Functional impact of PCSK9 variants on LDL uptake in a knockout hepatic model

Figures

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  Fig 1. Human PCSK9 and variants. (A) Schematic of the human PCSK9 gene on chromosome 1, indicating the 12 exons. The positions of the two guide RNAs (gRNAs) targeting exon 2 and exon 8 are shown. A predicted ~17.9 kb deletion will remove exons 2-8. This region encompasses the prodomain and the catalytic domain. (B) Diagram of the PCSK9 protein domains (Prodomain, Catalytic Domain, C-terminal Domain) with the locations of the clinically relevant amino acid variants investigated in this study. (C) A summary of variants to be investigated in this study.

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  Fig 2. Generation of PCSK9 knockout HepG2 cell clones. (A), Schematic of the CRISPR/Cas9 targeting strategy showing gRNAs targeting exons 2 and 8 of the PCSK9 gene. Comprehensive PCR screening of the clones using the three-primer strategy reveals homozygous or heterozygous clones. Region 1 and Region 2 primers amplify WT-specific fragments that are absent in both clones. Region 3 primers, designed to span the deletion, amplify a large ~18.9 kb band in WT cells and a distinct ~970 bp knockout-specific band in clones 1E4 and 1G9, confirming a homozygous deletion event. (B), Agarose gel electrophoresis of PCR products from the three-primer screening strategy for wild-type (WT) HepG2 cells and two representative single-cell clones (1E4 and 1G9). M: DNA ladder.

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  Fig 3. Validation of the PCSK9 knockout (KO) clone 1G9. (A) Normal epithelial-like morphology and contact inhibition for both 1E4 and 1G9 KO clones. Images were taken under 100x and 40x objectives using an Olympus SZX7 stereo microscope. (B) Western blot analysis of PCSK9 protein expression. The characteristic 62 kDa PCSK9 band is present in wild-type (WT) cells but completely absent in the 1G9 KO clone. The * indicates PCSK9 precursor (Pro-PCSK9), which migrates to a position higher than 70 kDa. The mature form of PCSK9 migrates around 62 kd. Clone 1E4 shows residual PCSK9 expression. Th protein ladder is Thermo Scientific PageRuler Prestained Protein Ladder (#26616). (C) Sanger sequencing chromatogram of the PCR product from clone 1G9, confirming a precise 17,920 bp genomic deletion between the gRNA target sites.

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  Fig 4. Functional validation of PCSK9 knockout via LDL uptake assay. (A) Flow cytometry analysis of DiI-Ac-LDL uptake in wild-type (WT) and PCSK9 knockout (KO) 1G9 clone. The histogram shows a rightward shift in fluorescence intensity for the KO clone (red) compared to WT cells (blue). (B) The mean fluorescence intensity of DiI-Ac-LDL was also presented. This result provides functional confirmation that loss of PCSK9 increases cellular LDL uptake. **p < 0.005. (C) Surface expression of LDLR on HepG2 and 1G9 cells was quantified by flow cytometry. *p < 0.05.

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  Fig 5. Validation of PCSK9 variants overexpression in 1G9 cells. (A) qPCR amplification for PCSK9 and then normalized to GAPDH reference gene is presented to confirm specific and robust transcript expression of PCSK9 variants in transfected cells. (B) Western blot analysis showing expression of wild-type (WT) PCSK9 and all tested variants at the expected molecular weight of ~62 kDa in transfected 1G9 KO cells. (C) Densitometric quantification of the Western blot bands, showing variable protein expression levels among the different variants.

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  Fig 6. Functional spectrum of PCSK9 variants on LDL uptake and LDLR surface expression. PCSK9 knockout (1G9) cells were transfected with a vector expressing wild-type (WT) PCSK9 or the indicated PCSK9 variants. (A) Quantification of DiI-LDL uptake by flow cytometry 24 hours after transfection. (B) Quantification of cell surface LDLR levels by flow cytometry 24 hours after transfection. Statistical significance was determined by one-way ANOVA test. (*P < 0.05, ***P < 0.001, ****P < 0.0001 compared to WT).