Posttranslational processing of the LDL receptor and its genetic disruption in familial hypercholesterolemia

Tolleshaug, Helge; Goldstein, Joseph L.; Schneider, Wolfgang J.; Brown, Michael S.

doi:10.1016/0092-8674(82)90276-8

Cited by 239 publications

(108 citation statements)

References 22 publications

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“…As expected for the LDL receptor (Ref. 35; see also Ref. 20), molecular weight differences were observed for fraction A(III) samples examined by SDS-PAGE under reducing (ϳ160 kDa) and non-reducing (ϳ130 kDa) conditions (Fig.…”

Section: Resultssupporting

confidence: 81%

Vesicle-reconstituted Low Density Lipoprotein Receptor

Jeon¹,

Shipley

2000

Journal of Biological Chemistry

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The low density lipoprotein (LDL) receptor is a key protein for maintaining cellular cholesterol homeostasis by binding cholesterol-rich lipoproteins through their apoB and apoE apoproteins. The LDL receptor is a transmembrane glycoprotein of M r ϳ115 kDa; based on its primary sequence, five distinct structural domains have been identified (Yamamoto, T., Davis, C. G., Brown, M. S., Schneider, W. J., Casey, M. L., Goldstein, J. L., and Russell, D. W. (1984) Cell 39, 27-38). As a first step toward providing a structural description of the intact LDL receptor, the receptor has been purified from bovine adrenal cortices, reconstituted into unilamellar egg yolk phosphatidylcholine vesicles, and imaged using cryoelectron microscopy (cryoEM). CryoEM has the advantage of providing images of the reconstituted LDL receptor in its frozen, fully hydrated state. LDL receptor molecules were visualized as elongated, stick-like projections from the vesicle surface with maximum dimensions ϳ120-Å length by ϳ45-Å width. In some of the images, a short arm (or arms) was visible at the distal end of the stick-like projections. The LDL receptor was labeled via accessible free cysteine residues, probably including that corresponding to Cys-431 of the known fulllength sequence of the human LDL receptor. The accessible cysteine was demonstrated using a maleimide-biotin⅐streptavidin conjugate and confirmed by labeling with monomaleimido-Nanogold. Images obtained by cryoEM showed that the extracellular stick-like domain of the reconstituted LDL receptor was labeled by Nanogold. This combined cryoEM-Nanogold labeling study has provided the first low resolution structural images of the reconstituted, full-length bovine LDL receptor. Low density lipoprotein (LDL)1 is the most abundant cholesterol-containing lipoprotein in human plasma (1). Since an elevated level of blood cholesterol is a major risk factor in developing atherosclerosis, the LDL receptor, which is responsible for the uptake of LDL into cells, has been studied extensively, notably by Brown and Goldstein (2). The LDL receptor is located in coated pit regions on the cell surface; it binds cholesterol-rich lipoproteins (LDL and ␤-VLDL) via receptor binding sub-domains of their apoB and apoE proteins, and the receptor-ligand complex is internalized by endocytosis. Acidification of the internal compartment of the endocytic vesicle leads to dissociation of the LDL receptor-LDL complex; the LDL receptor is recycled to the cell surface, and LDL is delivered to the lysosome. Release of cholesterol triggers a variety of intracellular metabolic and regulatory events, notably the regulation of cholesterol synthesis and expression of the LDL receptor and other genes (1-4). It should be stated that these studies of the LDL receptor were critical not only in providing a rationale for cholesterol homeostasis but also for laying the foundations for our understanding of intracellular trafficking mechanisms.The amino acid sequence of the LDL receptor has been reported for several species includin...

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Section: Resultssupporting

confidence: 81%

Vesicle-reconstituted Low Density Lipoprotein Receptor

Jeon¹,

Shipley

2000

Journal of Biological Chemistry

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“…As seen in Figure 4, the LDLR with the 27 amino-acid insert did not undergo oligosaccharide modifications in the Golgi apparatus, which increase the apparent molecular mass of the wild-type LDLR from B120 to 160 kDa. 10 …”

Section: Resultsmentioning

confidence: 99%

Analysis of LDLR mRNA in patients with familial hypercholesterolemia revealed a novel mutation in intron 14, which activates a cryptic splice site

