Tài liệu Báo cáo khoa học: Affinity and kinetics of proprotein convertase subtilisin ⁄ kexin type 9

Affinity and kinetics of proprotein convertase

subtilisin⁄ kexin type 9 binding to low-density lipoprotein

receptors on HepG2 cells

Seyed A. Mousavi1

, Knut E. Berge1

, Trond Berg2 and Trond P. Leren1

1 Unit for Cardiac and Cardiovascular Genetics, Department of Medical Genetics, Oslo University Hospital Rikshospitalet, Norway

2 Department of Molecular Biosciences, University of Oslo, Norway

Keywords

association; dissociation; dissociation

constants; low-density lipoprotein receptor;

proprotein convertase subtilisin ⁄ kexin 9

Correspondence

T. P. Leren, Unit for Cardiac and

Cardiovascular Genetics, Department of

Medical Genetics, Oslo University Hospital

Rikshospitalet, P.O. Box 4950 Nydalen,

NO-0424 Oslo, Norway

Fax: +47 23075561

Tel: +47 23075552

E-mail: [email protected]

(Received 29 March 2011, revised 7 June

2011, accepted 16 June 2011)

doi:10.1111/j.1742-4658.2011.08219.x

Proprotein convertase subtilisin ⁄ kexin type 9 (PCSK9) is a secreted protein

that regulates the number of cell surface low-density lipoprotein receptors

(LDLRs) and the levels of low-density lipoprotein cholesterol in plasma.

Intact cells have not previously been used to determine the characteristics

of binding of PCSK9 to LDLR. Using PCSK9 iodinated by the tyramine

cellobiose (TC) method ([125I]TC-PCSK9), we measured the affinity and

kinetics of binding of PCSK9 to LDLR on HepG2 cells at 4 C. The extent

of [125I]TC-PCSK9 binding increased as cell surface LDLR density

increased. Unlabeled wild-type and two gain-of-function mutants of

PCSK9 reduced binding of [125I]TC-PCSK9. The Scatchard plot of the

binding-inhibition curve was curvilinear, indicative of high-affinity and

low-affinity sites for PCSK9 binding on HepG2 cells. Nonlinear regression

analysis of the binding data also indicated that a two-site model better fit￾ted the data. The time course of [125I]TC-PCSK9 binding showed two

phases in the association kinetics. Dissociation of [125I]TC-PCSK9 also

occurred in two phases. Unlabeled PCSK9 accelerated the dissociation of

[

125I]TC-PCSK9. At low pH, only one phase of dissociation was apparent.

Furthermore, the dissociation of [125I]TC-PCSK9 under pre-equilibrium

conditions was faster than under equilibrium conditions. Overall, the data

suggest that PCSK9 binding to cell surface LDLR cannot be described by

a simple bimolecular reaction. Possible interpretations that can account for

these observations are discussed.

Introduction

Proprotein convertase subtilisin ⁄ kexin type 9 (PCSK9)

is a protein secreted by the liver that was recognized as

an important regulator of cholesterol homeostasis

through its link to autosomal dominant hypercholester￾olemia [1–3]. Central to its role as a cholesterol-regula￾tory protein is the ability of PCSK9 to downregulate the

low-density lipoprotein (LDL) receptor (LDLR) [4–6].

Hepatic LDLR seems to be particularly susceptible to

this effect of PCSK9. PCSK9-mediated downregulation

of hepatic LDLR inhibits LDL uptake from plasma,

thus increasing the concentrations of LDL cholesterol

in plasma [4,7]. Besides the effect on hepatic LDLR

levels, endocytosis of PCSK9 by the liver is also

responsible for clearance of PCSK9 from the circulation

[7].

The importance of PCSK9 in maintaining choles￾terol homeostasis is clinically evident, in that PCSK9

gain-of-function mutations are associated with elevated

Abbreviations

ECD, extracellular domain; EGF-A, epidermal growth factor-like repeat A; LDL, low-density lipoprotein; LDLR, low-density lipoprotein

receptor; LPDS, lipoprotein-depleted serum; PCSK9, proprotein convertase subtilisin ⁄ kexin type 9; PCSK9-WT, wild-type proprotein

convertase subtilisin ⁄ kexin type 9; TC, tyramine cellobiose.

2938 FEBS Journal 278 (2011) 2938–2950 ª 2011 The Authors Journal compilation ª 2011 FEBS