Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity - PubMed (original) (raw)

. 2007 Sep 1;406(2):203-7.

doi: 10.1042/BJ20070664.

Christine Tumanut, Julie-Ann Gavigan, Waan-Jeng Huang, Eric N Hampton, Rachelle Tumanut, Ka Fai Suen, John W Trauger, Glen Spraggon, Scott A Lesley, Gene Liau, David Yowe, Jennifer L Harris

Affiliations

Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity

Jun Li et al. Biochem J. 2007.

Abstract

PCSK9 (proprotein convertase subtilisin/kexin 9) is a secreted serine protease that regulates cholesterol homoeostasis by inducing post-translational degradation of hepatic LDL-R [LDL (low-density lipoprotein) receptor]. Intramolecular autocatalytic processing of the PCSK9 zymogen in the endoplasmic reticulum results in a tightly associated complex between the prodomain and the catalytic domain. Although the autocatalytic processing event is required for proper secretion of PCSK9, the requirement of proteolytic activity in the regulation of LDL-R is currently unknown. Co-expression of the prodomain and the catalytic domain in trans allowed for production of a catalytically inactive secreted form of PCSK9. This catalytically inactive PCSK9 was characterized and shown to be functionally equivalent to the wild-type protein in lowering cellular LDL uptake and LDL-R levels. These findings suggest that, apart from autocatalytic processing, the protease activity of PCSK9 is not necessary for LDL-R regulation.

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Figures

Figure 1

Figure 1. Production of the ta-S386A mutant

(A) Schematic diagram of the expression strategy for wt-PCSK9, ta-PCSK9 and ta-S386A proteins. The wt-PCSK9 protein is expressed from a single construct as described in the Methods section. The single-chain precursor is biosynthesized in the ER, auto-cleaved between Gln152 and Ser153, and secreted as a prodomain–catalytic domain bound complex. The ta-PCSK9 and ta-S386A proteins are produced with a two-plasmid co-transfection strategy, as described in the Methods section. The prodomain and catalytic domain of PCSK9, either wt or bearing the S386A mutation, are translated in the ER via separate secretion signal peptides, then folded in trans, assembled and secreted in the same complex form as wt-PCSK9. Segments on each construct are indicated as follows: SP, signal peptide; PRO, prodomain; CAT, catalytic domain; CRD, cysteine-rich domain; H, hexahistidine tag. (B) Electrophoretic analysis of purified PCSK9 variants. All PCSK9 variants were affinity purified via the C-terminal hexahistidine tag and were subjected to reducing SDS/PAGE (0.5 μg of protein per lane). The prodomains (14 kDa) and mature PCSK9 subunits (61 kDa) were visualized by Coomassie Blue staining. (C) Analytic size-exclusion chromatography of PCSK9 variants wt-PCSK9, ta-PCSK9 and ta-S386A. Purified samples (30 μg) were subjected to gel filtration on a Shodex KW-803 column equilibrated with 20 mM Tris/HCl (pH 7.6), 200 mM NaCl, 0.5 mM TCEP [tris-(2-carboxyethyl)phosphine]. Molecular mass references are indicated by downward arrows: thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa).

Figure 2

Figure 2. Ta-S386A is functionally equivalent to wt-PCSK9

(A) Effect on LDL uptake by HepG2 cells. HepG2 cells were incubated with purified wt-PCSK9, ta-S386A or ta-PCSK9 at the indicated concentrations for 3 h and subsequently exposed to 5 μg/ml DiI-LDL for an additional 1 h prior to flow-cytometric analysis. Geometric means of duplicates were nonlinearly fitted to a Sigmoidal dose–response equation and representative fitting curves were shown as: solid line, wt-PCSK9; large dashes, ta-S386A; small dashes, ta-PCSK9. The calculated EC50 values (concentration of PCSK9 required to reach 50% of the maximal reduction) were 4.4±1.1 μg/ml (wt-PCSK9), 6.1±1.1 μg/ml (ta-S386A) and 5.2±1.1 μg/ml (ta-PCSK9) (_n_=2). (B) Effect on cell-surface LDL-R level. HepG2 cells were incubated with ta-PCSK9, ta-S386A, gain-of-function mutant D374Y or control protein chitotriosidase for 4 h, stained with anti-LDL-R polyclonal antibody and subjected to FACS analysis. The averages of the geometric means (_n_=3) are plotted as percentage of buffer control. (C) Effect on cellular concentration of LDL-R. HepG2 cells were incubated with the indicated concentrations of ta-PCSK9, ta-S386A or chitotriosidase control for 4 h before harvesting. Soluble cell lysates (50 μg) were subjected to reducing SDS/PAGE and transferred on to nitrocellulose membranes for immunoblotting with anti-LDLR (top) and anti-PCSK9 (middle) antibodies. Approximately equal sample loading was demonstrated using an anti-actin antibody (bottom).

References

    1. Seidah N. G., Benjannet S., Wickham L., Marcinkiewicz J., Jasmin S. B., Stifani S., Basak A., Prat A., Chretien M. The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation. Proc. Natl. Acad. Sci. U.S.A. 2003;100:928–933. - PMC - PubMed
    1. Abifadel M., Varret M., Rabes J. P., Allard D., Ouguerram K., Devillers M., Cruaud C., Benjannet S., Wickham L., Erlich D., et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat. Genet. 2003;34:154–156. - PubMed
    1. Shioji K., Mannami T., Kokubo Y., Inamoto N., Takagi S., Goto Y., Nonogi H., Iwai N. Genetic variants in PCSK9 affect the cholesterol level in Japanese. J. Hum. Genet. 2004;49:109–114. - PubMed
    1. Timms K. M., Wagner S., Samuels M. E., Forbey K., Goldfine H., Jammulapati S., Skolnick M. H., Hopkins P. N., Hunt S. C., Shattuck D. M. A mutation in PCSK9 causing autosomal-dominant hypercholesterolemia in a Utah pedigree. Hum. Genet. 2004;114:349–353. - PubMed
    1. Leren T. P. Mutations in the PCSK9 gene in Norwegian subjects with autosomal dominant hypercholesterolemia. Clin. Genet. 2004;65:419–422. - PubMed

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