Activity-associated effect of LDL receptor missense variants located in the cysteine-rich repeats

Activity-associated effect of LDL receptor missense variants located in the cysteine-rich repeats A. Etxebarria1#, A. Benito-Vicente1#, M. Stef2, H. Ostolaza1, L. Palacios2, C. Martin1* 1Unidad de Biofísica (CSIC, UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, Apdo. 644, 48080 Bilbao, Spain. 2 Progenika Biopharma, a Grifols Company, Derio, Spain. * Corresponding Author E-mail: [email protected]. Tel. +34-94-601.80.53; Fax +3494-601.33.60 # A.E. and A.B-V. have equally contributed to this work Number of Tables: 2 Number of Figures: 7 Number of supplementary Figures: 6 Number of supplementary Tables: 1 This is the accepted manuscript of the article that appeared in final form in Atherosclerosis 238(2) : 304-312 (2015), which hasbeenpublished in final form at https://doi.org/10.1016/j.atherosclerosis.2014.12.026. © 2014 Elsevier under CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Abstract Background: The LDL receptor (LDLR) is a Class I transmembrane protein critical for the clearance of cholesterol-containing lipoprotein particles. The N-terminal domain of the LDLR harbours the ligand-binding domain consisting of seven cysteine-rich repeats of approximately 40 amino acids each. Mutations in the LDLR binding domain may result in loss of receptor activity leading to familial hypercholesterolemia (FH). In this study the activity of six mutations located in the cysteine-rich repeats of the LDLR has been investigated. Methods: CHO-ldlA7 transfected cells with six different LDLR mutations have been used to analyse in vitro LDLR expression, lipoprotein binding and uptake. Immunoblotting of cell extracts, flow cytometry and confocal microscopy have been performed to determine the effects of these mutations. In silico analysis was also performed to predict the mutation effect. Results and conclusion: From the six mutations, p.Arg257Trp turned out to be a nonpathogenic LDLR variant whereas p.Cys116Arg, p.Asp168Asn, p.Asp172Asn, p.Arg300Gly and p.Asp301Gly were classified as binding-defective LDLR variants whose effect is not as severe as null allele mutations. Keywords: LDLR, mutations, Familial hypercholesterolemia, ligand binding domain, mutation Class defect 1. Introduction Familial Hypercholesterolemia (FH; MIM#143890) is an autosomal dominant disorder causing premature coronary heart disease (CHD) [1] that is characterized by increased plasma LDL cholesterol, tendon xanthomas, deposits of cholesterol in peripheral tissues and accelerated atherosclerosis. FH has a homozygous frequency of 1:1,000,000, and its heterozygous frequency has recently been estimated to be as high as 1/200 in the general population [2,3], suggesting that the disease is heavily underdiagnosed and undertreated. FH is mainly due to mutations in the LDL receptor (LDLR; MIM# 606945) gene, which is responsible for the uptake of LDL particles into cells [1]. The LDLR is a modular protein that combines five different domains: the ligand binding domain, the EGF-like module that contain a 280 amino acid β-propeller, the Oglycosylated domain and the transmembrane and cytoplasmic domains. The binding domain of LDLR comprises ~40-amino acid long cysteine-rich repeats in tandem, structured in seven discrete extracellular modules (R1-R7), which are responsible for the binding and release of its lipoprotein ligands [4]. After binding, the LDLR-lipoprotein complex is internalized through clathrin-coated pits and traffics to endosomes, where lipoprotein cargo is released [5,6]. Lipoproteins are subsequently degraded in lysosomes, while the LDLR recycles back to the cell surface for further rounds of lipoprotein uptake. Nowadays more than 1300 different variants have been described in the LDLR gene [7], not all of them pathogenic. According to the nature and location of the mutations within the LDLR and to the phenotypic effects on the protein, mutations have been divided into five different classes [8]: Class 1: no detectable LDLR synthesis; Class 2: defective LDLR transport; Class 3: impaired LDL to LDLR binding; Class 4: no LDLR/LDL internalization due to defective clustering in clathrin-coated pits; and Class 5: no LDLR recycling. The fact that several missense variants of the LDLR found in FH patients have been shown not to be the actual cause of the disease [9,10] indicates that every detected variant needs to be functionally characterized in order to determine its severity, if any. The aim of this study was to analyse the impact on the LDLR activity of six missense variants located in the ligand binding domain of the protein, and previously found in FH patients. The sequence variations studied predict the following amino