691-693Crossref PubMed Scopus (299) Google Scholar, 6North C.L. Blacklow S.C. Biochemistry. 2000; 39: 2564-2571Crossref PubMed Scopus (68) Google Scholar, 7Jeon H. Meng W. Takagi J. Eck M.J. Springer T.A. Blacklow S.C. Nat. Struct. Biol. 2001; 8: 499-504Crossref PubMed Scopus (182) Google Scholar, 8Kurniawan N.D. Atkins A.R. Bieri S. Brown C.J. Brereton I.M. Kroon P.A. Smith R. Protein Sci. 2000; 9: 1282-1293Crossref PubMed Scopus (38) Google Scholar, 9Saha S. Boyd J. Werner J.M. Knott V. Handford P.A. Campbell I.D. Downing A.K. Structure. 2001; 9: 451-456Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 10Rudenko G. Henry L. Henderson K. Ichtchenko K. Brown M.S. Goldstein J.L. Deisenhofer J. Science. 2002; 298: 2353-2358Crossref PubMed Scopus (366) Google Scholar), the molecular defects associated with many of the >400 missense mutations that result in familial hypercholesterolemia remain unclear. Such information is vitally important for attempts to correlate protein defects with protein dysfunction and ultimately with phenotype. The LDLR consists of a series of discrete extracellular protein modules responsible for the binding and release of lipoprotein ligands. The ligand-binding region is composed of a series of seven LDL receptor-type A (LA) modules followed by a region with a homology to the EGF precursor, an O-glycosylation region, a transmembrane region, and a cytoplasmic tail (11Brown M.S. Herz J. Goldstein J.L. Nature. 1997; 388: 629-630Crossref PubMed Scopus (146) Google Scholar). The EGF precursor-homology region consists of two calcium-binding EGF domains, six YWTD repeats forming a β-propeller domain, and a third EGF domain. Calcium is required for ligand binding (12Davis C.G. Goldstein J.L. Sudhof T.C. Anderson R.G.W. Russell D.W. Brown M.S. Nature. 1987; 326: 760-765Crossref PubMed Scopus (300) Google Scholar, 13Kita T. Brown M.S. Watanabe Y. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2268-2272Crossref PubMed Scopus (172) Google Scholar). We have reported previously (9Saha S. Boyd J. Werner J.M. Knott V. Handford P.A. Campbell I.D. Downing A.K. Structure. 2001; 9: 451-456Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) the structure of the calcium-saturated EGF-AB pair from the LDL receptor in which the two domains adopt an extended rod-like arrangement. At present, over 60 FH mutations have been localized to the EGF-AB pair. Based on our structure, it was possible to predict the structural consequences of some missense mutations, for example G352D, which was suggested to domain the of other mutations were based on the structural data (9Saha S. Boyd J. Werner J.M. Knott V. Handford P.A. Campbell I.D. Downing A.K. Structure. 2001; 9: 451-456Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). this have the consequences of two mutations for which the structural consequences were and been classified previously 2B (partially or (4Hobbs H.H. Brown M.S. Goldstein J.L. Hum. Mutat. 1992; 1: 445-466Crossref PubMed Scopus (920) Google Scholar), a been to R329P L. V. Hum. Mutat. 1997; PubMed Scopus Google Scholar). two mutations were selected for a each the of the in the and are both in at of the of the possibility that the of the domain be the analysis of in vitro protein protein structure, and the calcium-binding of the EGF-AB pair that these two mutations are associated with of protein native LDL receptors are with receptors these mutations, the of receptors at the cell surface with the fraction of native-like receptors from in vitro the mutant receptors that fold and reach the cell surface retain the ability to bind LDL, and the ability of the mutant receptors to release bound LDL upon exposure to low pH is compared with native This provides important insight into the protein defect associated with two mutations to A of the in the of the two with to the blood cholesterol levels of suggests that the disease is by the of that results from each these results may enable the prediction of the molecular defects associated with disease-causing mutations to calcium-binding domains in the LDLR and other associated with M. J. 2000; PubMed Scopus Google Scholar). and for expression of the LDLR EGF-AB pair been previously S. R. S. R. S. Downing A.K. Biochemistry. 2001; PubMed Scopus Google Scholar). The for the full-length native LDL receptor and the mutant receptor, which were into the for cell which LDL receptors by M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google were with each and were selected with were in with and with and were at and R329P were introduced into both the LDLR EGF-AB and full-length LDLR PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). The used were and for LDLR EGF-AB and and for full-length LDLR The used were and The from the were and for the and and for the full-length and back into the the The of each was by and of EGF pair from LDLR was and a previously by S. R. S. R. S. Downing A.K. Biochemistry. 