Increased binding and degradation of LDLR(H306Y) by PCSK9. A, competition binding of EGF-AB and EGF-AB(H306Y) to PCSK9. Purified PCSK9 was prebound to slot-blotted anti-FLAG M2 monoclonal antibody (mAb). Blots were incubated for 90 min with 0.1 μg/ml infrared dye (DyLight800)-labeled LDLR-ECD at pH 7.4 in the presence of increasing concentrations of unlabeled EGF-AB (closed circles) or EGF-AB(H306Y) (open circles), and bound fluorophore-labeled LDLR was detected and quantified. B, HuH7 cells transiently expressing LDLR-WT or LDLR(H306Y) were cultured in sterol-supplemented medium (see “Experimental Procedures”) and treated for 1 h with the indicated amount of purified FLAG-tagged PCSK9. Cells were lysed, and cell extracts were subjected to SDS-PAGE and immunoblot analysis as described under “Experimental Procedures.” Internalized PCSK9 was detected with anti-FLAG M2 mAb. LDLR was detected with an anti-LDLR mAb (HL-1). TFR was detected as a control for equal protein loading. C, HuH7 cells transfected as in B were treated with the indicated amount of PCSK9 for 4 h. Following biotinylation, isolated cell surface protein lysates were subjected to SDS-PAGE and immunoblot analysis. LDLR and TFR were detected as in B. Secondary detection used an infrared dye (IRDye800)-labeled antibody. Blots were visualized and quantified using the LI-COR Odyssey infrared imaging system. LDLR levels were normalized to TFR expression. For these and subsequent data, results shown are from representative experiments repeated at least three times with similar results. Vec, vector.

EGF-AB(H306Y) blocks uptake of PCSK9 in HuH7 cells. HuH7 cells were treated 30 min with 5 μg/ml purified full-length FLAG-tagged PCSK9 alone or in combination with 5 μm EGF-AB(H306Y) blocking peptide or EGF-AB(L318A) negative-control peptide. A–D, cells were immunostained for PCSK9 (green) and nuclear stained with 4′,6-diamidino-2-phenylindole (magenta) as described under “Experimental Procedures.” E, cells were lysed and subjected to SDS-PAGE and immunoblotting for FLAG-tagged PCSK9 as described in the legend to Fig. 1B. Actin was detected as a control for equal protein loading. PCSK9 and actin levels were visualized and quantified using an IRDye800-labeled secondary antibody and the LI-COR Odyssey infrared imaging system. PCSK9 levels were normalized to actin expression.

EGF-AB(H306Y) treatment restores LDLR number and function in gain-of-function PCSK9 overexpressing cell lines. A and B, HepG2 cells stably overexpressing empty vector, PCSK9(S127R), or PCSK9(D374Y) were cultured in sterol-depleting medium (see “Experimental Procedures”) and treated for 18 h with 5 μm EGF-AB(H306Y) or negative-control EGF-AB(L318A) peptides. LDLR and TFR levels in cell surface and whole cell protein extracts were visualized and quantified as described in the legend to Fig. 4. C, empty vector, PCSK9-WT, PCSK9(S127R), or PCSK9(D374Y) overexpressing cells were treated for 18 h with the indicated concentration of EGF-AB(H306Y) or EGF-AB(L318A) and then incubated for 2 h with 100 μg/ml DiI-LDL. Cells were trypsinized, and the mean fluorescence intensity of each sample was measured by flow cytometry. Six replicate samples were used per condition. ^* indicates a statistical difference between columns with significance, p < 0.05, by one-way analysis of variance and post-test pairwise comparison using Tukey's method; ^**, p < 0.005; ^***, p < 0.001.

Structure of the PCSK9-EGF-A complex. The sigmaA weighted 2F_o - F_c electron density map contoured at 1σ shows the conformational change that occurs upon protonation of His-306. EGF-A and PCSK9 are represented as a stick model. Residues involved in the pH-dependent conformational change are colored according to element type as follows: nitrogen, blue; oxygen, red; EGF-A carbon, yellow; PCSK9 carbon, green. All other residues are colored gray. A, at acidic pH, His-306 of EGF-A forms a salt bridge with Asp-374 of PCSK9. B, at neutral pH, His-306 of EGF-A forms an intramolecular hydrogen bond with Ser-305. C, FH mutation H306Y of EGF-A is able to form a hydrogen bond with Asp-374 of PCSK9 at neutral pH.

EGF-AB(H306Y) treatment restores LDLR numbers in PCSK9 overexpressing cells. HepG2 cells stably overexpressing empty vector or wild-type PCSK9 (WT) were cultured in sterol-depleting medium (see “Experimental Procedures”) and treated for 18 h with 5 μm EGF-AB(H306Y) or negative-control EGF-AB(L318A) peptides. A, cell surface proteins were isolated following biotinylation, and whole cell and cell surface protein extracts were subjected to SDS-PAGE and immunoblot analysis for LDLR. LDLR and TFR levels were visualized as described in the legend to Fig. 1C. B, LDLR bands in A were quantified using the LI-COR Odyssey infrared imaging system. Values were normalized to TFR and graphed relative to untreated vector-transfected control cells.