Peptide sequencing,cDNA cloning, transfection-induced expression and determination of the molecular genetic basis for the A-B polymorphism of human serum paraoxonase.

Abstract

Paraoxonase is an aromatic esterase capable of hydrolyzing both organophosphate esters and aromatic esters of carboxylic acids. The esterase is widely distributed in mammals and is found in the serum and several organs, such as liver. Earlier studies from our laboratory and others established that two isozymic forms of human serum paraoxonase exist, and these are called the A and B isozymes, or allozymes, since it was presumed (and now has been clearly established) that these enzymes are allelic products which are very closely related, both in their structure and their function. Several years ago, our laboratory developed a simple method to distinguish the A, AB and B human serum paraoxonase phenotypes by measuring the ratio of the salt-stimulated paraoxonase activity of serum, divided by the rate of its arylesterase activity, using phenyl acetate as the substrate. The ratios obtained are characteristic and distinctive for each of the three phenotypes (A, 0.9-2.5; AB, 2.6-7.5, and B, 7.5-12). Such analyses of individual phenotypes show the expected codominance pattern and Mendelian autosomal inheritance in family pedigree analyses. However, some laboratories have disputed these conclusions about the paraoxonase polymorphism, and they claimed that different enzymes in serum were responsible for the paraoxonase and arylesterase activities. This thesis provides new biochemical and genetic information about the structural similarities and essential differences in the allelic human genes coding for the two allozymes of paraoxonase, the determination of the complete cDNA and amino acid sequence of the two forms of the esterase, the discovery of two polymorphic sites in the esterase structure at amino acid positions 54 (Met/Leu) and 191, (Gln/Arg) and convincing evidence that it is only the latter polymorphic site which determines the distinctive A and B allozymic properties of the esterase. Glutamine at position 191 is present in the A allozyme, and arginine is present in the B allozyme. Recombinant forms of A and B allozymes have both been expressed using the isolated coding regions of the respective genes. These experiments have established unequivocally that a single enzyme product (either A or B) possesses both paraoxonase and arylesterase activities. It is now clear that these dual activities should not be attributed to co-purification of two different proteins.