2010

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Familial hypercholesterolemia (FH) is caused by a defective low-density lipoprotein receptor (LDLR), and 41000 mutations in LDLR have been identified. However, in some patients with clinically defined FH, no mutation can be detected within the exons and adjacent intronic segments of the LDLR. We have analyzed RNA extracted from blood samples of patients with clinically defined FH and identified an aberrantly spliced mRNA containing an 81-bp insert from intron 14. The aberrant splicing was caused by a novel intronic mutation, c.2140+86C4G, which activated a cryptic splice site. Although the cryptic splice site does not completely surpass the normal splice site, the mutation was found to cosegregate with high cholesterol levels in a family, which supports the notion that c.2140+86C4G causes FH. The insertion of 81 bp in LDLR mRNA encodes an in-frame insertion of 27 amino acids in the LDLR. However, the insertion was found to hamper LDLR activity by preventing the receptor from leaving the endoplasmic reticulum, probably because of misfolding of the protein. In patients with clinically defined hypercholesterolemia, despite normal results from sequencing of exonic regions of the LDLR gene, characterization of the LDLR mRNA might identify the underlying genetic defect.

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“…Monolayers of fibroblasts were incubated with [35S~methionine as described (13). After labeling, cells were incubated in medium containing 200 ,uM methionine and harvested after various time intervals.…”

Section: Immunochemical Analysis Of [35s]methionine-labeled Ldlmentioning

confidence: 99%

Deletion in the first cysteine-rich repeat of low density lipoprotein receptor impairs its transport but not lipoprotein binding in fibroblasts from a subject with familial hypercholesterolemia.

Leitersdorf

Hobbs

Fourie

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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The ligand-binding domain of the low density lipoprotein (LDL) receptor is composed of seven cysteine-rich repeats, each -40 amino acids long. Previous studies by van Driel et al. [van Driel, I. R., Goldstein, J. L., Sudhof, T. C. & Brown, M. S. (1987) J. Biol. Chem. 262, 17443-17449] showed that if the first repeat of the ligand-binding domain (encoded by exon 2) is deleted, the receptor fails to bind an anti-LDL receptor monoclonal antibody (IgG-C7) but continues to bind LDL with high affinity. Cultured fibroblasts from a Black South African Xhosa patient (TT) with the clinical syndrome of homozygous familial hypercholesterolemia demonstrated highaffinity cell-surface binding of 125I-labeled LDL but not '25I-labeled IgG-C7. Previous haplotype analysis, using 10 restriction fragment length polymorphic sites, suggested that the patient inherited two identical LDL receptor alleles. The polymerase chain reaction technique was used to selectively amplify exon 2 of the LDL receptor gene from this patient. Sequence analysis of the amplified fragment disclosed a deletion of six base pairs that removes two amino acids, aspartic acid and glycine, from the first cysteine-rich ligand binding repeat. The mutation creates a new Pst I restriction site that can be used to detect the deletion. The existence of this mutant allele confirms that the epitope of IgG-C7 is located in the first cysteine-rich repeat and that this repeat is not necessary for LDL binding. The mutant gene produced a normally sized 120-kilodalton LDL receptor precursor protein that matured to the 160-kilodalton form at less than one-fourth the normal rate. Thus, deletion of two amino acids within the first cysteine-rich repeat retards receptor transport from the endoplasmic reticulum to the cell surface, in contrast to deletion of the entire first repeat, which has no effect on receptor maturation.Mutations in the low density lipoprotein (LDL) receptor gene result in the autosomal dominant disease familial hypercholesterolemia (FH) (1). The LDL receptor is a transmembrane protein that binds LDL and mediates its uptake into the cell. Individuals with two mutant LDL receptor alleles have profoundly fewer or no functional LDL receptors on their cell surfaces and this is associated with markedly elevated plasma LDL-cholesterol levels. These individuals develop premature coronary atherosclerosis that often leads to myocardial infarctions in early childhood. In homozygous FH, mutations in each LDL receptor allele can be either identical (a true homozygote) or different (a compound heterozygote).

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Posttranslational processing of the LDL receptor and its genetic disruption in familial hypercholesterolemia

Cited by 239 publications

References 22 publications

Vesicle-reconstituted Low Density Lipoprotein Receptor

Vesicle-reconstituted Low Density Lipoprotein Receptor

Analysis of LDLR mRNA in patients with familial hypercholesterolemia revealed a novel mutation in intron 14, which activates a cryptic splice site

Deletion in the first cysteine-rich repeat of low density lipoprotein receptor impairs its transport but not lipoprotein binding in fibroblasts from a subject with familial hypercholesterolemia.

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