acid changes in the LDLR: p.Cys116Arg, p.Asp168Asn, p.Asp172Asn, p.Arg257Trp, p.Arg300Gly and p.Asp301Gly. The effects on LDLR expression, binding capacity and uptake were studied by Western blot, flow cytometry and confocal microscopy in a transfected LDLRdefective Chinese hamster ovary (CHO) cell line. 2. Materials and Methods 2.1. Selection of Variants The selection of the six missense LDLR variants was based on two criteria: to have been previously found in FH patients and to be possibly associated to a binding defect. To cover the first criteria we selected variants previously described by other authors in FH patients that have also been found in at least one FH index case by LIPOchip® platform [11] or by SEQPRO LIPO RS® platform in Progenika Biopharma (Derio, Spain), both platforms with the CE mark. Just two of the variants have been found in large population studies, p.Asp168Asn and p.Arg257Trp, that were coded as rs200727689 and rs200990725, respectively in the NCBI SNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?geneId=3949). Both of them have a very low frequency, p.Asp168Asn has been found in the EVS database (http://evs.gs.washington.edu/EVS/) with a MAF(%)= 0.0154 and p.Arg257Trp in the 1000 genomes database (http://browser.1000genomes.org.) with a MAF(%)=0.001. The characteristics of the selected variants are compiled in Table 1 and their location within the protein is shown in Figure 1. 2.2. Site-directed mutagenesis Plasmids carrying the LDLR variants were constructed by Innoprot (Derio, Spain) as described in Online Supp. Data. 2.3. Cell culture and transfection LDLR-deficient CHO cell line ldlA7 (CHO-ldlA7) (kindly provided by Dr. Monty Krieger, Massachusetts Institute of Technology, Cambridge, MA) was cultured in Ham’s F-12 medium supplemented with 5% FBS, 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. CHO-ldlA7 cells were plated into 6or 24-well culture plates, and transfected with plasmids carrying the LDLR variants using Lipofectamine® LTX and PlusTM Reagent (Invitrogen) according to the manufacturer’s instructions. Transfected cells were maintained in culture during 48 h to achieve maximal LDLR expression. 2.4. Western blot analysis Cell lysates were prepared, protein concentration determined, and fractionated by electrophoresis as described in Online Supp. Data. 2.5. Lipoprotein isolation LDL and VLDL were isolated from blood samples of healthy individuals in a two step centrifugation as described in Online Supp. Data. 2.6. Lipoprotein labelling LDL and VLDL were labelled with FITC as previously described [12]. Briefly, lipoproteins (1mg/mL) in 0.1 M NaHCO3 (pH 9.0) was mixed with 10 µl/mL FITC (2 mg/mL in dimethyl sulfoxide). The mixture was gently mixed by slow rocking at room temperature for 2 h. The unreacted dye was removed by gel filtration on a Sephadex G25 column equilibrated with PBS EDTA-free buffer. All fractions were assayed for protein content with bovine serum albumin as standard (Pierce BCA protein assay, Pierce). 2.7. Quantification of LDLR activity by flow cytometry Transfected CHO-ldlA7 cells were grown in 24-well culture plates. 48 h after transfection, cells were incubated for 4 h, at 37ºC or at 4ºC with 20 µg/mL FITC-LDL to determine LDLR activity or LDL-LDLR binding, respectively. After incubation with FITC-LDL, CHO-ldlA7 cells were washed twice in PBS-1%BSA, fixed on 4% formaldehyde for 10 min and washed again twice with PBS-1%BSA. To determine the amount of internalized LDL, Trypan blue solution (Sigma-Aldrich, Steinheim, Germany) was added directly to the samples to a final concentration of 0.2%, eliminating the extracellular signal due to the non-internalized LDL-LDLR complexes. Measurement of VLDL was performed by incubation of cells with 20 µg/mL FITCVLDL for 4 h, at 37ºC as described for LDL. Fluorescence intensities were measured by FACS, in a Facscalibur Flow cytometer according to the manufacturer instructions as previously described [9]. For each sample, fluorescence of 10,000 events was acquired for data analysis. All measurements were performed at least in triplicate. 2.8. Quantification of LDLR expression by flow cytometry To determine LDLR cell surface expression by FACS, transfected CHO-ldlA7 cells grown during 48 h were incubated with a mouse primary antibody anti-LDLR (1:100; 2.5 mg/L; Progen Biotechnik GmbH) for 1 h, at room temperature, then washed twice with PBS-1%BSA and incubated with secondary antibody Alexa Fluor 488-conjugated goat anti-mouse IgG (1:100; Molecular Probes). For each sample, fluorescence of 10,000 events was acquired for data analysis. All measurements were performed at least in triplicate. 