Loss- and gain-of-function PCSK9 variants: cleavage specificity, dominant negative effects, and low density lipoprotein receptor (LDLR) degradation

Abstract

The proprotein convertase PCSK9 is a major target in the treatment of hypercholesterolemia because of its ability bind the LDL receptor (LDLR) and enhance its degradation in endosomes/lysosomes. In the endoplasmic reticulum, the zymogen pro-PCSK9 is first autocatalytically cleaved at its internal Gln(152)↓, resulting in a secreted enzymatically inactive complex of PCSK9 with its inhibitory prosegment (prosegment·PCSK9), which is the active form of PCSK9 on the LDLR. We mutagenized the P1 cleavage site Gln(152) into all other residues except Cys and analyzed the expression and secretion of the resulting mutants. The data demonstrated the following. 1) The only P1 residues recognized by PCSK9 are Gln > Met > Ala > Ser > Thr ≈ Asn, revealing an unsuspected specificity. 2) All other mutations led to the formation of an unprocessed zymogen that acted as a dominant negative retaining the native protein in the endoplasmic reticulum. Analysis of a large panoply of known natural and artificial point mutants revealed that this general dominant negative observation applies to all PCSK9 mutations that result in the inability of the protein to exit the endoplasmic reticulum. Such a tight quality control property of the endoplasmic reticulum may lead to the development of specific PCSK9 small molecule inhibitors that block its autocatalytic processing. Finally, inspired by the most active gain-of-function mutant, D374Y, we evaluated the LDLR degradation activity of 18 Asp(374) variants of PCSK9. All Asp(374) mutations resulted in similar gain-of-function activity on the LDLR except that D374E was as active as native PCSK9, D374G was relatively less active, and D374N and D374P were completely inactive.

FIGURE 1.

Short term expression and secretion of PCSK9 Gln¹⁵² mutants. HEK293 cells were transiently transfected with WT PCSK9-V5 and its Gln¹⁵² mutants. A, 48 h post-transfection, the cells were radiolabeled for 3 h with [³⁵S]Met/Cys. The media and cell lysates were then immunoprecipitated with mAb-V5, the immune complexes were resolved by SDS-PAGE on an 8% polyacrylamide Tricine gel, and the dried gel was autoradiographed. The migration positions of pro-PCSK9 and PCSK9 as well as that of the furin-cleaved form, PCSK9-ΔN²¹⁸, are shown. B, 24 h post-transfection, cells were washed and incubated for another 24 h in RPMI 1640 medium + 5% lipoprotein-deficient serum. PCSK9 in the media and cell lysates was then quantitated by ELISA. C, calculated percent ratio of PCSK9 in media/cells. This figure is representative of three independent experiments done in triplicate. The error bars above the mean value represent the range of values ± standard deviation from the mean.

FIGURE 2.

Steady-state expression and secretion of PCSK9 Gln¹⁵² mutants and their oligomerization. A, 24 h after transfection of HEK293 cells, the expression and secretion of PCSK9 Gln¹⁵² mutants were analyzed by Western blot (WB) using mAb-V5. B, HEK293 cells were transfected with V5-tagged PCSK9 and its C67A/C301A, Q152A, Q152H, and Q152W mutants with PCSK9-His₆ or without (control). 24 h post-transfection, cell lysates were immunoprecipitated (IP) with mAb-V5 or mAb-His₆, and the immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blot using mAb-V5-HRP. The migration positions of pro-PCSK9 and PCSK9 as well as that of the furin-cleaved form PCSK9-ΔN²¹⁸ are shown. This figure is representative of at least three independent experiments.

FIGURE 3.

Inhibition of zymogen processing by Gln¹⁵² mutants. HEK293 cells were transiently co-transfected with untagged PCSK9 (native PCSK9) and WT PCSK9-V5 or its Q152A, Q152D, Q152E, and Q152F mutants. 48 h post-transfection, the cells were radiolabeled for 3 h with [³⁵S]Met/Cys. The media and cell lysates were then immunoprecipitated with a PCSK9-specific antibody (9), the immune complexes were resolved by SDS-PAGE, and the dried gel was autoradiographed. This figure is representative of at least three independent experiments.

FIGURE 4.

Model of the dominant negative effect of PCSK9 mutants. In the ER, pro-PCSK9 undergoes an autocatalytic cleavage into PCSK9. The cleaved prosegment (light green) associates with the catalytic fragment (dark green) and functions as a chaperone, permitting the mature protein to move from the ER into the secretory pathway. Current evidence indicates that PCSK9 might work at two cellular sites. The first potential location is in a post-ER compartment, depicted here as the Golgi apparatus, where PCSK9 might target the LDLRs (blue) for degradation in an acidic compartment such as lysosomes. In the second possible pathway, the PCSK9 that is secreted binds to LDLRs on the cell surface. The LDLR·PCSK9 complex is then internalized in clathrin-coated (green bars) early endosomes together with the adaptor protein autosomal recessive hypercholesterolemia (ARH) (light blue). PCSK9 prevents the recycling of the LDLR from the endosome back to the cell surface by directing the LDLR to the lysosome where it is degraded. If pro-PCSK9 cannot be processed (red star in a gray background; e.g. because of a heterozygous natural mutation such as Q152H) it remains as a zymogen in the ER and binds the non-mutated WT zymogen and prevents its processing, thereby acting as a dominant negative, effectively preventing PCSK9 secretion. TGN, trans-Golgi network.

