Calcium Is Essential for the Structural Integrity of the Cysteine-Rich, Ligand-Binding Repeat of the Low-Density Lipoprotein Receptor

Atkins, Annette R.; Brereton, Ian M.; Kroon, Paulus A.; Lee, Huang T.; Smith, Ross

doi:10.1021/bi972529n

Cited by 57 publications

(52 citation statements)

References 31 publications

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“…Thermally and chemically denatured apoLR5 shows almost no near-UV CD signal at either pH. Overall, the effects of calcium in the far-and near-UV CD spectra are reminiscent of those reported for LR1 (21,49).…”

Section: Spectroscopic Characterization Of Apolr5 and Lr5-ca 2ϩmentioning

confidence: 57%

Mechanism of Low Density Lipoprotein (LDL) Release in the Endosome

Arias-Moreno

Velázquez‐Campoy

Rodríguez

et al. 2008

Journal of Biological Chemistry

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Uptake of low density lipoproteins (LDL) by their receptor, LDLR, is the primary mechanism by which cells incorporate cholesterol from plasma. Mutations in LDLR lead to familial hypercholesterolemia, a common disease affecting 1 in 500 of the human population. LDLR is a modular protein that uses several small repeats to bind LDL. The repeats contain around 40 residues, including three disulfide bonds and a calcium ion. Repeat 5 (LR5) is critical for LDL and ␤-migrating very low density lipoprotein binding. Based on the crystal structure of LDLR at endosomal pH (but close to extracellular calcium concentration), LR5 has been proposed to bind to the epidermal growth factor (EGF) precursor domain of LDLR in the endosome, thus releasing the LDL particles previously bound in extracellular conditions. We report here the conformational stability of LR5 as a function of temperature and calcium concentration under both extracellular and endosomal pH conditions. The repeat was very stable when it bore a bound calcium ion but was severely destabilized in the absence of calcium and even further destabilized at acidic versus neutral pH. The temperature and calcium concentration dependence of LR5 stability clearly indicate that under endosomal conditions the unfolded conformation of the repeat is largely dominant. We thus propose a new mechanism for LDL release in the endosome in which calcium depletion and decreased stability at acidic pH drives LR5 unfolding, which triggers LDL release from the receptor. Subsequent binding of LR5 to the EGF precursor domain, if it takes place at low calcium concentrations, would contribute to a further shifting of the equilibrium toward dissociation. Most plasma cholesterol in humans is transported by low density lipoprotein (LDL)4 (1). LDL receptors (LDLR) in cell membranes bind LDL, and the complexes enter the cell by endocytosis (2-5). At the acidic pH of the endosome, LDL is released allowing LDLR recycling to the membrane (1). Although LDLR was initially thought to play the single role of helping to achieve cholesterol homeostasis, its expression in neurons suggests that it may also perform other roles (6, 7). LDLR gives its name to the LDL receptor family of transmembrane receptors that additionally comprises the low density lipoprotein receptor-related protein and the very low density lipoprotein receptor among others (8). This family of receptors is involved in a variety of important biological functions related to cell uptake and signal transduction (9, 10). LDLR (11-13) is a transmembrane protein containing 839 amino acid residues organized in five domains: ligand binding, EGF precursor homologous, glycosylated, transmembrane, and cytoplasmatic (1, 14 -16). The ligand-binding domain, consisting of seven tandem, structurally-homologous repeats, is responsible for binding lipoproteins (17-21); the EGF precursor domain participates in receptor recycling to the cell surface and, together with the binding domain, in lipoprotein release in the endosome (18,(22)(23)(24)(25)(26)(27). Of t...

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Section: Spectroscopic Characterization Of Apolr5 and Lr5-ca 2ϩmentioning

confidence: 57%

Mechanism of Low Density Lipoprotein (LDL) Release in the Endosome

Arias-Moreno

Velázquez‐Campoy

Rodríguez

et al. 2008

Journal of Biological Chemistry

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“…Individual LDL-A repeats that were refolded in vitro without calcium adopted conformations with all possible cysteine pairings (39,40,51,52), indicative of a complete lack of stable native state. Accordingly, formation of the C7 epitope in LR1 requires calcium (29,53).…”

Section: Discussionmentioning

confidence: 99%

“…The structure of the EGF repeats, with their noncanonical calcium-binding site, appears less dependent on this cation (15). Once isolated LDL-A repeats are folded, calcium can be extracted by incubation without calcium or with EGTA or EDTA (39,41). Calcium affinity measurements demonstrate that bound calcium in a folded repeat indeed is in equilibrium with free calcium (54).…”

Section: Discussionmentioning

confidence: 99%

“…Most of these cations would not have sufficiently high concentrations to bind, because of affinities lower than millimolar. Mg 2ϩ , however, which is present in millimolar concentrations, should be sufficient to take care of the charge issue but cannot substitute for calcium in the native fold (39). This implies that calcium performs a specific role during the earliest stages of LDL receptor folding.…”

