Mapping of a Minimal Apolipoprotein(a) Interaction Motif Conserved in Fibrin(ogen) β- and γ-Chains

Regina Klose,Friedrich Fresser,Silvano Köchl,Walther Parson,Andreas Kapetanopoulos,Jamila Fruchart-Najib,Gottfried Baier,Gerd Utermann

doi:10.1074/jbc.m003640200

Abstract

Lipoprotein(a) (Lp(a)) is a major independent risk factor for atherothrombotic disease in humans. The physiological function(s) of Lp(a) as well as the precise mechanism(s) by which high plasma levels of Lp(a) increase risk are unknown. Binding of apolipoprotein(a) (apo(a)) to fibrin(ogen) and other components of the blood clotting cascade has been demonstrated in vitro, but the domains in fibrin(ogen) critical for interaction are undefined. We used apo(a) kringle IV subtypes to screen a human liver cDNA library by the yeast GAL4 two-hybrid interaction trap system. Among positive clones that emerged from the screen, clones were identified as fibrinogen beta- and gamma-chains. Peptide-based pull-down experiments confirmed that the emerging peptide motif, conserved in the carboxyl-terminal globular domains of the fibrinogen beta and gamma modules specifically interacts with apo(a)/Lp(a) in human plasma as well as in cell culture supernatants of HepG2 and Chinese hamster ovary cells, ectopically expressing apo(a)/Lp(a). The influence of lysine in the fibrinogen peptides and of lysine binding sites in apo(a) for the interaction was evaluated by binding experiments with apo(a) mutants and a mutated fibrin(ogen) peptid. This confirmed the lysine binding sites in kringle IV type 10 of apo(a) as the major fibrin(ogen) binding site but also demonstrated lysine-independent interactions.

Highlights

Lipoprotein(a) (Lp(a))1 from human plasma is composed of a low density lipoprotein core and the highly polymorphic apo(a), covalently linked to apo B-100 by a single disulfide bridge [1, 2]. apo(a) contains 10 distinct tandem repeats, named kringle IV types 1–10, closely resembling plg kringle IV followed by single plg kringle V-like and protease-like domains [3]
To identify cDNA clones encoding proteins that interact with apo(a) kringle IV type 6, we transformed the yeast host strain HF7c, carrying a GAL4-HIS3 selection and GAL4-␤-galactosidase reporter gene with the kringle IV type 6-expression plasmid as a bait and a human liver cDNA library with the cDNA fused to the GAL4 activation domain
To determine whether activation of the GAL4-dependent reporter genes reflects a specific interaction of the encoded proteins with the kringle IV type 6 bait, each cDNA clone was rescued from yeast colonies and retransformed into the same

Summary

Introduction

Lipoprotein(a) (Lp(a)) from human plasma is composed of a low density lipoprotein core and the highly polymorphic apo(a), covalently linked to apo B-100 by a single disulfide bridge [1, 2]. apo(a) contains 10 distinct tandem repeats, named kringle IV types 1–10, closely resembling plg kringle IV followed by single plg kringle V-like and protease-like domains [3]. The homology at the cDNA level between plg and apo(a) modules is 75– 85% for the kringle IV domains and 94% for the protease domain [3]. As a result of a size polymorphism in the apo(a) gene, more than 30 different apo(a) isoforms have been found in human plasma, differing in the number of the kringle IV type 2 repeat (4 – 6). Binding of Lp(a) to fibrin(ogen) has been hypothesized to underlie a postulated role of Lp(a) in wound healing. Lp(a) effectively competes with plg for binding sites on fibrin and fibrinogen and reduces the generation of active plasmin (18 –21). The aim of the study was to identify critical motifs in fibrin(ogen) interacting with Lp(a)/apo(a) for the understanding of the functions and pathophysiological properties of Lp(a). We present fibrin(ogen) ␤- and ␥-chain sequences interacting with apo(a) in the yeast two-hybrid system and the identification of a conserved 30-amino acid fibrin(ogen) minimal peptide motif that is sufficient for binding to apo(a)/Lp(a) in vitro

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