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Annexin A2 reduces PCSK9 protein levels via a translational mechanism and interacts with the M1 and M2 domains of PCSK9 - PubMed

  • ️Wed Jan 01 2014

Annexin A2 reduces PCSK9 protein levels via a translational mechanism and interacts with the M1 and M2 domains of PCSK9

Kévin Ly et al. J Biol Chem. 2014.

Abstract

Annexin A2 (AnxA2) was reported to be an extracellular endogenous inhibitor of proprotein convertase subtilisin kexin type 9 (PCSK9) activity on cell-surface LDL receptor degradation. In this study, we investigated the effect of silencing the expression of AnxA2 and PCSK9 in HepG2 and Huh7 cells to better define the role of AnxA2 in PCSK9 regulation. AnxA2 knockdown in Huh7 cells significantly increased PCSK9 protein levels as opposed to AnxA2 knockdown in HepG2 cells. However, HepG2 cells overexpressing AnxA2 had lower levels of PCSK9 protein. Overall, our data revealed a plausible new role of AnxA2 in the reduction of PCSK9 protein levels via a translational mechanism. Moreover, the C-terminal Cys/His-rich domain of PCSK9 is crucial in the regulation of PCSK9 activity, and we demonstrated by far-Western blot assay that the M1 and M2 domains are necessary for the specific interaction of PCSK9's C-terminal Cys/His-rich domain and AnxA2. Finally, we produced and purified recombinant PCSK9 from humans and mice, which was characterized and used to perform 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate LDL cell-based assays on the stable knockdown HepG2 and Huh7 cells. We also demonstrated for the first time the equipotency of human and mouse PCSK9 R218S on human cells.

Keywords: Annexin; Cycloheximide; Drosophila; HepG2 Cells; Huh7 Cells; LDLR; Low Density Lipoprotein (LDL); Proprotein Convertase PCSK9; Short Hairpin RNA (shRNA); Translation Regulation.

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figures

FIGURE 1.
FIGURE 1.

Stable knockdown of AnxA2 and PCSK9. QPCR analysis on RNA extracts from Huh7 and HepG2 cells expressing a nonspecific shRNA control (nontarget) or an shRNA targeting AnxA2 (AnxA2-KD) (A) and PCSK9 (PCSK9-KD) (B). Results are the average of three independent experiments relative to Huh7 control nontarget with the mean ± S.E. shown as error bars. Results are normalized using actin housekeeping gene.

FIGURE 2.
FIGURE 2.

Western blot of stable Huh7 and HepG2 AnxA2 knockdown cells. Total cell lysate (30 μg of protein) and cell media from stable duplicate Huh7 (A) and HepG2 (B) nontarget and AnxA2-KD cells were resolved by SDS-PAGE, blotted onto a nitrocellulose membrane, and incubated with LDLR, LRP1, and PCSK9 primary antibodies. Protein levels were quantitated relative to β-actin.

FIGURE 3.
FIGURE 3.

Cycloheximide time course on stable Huh7 AnxA2 knockdown, HepG2 pIRES, and pIRES-AnxA2 cells. A and D, QPCR analysis of PCSK9 and LDLR mRNAs in Huh7 nontarget and AnxA2-KD (A) and in HepG2 pIRES and HepG2 pIRES-AnxA2 (D). B–F, Western blot and degradation of PCSK9 from Huh7 nontarget and AnxA2-KD (B and C) and HepG2 pIRES and pIRES-AnxA2 (E and F) total cell lysate (30 μg) treated with cycloheximide for 0 h (control) and 1, 2, and 4–6 h. Results are the average of three independent experiments with the mean ± S.E. shown as error bars.

FIGURE 4.
FIGURE 4.

RIP assay using annexin A2 antibody immunoprecipitates PCSK9 mRNA. A, schematic representation of hPCSK9 mRNA with the five sequences possibly recognized by annexin A2 and their position in the 3′UTR. B, RIP assay performed on endogenous PCSK9 mRNA using Huh7 nontarget and AnxA2-KD total cell lysate (500 μg). Immunoprecipitation of AnxA2 was performed and followed by RNA extraction/RT-PCR on the immunoprecipitate (IP AnxA2 lane) and the supernatant (Sup. lane). C, schematic diagram of hPCSK9 Δ3′UTR and hPCSK9 3′UTR constructs and Western blot of HEK293 cell lysates expressing those two vectors. D, RIP assay performed on HEK293 cells transiently transfected with hPCSK9 Δ3′UTR and hPCSK9 3′UTR. Immunoprecipitation was carried out without AnxA2 antibody (Mock IP/Mock Sup.) as a control and with antibody (IP AnxA2/Sup.).

FIGURE 5.
FIGURE 5.

