Solubilization and Humanization of Paraoxonase-1

Abstract

Paraoxonase-1 (PON1) is a serum protein, the activity of which is related to susceptibility to cardiovascular disease and intoxication by organophosphorus (OP) compounds. It may also be involved in innate immunity, and it is a possible lead molecule in the development of a catalytic bioscavenger of OP pesticides and nerve agents. Human PON1 expressed in E. coli is mostly found in the insoluble fraction, which motivated the engineering of soluble variants, such as G2E6, with more than 50 mutations from huPON1. We examined the effect on the solubility, activity, and stability of three sets of mutations designed to solubilize huPON1 with fewer overall changes: deletion of the N-terminal leader, polar mutations in the putative HDL binding site, and selection of the subset of residues that became more polar in going from huPON1 to G2E6. All three sets of mutations increase the solubility of huPON1; the HDL-binding mutant has the largest effect on solubility, but it also decreases the activity and stability the most. Based on the G2E6 polar mutations, we “humanized” an engineered variant of PON1 with high activity against cyclosarin (GF) and found that it was still very active against GF with much greater similarity to the human sequence.

Pharmacogenetics of Paraoxonase Activity: Elucidating the Role of High-Density Lipoprotein in Disease

“…These two approaches are informing each other. Efforts are underway to make the rEngPON1 more human-like with increased catalytic efficiency for agent hydrolysis and to minimize immunological responses to injected rEngPON1 [45] and separately, to engineer the rHuPON1 for higher catalytic efficiency with as few changes as possible from the native human sequence.…”

Serum paraoxonase type-1 activity in pigs: Assay validation and evolution after an induced experimental inflammation

“…This enzyme appears mainly in serum, where it is associated with high-density lipoprotein (HDL) (James and Deakin, 2004). Although the exact function of PON1 is not known (Sarkar et al, 2012), it is believed to function as an enzyme that protects lipoproteins from peroxidation, and possesses anti-inflammatory properties limiting the production of pro-inflammatory mediators (Watson et al, 1995).…”

Characterization of human paraoxonase 1 variants suggest that His residues at 115 and 134 positions are not always needed for the lactonase/arylesterase activities of the enzyme

“…identified amino acid substitutions that significantly increased the activity of Chi‐PON1 variant (4E9) against some G‐type nerve agents . However, since Chi‐PON1 is considerably different than h‐PON1, it is proposed that this engineered variant of Chi‐PON1 may not be a good catalytic bioscavenger candidates for the development of antidote against OP‐poisoning in humans . Thus, it is essential to engineer recombinant PON1 whose amino acid is as close as possible to the sequence of h‐PON1.…”

“…16 However, since Chi-PON1 is considerably different than h-PON1, 15,[17][18][19] it is proposed that this engineered variant of Chi-PON1 may not be a good catalytic bioscavenger candidates for the development of antidote against OP-poisoning in humans. [14][15][16]32 Thus, it is essential to engineer recombinant PON1 whose amino acid is as close as possible to the sequence of h-PON1.…”