2001; PubMed Scopus Google Scholar) with The protein was a in either in LB or in the The protein was pH being to a containing for purification and into pH containing The was reduced with in pH to purification by of the analysis was on a A was used with an of of a containing at a of a of to The was previously S. R. S. R. S. Downing A.K. Biochemistry. 2001; PubMed Scopus Google Scholar). protein was by a containing and in pH at a of The protein for analysis was by two of HPLC, and and analyzed by to the of a of the The was on a at pH and a of at a of at a of The protein with a native or native-like fold and This was and by a and an of of a containing at a of with a of to of a previously S.C. PubMed Scopus Google Scholar), were the The of was analyzed by a with an of of a containing at a of a of The for analysis were the of to after to were analyzed by the and in NMR of the NMR data were with at Protein were in at pH and WT, and R329P were at and The at of and of and of both sites at a of T. J. 1992; Scopus Google Scholar) were for each with by and with of in and in The data were a in and a with an in The were with to the and in the to with a resolution of A. J. Scholar) were with a of for the of the and was with of and with a of The data were a in and a in The were with to the and in the to with a resolution of in the were the the in the and the in the of the and changes were the with by to of calcium-binding of the EGF-AB pairs were previously S. R. S. R. S. Downing A.K. Biochemistry. 2001; PubMed Scopus Google Scholar, S. C. T. T. S. Biochemistry. PubMed Scopus Google Scholar). protein previously by NMR in the of were to the the were into a containing and pH through a with the of the were possible to and were a were the containing Molecular and protein in a of Protein were by The was to be on the of and for containing or Protein and were of the of a by the with a of of to The of the was the S. 2002; PubMed Scopus Google Scholar) data from for the two binding sites were the for each in the were the binding and and cell of with native and LDL receptors were analyzed for expression by on and to receptors were either with receptor or followed by the of a to were by of and was a data with the were into the for The of receptor expression was by with receptor followed by the of a to was by LDL binding and native or mutant receptors were from and with at for The ligand was at a of in pH and was with the for at for low ligand the pH was to by the with pH containing and followed by at for was by of and R329P Mutations on and Protein and R329P of the EGF-AB pair were in and from the of protein from is to a of both and R329P in reduced expression levels of the EGF-AB pairs compared with and The in vitro ability of each EGF-AB pair was analyzed by of each were established for the of the EGF-AB pair. were to and the were analyzed by The of protein for each that the the EGF-AB pair to a of was with to The of after the of for NMR and calcium-binding analysis levels for the two The from the analysis an of for both and of and R329P Mutations on the EGF-AB by of the and R329P mutations on the protein fold were by NMR of the mutant used from the the to the An that the analysis was on protein a to the native for The analysis of these the of a with of the A of the of the calcium-saturated that the of for each of the is to the that for the of NMR data for the and mutant EGF-AB selected region of of the LDLR EGF-AB pair at The of the is of calcium-saturated WT, and R329P EGF-AB pairs at pH and are to for and to domain for and for The are or by for the of the domain pair (9Saha S. Boyd J. Werner J.M. Knott V. Handford P.A. Campbell I.D. Downing A.K. Structure. 2001; 9: 451-456Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) were compared with in the of the two Both a to the EGF-AB pair. the of structural of the mutations, were in the to the in the mutant The are in are in The results in for in the of the R329P results in changes for the of the in a of the in may in the the and are in the to a EGF domain arrangement. The analysis for the R329P mutant was possible of the low of the The of of the protein in be to to a of The of is reduced from the for both and R329P that both are the range of the NMR to a in the EGF-AB pair of of native The with the R329P mutant for the of the and the first of the are The and the and results that the of the R329P mutant native changes for the A domain and in the R329P mutant may result from either localized structural and R329P Calcium in were for of the of and the EGF-AB A of was for in and at pH which is in with previously of the S. R. S. R. S. Downing A.K. Biochemistry. 