2.9. Confocal Laser Scanning Microscopy (CLSM) CLSM was used to analyse LDL-LDLR binding and LDL uptake in LDLR transfected CHO-ldlA7 cells. Briefly, cells were plated in coverslips and then transfected with the LDLR containing plasmids and cultured for 48 h, at 37ºC in 5% CO2. Then the medium was removed and coverslips washed twice with PBS-1%BSA. To determine LDL-LDLR binding and LDL uptake, non-labelled lipoproteins (20 µg/mL LDL) were added and cells were incubated for additional 4 h at 4ºC or 37ºC, respectively. Cells were fixed with 4% paraformaldehyde during 10 min, washed three times with PBS-1%BSA and permeabilised with 1% Triton X-100 for 30 min at room temperature. Samples were then washed and blocked in PBS-10%FBS for 1 h and washed in PBS-1%BSA three times. Then samples were incubated with the appropriate primary antibodies for 16 h at 4ºC, followed by incubation with the appropriate fluorescent secondary antibodies. Coverslips were mounted on a glass slide and samples were visualised using a confocal microscope (Olympus IX 81) with sequential excitation and capture image acquisition with a digital camera (Axiocam NRc5, Zeiss). Images were processed with Fluoview v.50 software. Image analysis to quantify the fluorescence intensities was accomplished using the public domain software ImageJ (available at http://rsb.info.nih.gov/ij) running on a standard PC. 2.10. In silico predicted effect of molecular event on LDLR The possible impact of amino acid substitutions on the structure and function of missense variants was predicted by using four different softwares as described in Online Supp. Data. 2.11. Conservation analysis Conservation analysis among species for nucleotide and amino acid was carried out as described in Online Supp. Data. 2.12. Statistical analysis All measurements were performed at least 3 times, with n=3 unless otherwise stated, and results are presented as mean ± s.d. Levels of significance were determined by a two-tailed Student's t-test, and a confidence level of greater than 95% (p<0.05) was used to establish statistical significance. 3. Results 3.1. In silico analysis The results obtained by different software packages are presented in Table 2. Depending on the program, especially Align GVGD compared to the other ones, the prediction of the effect caused by the variants is different. All the variants except p.Arg257Trp and p.Arg300Gly were classified as pathogenic by the majority of the prediction programs. These 2 variants are the less conserved ones and, as the prediction algorithms are mainly based on conservation analysis, the results are the expected ones. No splicing defects were predicted for any of the studied variants (data not shown). 3.2. Expression of LDLR variants in CHO-ldlA7 cells CHO-ldlA7 cells were transfected with plasmids carrying the different variants and LDLR expression was assayed by immunoblotting as described in Materials and Methods. As shown in Figure 2A (upper panel), all the mutated LDLR are expressed at similar levels as wt 48 h after transfection. Equal loading of protein was confirmed in each blot by membrane stripping and further incubation with antibodies to visualise cytosolic GAPDH protein (Figure 2A, lower panel). The extent of protein expression was determined by quantitative densitometric analysis (Figure 2B). The data obtained by flow cytometry confirmed these results, the LDLR expression of all the variants being similar to the one in the wt (Figure 3A). 3.3. Analysis of LDLR activity by FACS CHO-ldlA7 cells expressing wt LDL receptor or LDLR p.Cys116Arg, p. Asp168Ans, p.Asp172Asn, p.Arg257Trp, p.Arg300Gly and p.Asp301Gly variants were assayed for LDL binding and uptake by flow cytometry. For internal method validation two controls were used because their effects on expression, binding and uptake are optimal for comparison with the experimental results obtained by FACS. One of the control is p.Trp87*, a null allele mutant, that does not produce LDLR. The other internal control is Ex3_4del mutant that produces a defective binding protein because the mRNA contains an in frame deletion of exons 3 and 4, essential for LDL binding [13]. As shown in Figure 3B, LDL-LDLR binding activities were similar in wt and p.Arg257Trp. However, binding activities of the other 5 variants were diminished as compared to wt (Figure 3B). As shown in Figure 3C and in agreement with LDLR expression and binding results, LDL internalisation in p.Arg257Trp was similar to wt, and LDL uptake determined in the other 5 variants was diminished when compared to wt. Values of LDLR expression, LDL binding and LDL uptake are shown in Supplementary Table 1. 