FIGURE 5.

FACS analysis of LDLR following PCSK9 incubations. HuH7 cells were incubated for 24 h with 750 ng/ml WT PCSK9 or its Q152A, Q152M, and Q152S mutants. The levels of cell surface LDLR were then quantitated by FACS analysis. Although WT PCSK9 and its Q152A, Q152M, and Q152S mutants significantly and similarly reduce cell surface LDLR levels by ∼50%, their individual LDLR-reducing activities are not statistically different. This figure is representative of three independent experiments done in triplicate. The error bars above the mean value represent the range of values ± standard deviation from the mean.

FIGURE 6.

Exchangeability of the prosegment of PCSK9. A, HEK293 cells were transiently co-transfected with 0.5 μg of untagged PCSK9 (native PCSK9) and increasing amounts (0.3, 0.6, and 0.9 μg) of a cDNA coding for the WT V5-prosegment of PCSK9. 48 h post-transfection, the cells were radiolabeled for 4 h with [³⁵S]Met/Cys. The media and cell lysates were then immunoprecipitated with mAb-V5 or a PCSK9-specific antibody, and the immune complexes were resolved by SDS-PAGE. B, HEK293 cells were transiently co-transfected with 0.5 μg of pIRES vector, untagged PCSK9-Δpro (native PCSK9-Δpro), or native PCSK9 together with 0.5 μg of a cDNA coding for the WT V5-prosegment of PCSK9 or its Q152H and Q152W mutants. 48 h post-transfection, the cells were radiolabeled for 4 h with [³⁵S]Met/Cys. The media and cell lysates were then immunoprecipitated with mAb-V5, and the immune complexes were resolved by SDS-PAGE. The migration positions of pro-PCSK9 and PCSK9 as well as that of the furin-cleaved form, PCSK9-ΔN²¹⁸, and those of the V5- and untagged prosegments are emphasized. The control consisted of replacing the native PCSK9 by the empty vector pIRES (pIR). This figure is representative of two independent experiments. Ab, antibody.

FIGURE 7.

Expression and secretion of PCSK9 mutants. HEK293 cells were transiently transfected with 0.5 μg of PCSK9-V5 mutants in the prosegment (Y78A, V79A, V80A, L82A, A102A103R, M126A, P138Y, and I143P), catalytic domain (active site mutant H226A and H229R), or CHRD (C679X, D480N, E498K, and L455X) or with an empty vector control pIRES. A, 24 h post-transfection, cells were washed and incubated for another 24 h in RPMI 1640 medium + 5% lipoprotein-deficient serum. PCSK9 in the media and cell lysates was then quantitated by ELISA. B, calculated percent ratio of PCSK9 in media/cells. This figure is representative of three independent experiments done in triplicate. The error bars above the mean value represent the range of values ± standard deviation from the mean.

FIGURE 8.

Expression and secretion of PCSK9 Asp³⁷⁴ mutants. HEK293 cells were transiently transfected with WT PCSK9-V5 and its Asp³⁷⁴ mutants. 48 h post-transfection, the cells were radiolabeled for 3 h with [³⁵S]Met/Cys. The media and cell lysates were then immunoprecipitated with mAb-V5, the immune complexes were resolved by SDS-PAGE on an 8% polyacrylamide Tricine gel, and the dried gel was autoradiographed. The migration positions of pro-PCSK9 and PCSK9 as well as that of the furin-cleaved form, PCSK9-ΔN²¹⁸, and the prosegment are shown. This figure is representative of three independent experiments.

FIGURE 9.

Expression, secretion, and activity of PCSK9 Asp³⁷⁴ mutants. HEK293 cells were transiently transfected with WT PCSK9-V5 and its Asp³⁷⁴ mutants. 24 h post-transfection, cells were washed and incubated for another 24 h in RPMI 1640 medium + 5% lipoprotein-deficient serum. A, PCSK9 in the media and cell lysates was then quantitated by ELISA. B, calculated percent ratio of PCSK9 in media/cells. C, HuH7 cells were incubated for 4 h with 800 ng/ml WT PCSK9, its Asp³⁷⁴ mutants, or a control vector (pIRES). The levels of cell surface LDLR were then quantitated by FACS analysis. The stars point to the two known natural mutants, D374Y and D374H. This figure is representative of three independent experiments done in triplicate. The error bars above the mean value represent the range of values ± standard deviation from the mean.