2001; PubMed Scopus Google Scholar, A. J. J. Protein Sci. 1997; PubMed Scopus Google Scholar). Two were analyzed for each of the and with the to the data for both sites The for the EGF-AB pair are in with those previously at and pH of for and for S. R. S. R. S. Downing A.K. Biochemistry. 2001; PubMed Scopus Google Scholar). the calcium-binding the for both and R329P are to the The for the is to for The R329P mutant a from the analysis Calcium for the two sites in the EGF-AB pair were S. 2002; PubMed Scopus Google Scholar) from two for each and the for the used with protein in a and LDL of and the of cell-surface expression of the native LDLR and the two mutants, a was used to receptors with the which the first ligand-binding of the receptor and in the of to the native receptor, expression at the cell surface is reduced and R329P expression is reduced by an of the receptors that reach the cell surface retain the ability to bind and release LDL, a binding and analyzed the of bound LDL by The of LDL bound to the mutant receptors with cell-surface the EGF domain are to release ligand at pH (12Davis C.G. Goldstein J.L. Sudhof T.C. Anderson R.G.W. Russell D.W. Brown M.S. Nature. 1987; 326: 760-765Crossref PubMed Scopus (300) Google Scholar), and the been used a for the the bound LDL is released from the mutant receptors upon exposure to low pH it is released from the native receptor, that the receptors that are at the cell surface are and that the defect resulting from these mutations is a The two mutations selected for (G322S and R329P) have been in a of FH (4Hobbs H.H. Brown M.S. Goldstein J.L. Hum. Mutat. 1992; 1: 445-466Crossref PubMed Scopus (920) Google Scholar, L. V. Hum. Mutat. 1997; PubMed Scopus Google Scholar, C. A.K. J. Full Text PDF PubMed Google Scholar, A. H. L. D. R. M. Hum. PubMed Scopus Google Scholar, J.P. Hum. 2001; PubMed Scopus Google Scholar, R. Varret M. C.J. L. Boileau C. Kotze M.J. 2000; PubMed Scopus Google Scholar), and both of result in cholesterol levels associated with FH for FH the molecular of the LDLR is a the available for the of LDLR are and J. J. 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Biochemistry. 2001; PubMed Scopus Google Scholar). this have characterized the consequences of the and R329P mutations in vitro based on the analysis of structure, and calcium-binding of the EGF-AB pair and have compared these results with an in vivo analysis of the expression and of the mutations in the full-length The results that both mutations result in misfolding of the EGF-AB pair to a The in the that are be or The may that are by the and which result from R329P Based on the analysis of both an increased of upon in vitro of the pair. for each mutant the of the native-like was reduced with R329P the most decrease. This was by analysis of after refolding. of the wild and the of of the of each the it was possible to the of the in A of and result from protein misfolding either by mutations that the of a protein or from Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). to the in are characterized by either the of protein or of the protein that in the on L. Hum. Mutat. PubMed Scopus Google Scholar, R. Nature. PubMed Scopus Google Scholar). An analysis of the in vivo expression data of the full-length receptors the of protein on the cell surface for both at a reduced from R329P again the most effect to the of misfolding by R329P also the in the expression levels of the EGF-AB pair in in with the in vivo The of protein in be by a of the a analysis of the levels of a series of expression the of the with to levels of mutant protein these L. Hum. Mutat. PubMed Scopus Google Scholar). the mutant receptors at the cell data for both and R329P that these are in both the binding and release of results are with LDLR studies on a FH with the that showed reduced uptake of LDL R. Varret M. C.J. L. Boileau C. Kotze M.J. 2000; PubMed Scopus Google Scholar). The that receptors for both are to reach the cell surface is of a native-like fold in the and that structural to the significantly into the ligand-binding domains or β-propeller domain. The of an in the of for both and R329P after is with the mutations resulting in domain a fraction at the wild type. This fraction was for both to those used for the EGF-AB pair and was to to a to and NMR Solution NMR data were used on this to the structural changes to the EGF-AB pair resulting from the mutations. The of with the of misfolding associated with each of the mutations, again with the of the R329P more through the with those of the results for a of the the mutations result in local changes in Calcium is to an important in ligand binding to LDLR (12Davis C.G. Goldstein J.L. Sudhof T.C. Anderson R.G.W. Russell D.W. Brown M.S. Nature. 