Section: Discussionmentioning

confidence: 99%

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Calcium as a Crucial Cofactor for Low Density Lipoprotein Receptor Folding in the Endoplasmic Reticulum

Pena¹,

Jansens²,

Zadelhoff

et al. 2010

Journal of Biological Chemistry

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The family of low density lipoprotein (LDL) receptors mediate uptake of a plethora of ligands from the circulation and couple this to signaling, thereby performing a crucial role in physiological processes including embryonic development, cancer development, homeostasis of lipoproteins, viral infection, and neuronal plasticity. Structural integrity of individual ectodomain modules in these receptors depends on calcium, and we showed before that the LDL receptor folds its modules late after synthesis via intermediates with abundant non-native disulfide bonds and structure. Using a radioactive pulsechase approach, we here show that for proper LDL receptor folding, calcium had to be present from the very early start of folding, which suggests at least some native, essential coordination of calcium ions at the still largely non-native folding phase. As long as the protein was in the endoplasmic reticulum (ER), its folding was reversible, which changed only upon both proper incorporation of calcium and exit from the ER. Coevolution of protein folding with the high calcium concentration in the ER may be the basis for the need for this cation throughout the folding process even though calcium is only stably integrated in native repeats at a later stage.

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“…This, obviously, precluded CD81 from taking any part in HCV-SP binding or entry in HepG2 cells. The low-density lipoprotein receptor (LDL-R) has been implicated in the binding and entry of HCV into cells (1,42) and also requires calcium for ligand binding (3). The HCV virion has been reported to complex with low-density lipoprotein and very-low-density lipoprotein in the serum (54,64,65).…”

Section: Discussionmentioning

confidence: 99%

Role of the Asialoglycoprotein Receptor in Binding and Entry of Hepatitis C Virus Structural Proteins in Cultured Human Hepatocytes

Saunier

Triyatni

Ulianich

et al. 2003

J Virol

118

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We used a baculovirus-based system to prepare structural proteins of hepatitis C virus (HCV) genotype 1a. Binding of this preparation to cultured human hepatic cells was both dose dependent and saturable. This binding was decreased by calcium depletion and was partially prevented by ligands of the asialoglycoprotein receptor (ASGP-R), thyroglobulin, asialothyroglobulin, and antibody against a peptide in the carbohydrate recognition domain of ASGP-R but not preimmune antibody. Uptake by hepatocytes was observed with both radiolabeled and dye-labeled HCV structural proteins. With hepatocytes expressing the hH1 subunit of the ASGP-R fused to green fluorescent protein, we could show by confocal microscopy that dye stain cointernalized with the fusion protein in an area surrounding the nucleus. Internalization was more efficient with a preparation containing p7 than with one that did not. The two preparations bound to transfected 3T3-L1 cells expressing either both (hH1 and hH2) subunits of the ASGP-R (3T3-22Z cells) or both hH1 and a functionally defective variant of hH2 (3T3-24X cells) but not to parental cells. Additionally, uptake of dye-labeled preparation containing p7 was observed with 3T3-22Z cells but not with 3T3-L1 or 3T3-24X cells or with the preparation lacking p7, suggesting that p7 regulates the internalization properties of HCV structural proteins. Our observations suggest that HCV structural proteins bind to and cointernalize with the ASGP-R in cultured human hepatocytes.Hepatitis C virus (HCV) infection has become a major health problem affecting an estimated 170 million people worldwide. Persistent infection occurs in more than 70% of people infected with HCV, which may be complicated by cirrhosis and/or hepatocellular carcinoma (24, 33). Despite highly competitive and extensive research in this field, a highly effective treatment is not yet available. The mainstay of anti-HCV therapy, alpha interferon (IFN-␣) or pegylated IFN-␣, together with ribavirin leads, at best, to viral clearance in ca. 41 to 54% of patients infected with HCV genotype 1a (34,38). Since mechanisms of HCV infection remain unclear, characterization of these mechanisms is now a major issue for the development of new strategies for anti-HCV treatment and prevention.

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Calcium Is Essential for the Structural Integrity of the Cysteine-Rich, Ligand-Binding Repeat of the Low-Density Lipoprotein Receptor

Cited by 57 publications

References 31 publications

Mechanism of Low Density Lipoprotein (LDL) Release in the Endosome

Mechanism of Low Density Lipoprotein (LDL) Release in the Endosome

Calcium as a Crucial Cofactor for Low Density Lipoprotein Receptor Folding in the Endoplasmic Reticulum

Role of the Asialoglycoprotein Receptor in Binding and Entry of Hepatitis C Virus Structural Proteins in Cultured Human Hepatocytes

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