In vitro binding of PCSK9 mutants to annexin A2. A, schematic diagram of PCSK9, PCSK9-L455X, CHRD, and two PCSK9 mutants E482Q and E482R suspected to interfere in the PCSK9·ANX2 binding. B, Western blot of HEK293 conditioned media containing PCSK9, CHRD, PCSK9ΔCHRD (L455X) and new mutants (E482Q and E482R). C, cell lysates of HEK293 expressing AnxA1-HA or AnxA2-HA. D and E, direct binding of PCSK9 and AnxA2 was analyzed by far-Western blot assay. Thirty micrograms of total cell lysates from HEK293 cells expressing AnxA1-HA or AnxA2-HA were resolved by SDS-PAGE, transferred onto a PVDF membrane (bait proteins), and incubated 4 h with concentrated (10–15-fold) conditioned media from HEK293 cells containing 2 μg of PCSK9 or its derivatives (prey proteins). Specific binding of prey proteins on AnxA2 (bait) was detected with HRP-conjugated anti-V5 mAb. Cell lysates β-actin levels are shown. n.s. for nonspecific band.

FIGURE 6.
FIGURE 6.

PCSK9 domain M1 and M2 interact with annexin A2. A, schematic diagram of different PCSK9 domain deletants. B, Western blot on HEK293 cell lysates showing intracellular expression and autoprocessing levels of PCSK9 or its module deletants. C, HEK293 conditioned media containing PCSK9, CHRD, L455X, and its module deletants. D, direct binding of PCSK9 variants and AnxA2 was analyzed by far-Western blot assay as described in Fig. 5. Cell lysates β-actin levels are shown. n.s. indicates nonspecific band.

FIGURE 7.
FIGURE 7.

Purified recombinant human and mouse PCSK9. A, Coomassie staining of purified recombinant hPCSK9 WT (10 μg) showing two distinct PCSK9 forms (PCSK9 WT mature and PCSK9-ΔN218 cleaved form). B, purified recombinant human (10 μg) PCSK9-RS, -RSDY, and mouse PCSK9-RS resulting from the last purification step by size exclusion chromatography.

FIGURE 8.
FIGURE 8.

Mass spectrometric analysis of recombinant hPCSK9-RS and -RSDY propeptide. A, schematic representation of the different hPCSK9 domains and the known PTMs. Scissors indicate the cleavage site of signal peptidase and PCSK9's prodomain autocleavage. B, MS spectra of hPCSK9-RS (upper panel) and preincubated with SAP (lower panel). Two different molecular forms identified as SO42−/ propeptide form with sulfation at Tyr38 and SO42− + PO42−/propeptide partially phosphorylated. Values in red below each form correspond to observed masses compared with calculated masses in black. C, graphical representation of the different propeptide forms based on area under curves (AUC) from the MS analyses without SAP treatment. Analyses were conducted on at least three independent experiments.

FIGURE 9.
FIGURE 9.

Mass spectrometric analysis of recombinant hPCSK9-RS mature form. A, MS analysis of recombinant hPCSK9-RS in the absence (upper panel) and presence of PNGase F (lower panel). First peak corresponds to hPCSK9 mature form without its prodomain, and the second peak represents hPCSK9 in complex with its prodomain. Values in red below each form correspond to the observed masses versus calculated masses in black. B, DiI-LDL uptake in HepG2 cells was measured in absence or presence of hPCSK9-RS (5 μg/ml) and with hPCSK9-RS preincubated with PNGase F. Results are the average of two independent experiments with the mean ± S.E. shown as error bars.

FIGURE 10.
FIGURE 10.

Comparison between purified recombinant PCSK9 mutants on DiI-LDL uptake in HepG2 cells. Purified recombinant PCSK9 was added to HepG2 cells medium at a final concentration of 0.01 to 20 μg/ml followed by the addition of DiI-LDL. DiI-LDL raw fluorescence units (RFUs) were normalized with Cyquant fluorescence units for each well in relation to different concentration of hPCSK9-RS, RSDY, and mPCSK9-RS. Results are the average of three independent experiments with the ± S.E. shown as error bars. ***, p < 0.001.

FIGURE 11.
FIGURE 11.

DiI-LDL uptake in HepG2 and Huh7 PCSK9 and AnxA2 mRNAs knockdown cells at different concentrations of recombinant hPCSK9-RSDY. DiI-LDL uptake was in HepG2 cells nontarget and PCSK9 (A), AnxA2 knockdown (B) and in Huh7 cells, PCSK9 (D), or AnxA2 knockdown (E) in a dose-response curve of exogenous recombinant hPCSK9-RSDY. C, DiI-LDL uptake in HepG2 cells overexpressing AnxA2 versus control cells stably transfected with empty pIRES vector. DiI-LDL incorporation is normalized with Cyquant raw fluorescence units (RFUs) relative to maximal DiI-LDL incorporation observed within the experiment. Results are the average of three independent experiments with the mean ± S.E. shown as error bars. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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