Isolation and complete covalent structure of liver microsomal paraoxonase

Paraoxonase (PON1) is a serum esterase exclusively associated with high-den sity lipoproteins; it might confer protection against coronary artery disea se by destroying pro-inflammatory oxidized lipids in oxidized low-density l ipoproteins. Here I show that rabbit liver microsomes contain a PON analogu e (MsPON) and report the isolation and complete covalent structure of MsPON , In detergent-solubilized microsomes, MsPON co-purifies with the microsoma l triacylglycerol transfer protein (MTP) complex. MsPON was separated from the complex and purified to homogeneity under non-denaturing conditions. Au tomated sequence analysis of intact MsPON and peptides obtained from enzymi c and chemical cleavages led to the elucidation of the complete covalent st ructure of MsPON, The protein is a single polypeptide consisting of 350 res idues. The sequence of rabbit liver microsomal MsPON is 60% identical with that of rabbit serum PON1, and 84% identical with the sequence predicted by a human cDNA of unknown function, designated PON3. MsPON has a hydrophobic segment at the N-terminus that might serve to anchor the protein to the mi crosomal membrane or to the MTP complex. Unlike in the serum enzyme, two po tential N-glycan acceptor sites in MsPON are not glycosylated. An absence o f N-glycans was also indicated in the rabbit liver MTP. MsPON has a single free cysteine residue at position 38 and a disulphide bond between Cys-279 and Cys-348. The microsomal enzyme lacks three residues at the N-terminus t hat are present in the serum protein. MsPON lacks four residues at the C-te rminus that are present in the rabbit serum protein but absent from human s erum PON1. On the basis of the observation that MsPON displays a high degre e of similarity with serum PON1, it is proposed that MsPON might have a fun ction related to that of PON1 in serum high-density lipoprotein complexes.