1987; 326: 760-765Crossref PubMed Scopus (300) Google Scholar, 13Kita T. Brown M.S. Watanabe Y. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2268-2272Crossref PubMed Scopus (172) Google Scholar, Protein 8: PubMed Scopus Google Scholar) and is to the fold of the EGF-AB pair S. R. S. R. S. Downing A.K. Biochemistry. 2001; PubMed Scopus Google Scholar). The of the for the of for and R329P for the EGF-AB pair the of a calcium-binding and a domain for S. R. S. R. S. Downing A.K. Biochemistry. 2001; PubMed Scopus Google Scholar). Calcium binding to both sites also was of the binding The for both mutant are to those previously for the EGF-AB pair S. R. S. R. S. Downing A.K. Biochemistry. 2001; PubMed Scopus Google Scholar), for both sites intact and for a native-like The sum of our results that both mutations result in the misfolding of the domain pair with R329P the more severe both is a of which a fraction maintains a native-like fold and is to bind to both domains, that is the intact receptor, is also the possibility that the of in domain results in more global misfolding suggested by the that LDLR in the cell range that are into native range pairs A. Science. 2002; 298: PubMed Scopus Google Scholar). misfolding be with the low surface expression of the receptors that have be to the cell surface more the misfolding was localized to a domain. A of our results and LDL levels suggests for these two mutations, is a protein defect and disease phenotype. The data from the studies in for both and the full-length which reach the cell are and the analysis of reduced uptake of LDL may be by reduced levels of receptor on the cell to this of these mutations been characterized in of effect or FH Based on the results and the available that both mutations be classified This is the more of a of the receptor at a reduced to the and to the cell and 2B are to missense mutations or that or the of the receptor (2Goldstein J.L. Hobbs H.H. Brown M.S. Scriver C.R. Sly W.S. Childs B. Beaudet A.L. Valle D. Kinzler K.W. Vogelstein B. 8th Ed. The Metabolic and Molecular Bases of Inherited Disease. II. McGraw-Hill, New York2001: 2863-2913Google Scholar). A studies have that levels and cardiovascular disease may be by the of that FH M. W. W. H. D. Full Text PDF PubMed Scopus Google Scholar, V. PubMed Scopus Google Scholar). the available data for the two mutations, is a significant in the levels of cholesterol in the data a more severe effect for R329P on in vitro and structure and on expression in may be by on blood cholesterol is that the of blood cholesterol and the of coronary with FH, those with the for the may be by (2Goldstein J.L. Hobbs H.H. Brown M.S. Scriver C.R. Sly W.S. Childs B. Beaudet A.L. Valle D. Kinzler K.W. Vogelstein B. 8th Ed. The Metabolic and Molecular Bases of Inherited Disease. II. McGraw-Hill, New York2001: 2863-2913Google Scholar). to the involved in protein a molecular and have been to be for the LDLR of proteins. is that and most with the EGF region the protein with the ligand-binding repeats of LDL receptor proteins. the of protein the misfolding of FH that in the repeats of the LDLR Y. W. A.L. G. Biochemistry. 2002; PubMed Scopus Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google Scholar). The of in vitro the of protein to some through of protein This is available in vivo of molecular and molecular are required to the and the sum of our in vivo data and the data available for R329P and that the of these mutations be by the for the The data for and R329P to a of the structural consequences of mutations to calcium-binding EGF domains that more in the A to in the calcium-binding EGF domain of is associated with U. K. T. W. T. C. H. J. Hum. Google Scholar, PubMed Google Scholar), and the in results in B. The in of the and been to result in of the domain in The in the calcium-binding EGF in the also the misfolding of the domain, the domain, a of the a to the domain Downing A.K. R. Handford P.A. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). these results that mutations may result in defects.