Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Stability of highly purified human paraoxonase (PON1): Association with human phosphate binding protein (HPBP) is essential for preserving its active conformation(s)
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.
Section snippets
Reagents and materials
DMF, used as the electroosmotic flow (EOF) marker, was from Pierce (Rockford, IL, U.S.A.). Protein standards (HMW and LMW Marker Kits, 14.4–97 kDa) were purchased from Amersham Biosciences (Uppsala, Sweden). Hydroxyapatite media was obtained from Bio-Rad Laboratories (Munich, Germany) as Bio-Gel HTP Hydroxyapatite powder. All other chemicals were of analytical grade, from Sigma-Aldrich Chimie (L'Isle-d'Abeau, France). Macrosep 30 concentrators were obtained from Pall Life Science (Ann Arbor,
Effect of the presence of HPBP on oligomeric state of human PON1
In order to investigate the stability of human PON1, a preliminary task was carried out to verify the oligomeric state of the enzyme subsequent to its purification from plasma. Capillary electrophoresis (CE) was a pertinent tool since it allows performing purity assays, structural, binding and stability studies, process analysis, in very small volumes. Human PON1 was submitted to CE before and after separation of HPBP. The high electric fields used in CE and the very different pI of PON1 and
Discussion
Among the multiple requirements to be an effective OPs scavenger in vivo, PON1 must display a shelf life well suited for use under various climate conditions. To achieve this status, human PON1-based pharmaceuticals should have to be preferentially lyophilized. But prior to this ultimate packaging step, it is important to know the stability properties of the protein. The identification of intrinsic and extrinsic (not primary structure-related) factors must provide valuable information for
Acknowledgment
The authors thank Drs. Dan S. Tawfik and Amir Aharoni (Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel) for providing the evolved mammalian recombinant PON1-G3C9. This work was supported by DGA contract (PEA 010807) and EMA (LR2006) to P.M. D.R. is under contract with the German Bundesministerium der Verteidigung (M/SAB 1/3/A010 and M/SAB 1/6/A002).
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