Stability of highly purified human paraoxonase (PON1): Association with human phosphate binding protein (HPBP) is essential for preserving its active conformation(s)

Abstract

The biological role of human paraoxonase (PON1) remains unclear, whilst there is a consensus that the enzyme has a protective influence. A toxicological role, protecting from environmental poisoning by organophosphate derivatives drove earlier works, and more recently, clinical interest has focused on a protective role in vascular disease. PON1 resides essentially on HDL particles, a complex and dynamic molecular environment. Our recent discovery of the human phosphate binding protein (HPBP), displaying a firm propensity to associate with PON1, has steered new directions for characterizing PON1 functional state. Here, we report investigations on the effect of HPBP on oligomerization, storage and thermal stability of PON1. We found that purified PON1 is as a mixture of at least two states, and that the absence of HPBP favors homo-oligomerization of PON1 into state(s) of higher molecular size. We showed that HPBP allows stabilizing active conformation(s) of PON1 disencumbered of its natural environment. We also showed that PON1 exhibits intrinsically a remarkable thermal stability, and that the association of HPBP strongly contributes to slow the denaturation rate. A hybrid recombinant PON1 was shown more thermostable than the human enzyme, and its stability was unaffected by the presence of HPBP. Altogether, the results strongly encourage further study of the human enzyme.

Introduction

Human plasma paraoxonase (PON1; aryldialkylphosphatase; EC 3.1.8.1) is a calcium-dependent enzyme displaying hydrolase activity toward a broad range of esters and lactones [1], [2], and at much lower rates toward various organophosphates (OPs) [3]. Thus, albeit its physiological activity is suggested to be lactonase [4], PON1 is considered like an enzyme with promiscuous functions [5]. However, certain activities attributed to PON1 are largely debated. PON1 resides on the cholesterol-carrying HDL particles, and is involved in the prevention of atherosclerosis [6]. The catalytic mechanism and the physiological function of human PON1 are still unclear. As a phosphotriesterase, PON1 is regarded as a promising catalytic scavenger for the pre-treatment and therapy of poisoning by OPs including chemical warfare nerve agents and pesticides [7].

The PON1 anti-atherosclerotic activity is closely linked to its localization on HDL. Besides the promiscuous activities of PON1, other activities can be due to contaminants and/or enzyme partners present in purified preparations. Recent works highlighted the importance of the HDL-macromolecular environment for the stability and activity of PON1 [8], [9], but the mode of binding of PON1 and other HDL-associated proteins to HDL is still unknown. In order to solve the 3D structure of human PON1, we used apparently pure enzyme and obtained crystals, providing a structure that did not match with the amino-acid sequence of PON1 [10], [11]. The solved structure was that of a human phosphate binding protein (HPBP), a lipoprotein having a MW similar to that of PON1 [12]. This unknown protein was co-purified with PON1, and hypothesized to be a stabilizing partner [13]. The presence of HPBP in allegedly pure PON1 preparations was not regarded as questionable. Indeed, following our first report [10], several authors reported the presence of various contaminants in PON1 preparations, afterwards found to be responsible of certain catalytic activities previously attributed to PON1 [14], [15], [16]. Our results corroborate current literature data indicating that activities and stability of PON1 depend decisively on the enzyme molecular environment. Because PON1 is a promising catalytic OP scavenger, it will be important to develop media that mimic its in vivo environment. To date, very few kinetic studies on plasma PON1 have been carried out under ‘physiological conditions’. The biological life of PON1 is not known. However HDL particles have a fairly short half-life (2–2.5 days) which may preclude a major impact of inactivation on PON1 during the residence of the enzyme in blood [17]. Since HDL are in continuous rearrangement with time, knowledge of partner lipoprotein(s) or/and hydrophobic cofactor(s) ability to surrogate a stabilizing environment for active PON1 is essential.

In the present work, our goal was to assess whether these functional distinctions could be attributed to the presence or absence of HPBP. Highly purified PON1 was intended for study of its functional stability and activity in the absence of its partner. The storage stability of highly purified PON1 in solution was investigated by following its progressive loss of activity, changes in its oligomeric structure and alteration in its conformational stability. The thermal stability of PON1 was analysed using capillary electrophoresis (CE) at varying temperatures and differential scanning calorimetry (DSC). The thermal inactivation of the arylesterase activity of PON1 was also studied. All approaches showed that natural human PON1 exhibits a high thermal stability. Finally, stability of rPON1 was compared to that of human PON1. Heat stability is a crucial property for a prototypic OP catalytic scavenger that has to be stored over long periods of time at ambient temperatures and used under extreme climates without loss of activity. The current data will provide reference values for optimising the stability of future human rPON1-based mutants with operational catalytic activity against OPs.