Proteomics of Lipoprotein(a) identifies a protein complement associated with response to wounding

Abstract

Lipoprotein(a) [Lp(a)] is a major independent risk factor for cardiovascular disease. Twenty percent of the general population exhibit levels above the risk threshold highlighting the importance for clinical and basic research. Comprehensive proteomics of human Lp(a) will provide significant insights into Lp(a) physiology and pathogenicity. Using liquid chromatography-coupled mass spectrometry, we established a high confidence Lp(a) proteome of 35 proteins from highly purified particles. Protein interaction network analysis and functional clustering revealed proteins assigned to the two major biological processes of lipid metabolism and response to wounding. The latter includes the processes of coagulation, complement activation and inflammatory response. Furthermore, absolute protein quantification of apoB-100, apo(a), apoA1, complement C3 and PON1 gave insights into the compositional stoichiometry of associated proteins per particle. Our proteomics study has identified Lp(a)-associated proteins that support a suggested role of Lp(a) in response to wounding which points to mechanisms of Lp(a) pathogenicity at sites of vascular injury and atherosclerotic lesions. This study has identified a high confidence Lp(a) proteome and provides an important basis for further comparative and quantitative analyses of Lp(a) isolated from greater numbers of plasma samples to investigate the significance of associated proteins and their dynamics for Lp(a) pathogenicity.

Introduction

Lipoprotein(a) [Lp(a)] is gaining increasing clinical interest as an emerging and major independent risk factor for cardiovascular disease (CVD), with 20% of the general population having at risk plasma Lp(a) levels [1]. Elevated Lp(a) levels are associated with an increased possibility of developing CVD [2], [3] and extreme Lp(a) levels with a high risk [4]. New guidelines have been recommended for Lp(a) screening and niacin treatment has been advised for individuals at risk [1].

Lp(a) is characterized by apolipoprotein(a) [apo(a)], a high molecular weight glycoprotein associated with a low density lipoprotein (LDL) particle. Apo(a) is covalently bound via a single disulfide bond to the single molecule of apoB-100 on the surface of the lipoprotein particle [5] which provides Lp(a) with a unique apolipoprotein makeup. It is the only lipoprotein particle with two non-exchangeable covalently linked apolipoproteins [apoB-100 and apo(a)] in a one to one ratio. Apo(a) evolved from plasminogen and consists of plasminogen kringle 4 (KIV) and 5 (KV) domains plus an inactive protease domain at the C-terminus. A size polymorphism in the LPA gene locus gives rise to a variable number of KIV-2 repeats, a subtype of KIV [6] which determines the size of the apo(a) isoform between ~ 400 and 800 kDa [7] and shows an inverse correlation with Lp(a)-plasma levels [8].

Despite much research investigating the clinical role of Lp(a) in CVD, its physiological function and catabolic fate remains mostly elusive. LDL, the main transporter of cholesterol in plasma, is cleared via the LDL receptor. In contrast, the LDL receptor is not a major pathway of Lp(a) clearance [9]. Just like LDL, Lp(a) can be retained at the arterial wall and get oxidized which promotes atherosclerosis and leads to the formation of plaques. The apo(a) component clearly distinguishes Lp(a) from LDL and may contribute to its proatherogenic and prothrombotic properties.

Proteome studies of LDL and HDL [10], [11], [12] have uncovered a more complex functionality of the two main plasma lipoproteins besides their well known roles in lipid transport and metabolism. They described a dynamic proteome of LDL and HDL with a constant set of apolipoproteins and variations in the lower abundant protein species between different studies. Surprisingly Lp(a) has been widely ignored as a target for proteomics analysis so far.

Here, we report the first comprehensive profile of proteins associated with highly purified Lp(a) and the classification of their functions. The identified proteins classify Lp(a) as a distinct particle involved in processes such as coagulation, complement activation, inflammatory response and response to wounding. Furthermore, absolute quantification (AQUA) of proteins by mass spectrometry [13] revealed direct insights into the stoichiometry and dynamics of some Lp(a)-associated proteins.