3.4. Analysis of LDLR activity by confocal microscopy Confocal microscopy was used to confirm the activities and phenotypes of the analysed LDLR variants. CHO-ldlA7 cells expressing either wt or the LDLR variants were incubated with LDL for 4 h and then immunostained with the appropriate antibodies to determine LDLR and LDL localisation within the cell. LDL incubation was performed at 4ºC to determine LDL binding, or at 37ºC to determine LDL uptake. FITC or Texas Red® -conjugated secondary antibodies were used to visualise LDLR and LDL respectively. Figures S1 and S2 show LDL binding to CHO-ldlA7 transfected cells at low magnification in order to show a wide field with multiple cells. As shown in Figure 4A, LDLR expression and LDL binding, determined at 4ºC, were similar in wt and in p.Arg257Trp. Quantification of fluorescence intensities of the images obtained by confocal microscopy showed no statistically significant differences between wt and p.Arg257Trp (Figure 4C upper: LDL expression, and Figure 4C middle: LDL binding). 16. 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FIGURE LEGENDS Figure 1: Three-dimensional model of the structure of the LDLR ectodomain from the first cysteine-rich repeat to the EGF domain (black). Location of the variants analysed in this study is shown in each corresponding R module by arrows. This figure was prepared with PyMOL (DeLano scientifics). Figure 2: Expression of wt LDLR and LDLR mutations in CHO-ldlA7 transfected cells. Cells were transfected with the corresponding plasmids carrying the mutations of interest, LDLR was overexpressed for 48 h and then cells were lysed and analysed by Western blot. Whole cell extracts (20 µg) were fractioned in non reducing 8.5% SDSPAGE, transferred onto nitrocellulose membranes for incubation with a rabbit polyclonal anti-hLDLR antibody and detected by chemioluminiscence as described in Materials and Methods section. The relative band intensity of mature LDLR protein expression was calculated as the ratio of 160 kDa LDLR band intensity to that of GAPDH. A representative experiment from three independently performed assays is shown in upper panel. Levels of significance were determined by a two-tailed Student’s t-test, and a confidence level of greater than 95% (p<0.05) was used to establish statistical significance. No statistically significant differences were found among the LDLR expression. Figure 3: Functional characterization of LDLR variants. A: LDLR expression at cellular membrane; B: LDL-LDLR binding after 4 h incubation at 4 ºC; and C: LDL internalisation efficiency after 4 h incubation at 37ºC. 10,000 cells were acquired in a Facscalibur and values of LDL uptake, binding and LDLR expression were calculated as described in Materials and Methods. The values represent the mean of triplicate determinations (n = 3); error bars represent ±SD. *P < 0.001 compared to the wt using a Student’s t-test. Figure 4: Analysis of wt and p.Arg257Trp LDLR activity by confocal microscopy. A: LDLR expression and LDL binding determined at 4ºC; B: LDL uptake determined at 37ºC; C: Quantification of LDLR expression, LDL binding and LDL uptake in wt and p.Arg257Trp. For LDLR expression and LDL binding assays, transfected cells were incubated with non labelled LDL for 4 h at 4ºC, and for LDL uptake, cells were incubated with non labelled LDL for 4 h at 37ºC as described in Materials and Methods. Anti- hLDLR and anti-ApoB100 primary antibodies followed by Alexa Fluor® 488 and Texas Red® labelled secondary antibodies were used to visualize LDLR or LDL, respectively. Dapi was used to visualize the nuclei of non transfected cells or p.Trp87* transfected cells. The images show a representative individual cell of n=30. The histograms represent the mean ± standard deviation (n=30 cells), Student’s t-test was performed showing no statistical significant differences between wt and p.Arg257Trp. Figure 5: Analysis of LDLR expression and LDL binding in wt, p.Cys116Arg, p. Asp168Ans, p.Asp172Asn, p.Arg300Gly and p.Asp301Gly LDLR variants. A: LDLR expression and LDL binding determined at 4ºC; B: Quantification of LDLR expression and LDL binding. For LDLR expression and LDL binding assays, transfected cells were incubated with non labelled for 4 h at 4ºC. Anti-hLDLR and anti-ApoB100 primary antibodies followed by Alexa Fluor® 488 and Texas Red® labelled secondary antibodies were used to visualize LDLR or LDL, respectively. The images show a representative individual cell of n=30. The histograms represent the mean ± standard deviation (n=30 cells), *p< 0.001 compared to the wild-type (wt) using a Student’s t-test. Figure 6: Analysis of LDL uptake in wt, p.Cys116Arg, p. Asp168Ans, p.Asp172Asn, p.Arg300Gly and p.Asp301Gly LDLR variants. A: LDL uptake determined at 37ºC; B: Quantification of LDL uptake. For LDL uptake, cells were incubated with non labelled LDL for 4 h at 37ºC as described in Materials and Methods. Anti-hLDLR and anti-