Model System for Phenotypic Characterization of Sequence Variations in the LDL Receptor Gene

Clinical Chemistry - Tập 52 Số 8 - Trang 1469-1479 - 2006
Trine Ranheim1, Mari Ann Kulseth1, Knut Erik Berge1, Trond P. Leren
1Department of Medical Genetics, Rikshospitalet-Radiumhospitalet Medi-cal Center, N-0027 Oslo, Norway.

Tóm tắt

AbstractBackground: Sequence variations in the LDL receptor (LDLR) gene cause defects of LDLR protein production and function through different molecular mechanisms. Here we describe a cell model system for the phenotypic characterization of sequence variations in the LDLR gene. Well-known sequence variations belonging to LDLR classes 2 to 5 (p.G565V, p.I161D, p.Y828C, and p.V429M) were studied in CHO and HepG2 cells.Methods: Expression of LDLR protein on the cell surface was detected by use of fluorescence-conjugated antibodies against the LDLR and the LDLR activity was measured by incubating the cells with fluorescently labeled and radiolabeled LDL. The intracellular locations of the LDLR mutants and wild-type were also investigated.Results: The class 2A p.G565V sequence variant exhibited an intracellular distribution of LDLR with no active receptors on the cell surface. Both the class 3 p.I161D and class 4 p.Y828C sequence variants gave surface staining but had a reduced ability to bind or internalize LDL, respectively. By determining the intracellular locations of the receptors we were able to visualize the accumulation of the class 5 p.V429M sequence variant in endosomes by means of a specific marker, as well as confirming that the class 4 p.Y828C variant was not localized in clathrin-coated pits. Flow cytometry allowed us quantitatively to determine the amount and activity of receptors. To confirm the results of binding and cell association of fluorescently labeled LDL analyzed by flow cytometry, assays using 125I-labeled LDL were performed. In addition to a useful and valid alternative to radiolabeled LDL, the unique properties of fluorescently labeled LDL allowed a variety of detection technologies to be used.Conclusions: This new approach enables phenotypic characterization of sequence variations in the LDLR gene. The assays developed may be valuable for confirming the pathogenicity of novel missense sequence variations found throughout the LDLR gene.

Từ khóa


Tài liệu tham khảo

Goldstein JL, Hobbs HH, Brown MS. Familial hypercholesterolemia. Scriver CR Beaudet AL Sly WS Valle D eds. The Metabolic Basis of Inherited Disease2001:2863-2913 McGraw-Hill New York. .

Division of Cardiovascular Genetics. Department of Medicine, University College London Medical School. The low density lipoprotein receptor (LDLR) gene in familial hypercholesterolemia.http://www.ucl.ac.uk/fh (accessed ???)..

Laboratory of Human Genetics. Universal mutation database (UMD). http://www.umd.necker.fr (accessed ????).

Tolleshaug H, Goldstein JL, Schneider WJ, Brown MS. Posttranslational processing of the LDL receptor and its genetic disruption in familial hypercholesterolemia. Cell1982;30:715-724.

Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science1986;232:34-47.

Davis CG, Lehrman MA, Russell DW, Anderson RG, Brown MS, Goldstein JL. The J.D. mutation in familial hypercholesterolemia: amino acid substitution in cytoplasmic domain impedes internalization of LDL receptors. Cell1986;45:15-24.

Hobbs HH, Brown MS, Goldstein JL. Molecular genetics of the LDL receptor gene in familial hypercholesterolemia. Hum Mutat1992;1:445-466.

Bertolini S, Pisciotta L, Di SL, Langheim S, Bellocchio A, Masturzo P, et al. Genetic polymorphisms affecting the phenotypic expression of familial hypercholesterolemia. Atherosclerosis2004;174:57-65.

Hobbs HH, Russell DW, Brown MS, Goldstein JL. The LDL receptor locus in familial hypercholesterolemia: mutational analysis of a membrane protein. Annu Rev Genet1990;24:133-170.

Hobbs HH, Leitersdorf E, Goldstein JL, Brown MS, Russell DW. Multiple crm− mutations in familial hypercholesterolemia. Evidence for 13 alleles, including four deletions. J Clin Invest1988;81:909-917.

Goldstein JL, Brown MS, Anderson RG, Russell DW, Schneider WJ. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol1985;1:1-39.

