Paraxonase-1 Variants Alter Stability and Activity

Abstract

Paraoxonase-1 (PON1) is a high-density lipoprotein-associated enzyme that has been extensively studied due to its ability to hydrolyze and inactivate toxic compounds such as organophosphate (OP) pesticide metabolites and its anti-atherogenic properties. However, the physiological substrate of PON1 is still unknown. Protein therapeutics have been proposed as a treatment for OP intoxication and PON1 is a major candidate. Production in bacterial cells would allow for expression of an unglycosylated enzyme which would reduce the possibility of an immunogenic response. It would also allow for easy mutagenesis of PON1 and scale-up production. We developed a system for expression of recombinant untagged native and variant human PON1s (rHuPON1s) in E. coli cells. The proteins were purified using multiple column chromatographic steps. This was the first expression, purification, and characterization of active native human PON1 from bacterial cells. A variant PON1, rHuPON1K192, had increased catalytic efficiency of hydrolysis for the OPs diazoxon (DZO), chlorpyrifos oxon, and paraoxon. Catalytic efficiency is an important characteristic as previous experiments demonstrated that it determines whether PON1 will protect against a specific OP in vivo. The rHuPON1K192 variant was injected in PON1 knockout (PON1-/-) mice and was found to be long-lasting in plasma, nontoxic, and protect against a 1 LD50 dose of dermal DZO and prevent the lethality of high doses (2-3 LD50) of DZO. Other rHuPON1 variants were also expressed, purified, and characterized. Some variants with a single amino acid change had increased protein stability and calcium binding. On the other hand, the disease-associated variant rHuPON1V109I had decreased protein stability. While PON1's physiological substrate is still unclear, homocysteine thiolactone (HCTL), a toxic, reactive metabolite of the amino acid homologue homocysteine, has been proposed as the physiological substrate of PON1. Bleomycin hydrolase (Blmh), another enzyme, was described as the HCTLase in tissues. We purified another HCTLase from liver and identified it by MS as biphenyl hydrolase-like protein (BPHL). Recombinant BPHL (rBPHL) was cloned, expressed, and characterized. BPHL's high catalytic efficiency for HCTL, which is 7700 times greater than that of PON1, total HCTLase activity calculations, and liver HCTLase inhibition assays suggest that BPHL is the most physiologically-relevant HCTLase isolated to date.