Lehrman MA, Goldstein JL, Brown MS, Russell DW, Schneider WJ. Internalization-defective LDL receptors produced by genes with nonsense and frameshift mutations that truncate the cytoplasmic domain. Cell1985;41:735-743.

Beglova N, Jeon H, Fisher C, Blacklow SC. Cooperation between fixed and low pH-inducible interfaces controls lipoprotein release by the LDL receptor. Mol Cell2004;16:281-292.

Sorensen S, Ranheim T, Bakken KS, Leren TP, Kulseth MA. Retention of mutant low density lipoprotein receptor in ER leads to ER stress. J Biol Chem2006;281:468-476.

Church GM, Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A1984;81:1991-1995.

Peacock SL, Bates MP, Russell DW, Brown MS, Goldstein JL. Human low density lipoprotein receptor expressed in Xenopus oocytes. Conserved signals for O-linked glycosylation and receptor-mediated endocytosis. J Biol Chem1988;263:7838-7845.

Beisiegel U, Schneider WJ, Goldstein JL, Anderson RG, Brown MS. Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia. J Biol Chem1981;256:11923-11931.

Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest1955;34:1345-1353.

Pitas RE, Innerarity TL, Weinstein JN, Mahley RW. Acetoacetylated lipoproteins used to distinguish fibroblasts from macrophages in vitro by fluorescence microscopy. Arteriosclerosis1981;1:177-185.

Pittman RC, Carew TE, Glass CK, Green SR, Taylor CA, Jr, Attie AD. A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo. Biochem J1983;212:791-800.

Goldstein JL, Basu SK, Brown MS. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol1983;98:241-260.

Gillian-Daniel DL, Bates PW, Tebon A, Attie AD. Endoplasmic reticulum localization of the low density lipoprotein receptor mediates presecretory degradation of apolipoprotein B. Proc Natl Acad Sci U S A2002;99:4337-4342.

Mu FT, Callaghan JM, Steele-Mortimer O, Stenmark H, Parton RG, Campbell PL, et al. EEA1, an early endosome-associated protein. EEA1 is a conserved α-helical peripheral membrane protein flanked by cysteine “fingers” and contains a calmodulin-binding IQ motif. J Biol Chem1995;270:13503-13511.

Goldstein JL, Brown MS, Stone NJ. Genetics of the LDL receptor: evidence that the mutations affecting binding and internalization are allelic. Cell1977;12:629-641.

Robinson MS. The role of clathrin, adaptors and dynamin in endocytosis. Curr Opin Cell Biol1994;6:538-544.

Stephan ZF, Yurachek EC. Rapid fluorometric assay of LDL receptor activity by DiI-labeled LDL. J Lipid Res1993;34:325-330.

Schmitz G, Bruning T, Kovacs E, Barlage S. Fluorescence flow cytometry of human leukocytes in the detection of LDL receptor defects in the differential diagnosis of hypercholesterolemia. Arterioscler Thromb1993;13:1053-1065.

Esser V, Russell DW. Transport-deficient mutations in the low density lipoprotein receptor. Alterations in the cysteine-rich and cysteine-poor regions of the protein block intracellular transport. J Biol Chem1988;263:13276-13281.

Russell DW, Brown MS, Goldstein JL. Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins. J Biol Chem1989;264:21682-21688.

Seidah NG, Benjannet S, Wickham L, Marcinkiewicz J, Jasmin SB, Stifani S, et al. The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation. Proc Natl Acad Sci U S A2003;100:928-933.

Park SW, Moon YA, Horton JD. Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin/kexin type 9a in mouse liver. J Biol Chem2004;279:50630-50638.

Davis CG, Goldstein JL, Sudhof TC, Anderson RG, Russell DW, Brown MS. Acid-dependent ligand dissociation and recycling of LDL receptor mediated by growth factor homology region. Nature1987;326:760-765.

Goldstein JL, Anderson RG, Brown MS. Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature1979;279:679-685.

Chang JH, Pan JP, Tai DY, Huang AC, Li PH, Ho HL, et al. Identification and characterization of LDL receptor gene mutations in hyperlipidemic Chinese. J Lipid Res2003;44:1850-1858.

Jensen TG, Andresen BS, Jensen HK, Jensen LG, Heath F, Pedersen S, et al. Rapid characterization of disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene by overexpression in COS cells. Z Gastroenterol1996;34(Suppl 3):9-11.

Thông tin
Thông tin xuất bản

Clinical Chemistry

Tập 52 Số 8

1469-1479

Thông tin tác giả