Characterization of human and murine PMP20 peroxisomal proteins that exhibit antioxidant activity in vitro

We have isolated the cDNAs encoding human and mouse homologues of a yeast p rotein, termed peroxisomal membrane protein 20 (PMP20). Comparison of the a mino acid sequences of human (HsPMP20) and mouse (MmPMP20) PMP20 proteins r evealed a high degree of identity (93%), whereas resemblance to the yeast C andida boidinii PMP20A and PMP20B (CbPMP20A and CbPMP20B) was less (30% ide ntity). Both HsPMP20 and MmPMP20 lack transmembrane regions, as do CbPMP20A and CbPMP20B. HsPMP20 mRNA expression was low in human fetal tissues, espe cially in the brain, in adult tissues, HsPMP20 mRNA was expressed in the ma jority of tissues tested. HsPMP20 and MmPMP20 contained the C-terminal trip eptide sequence Ser-Gln-Leu (SQL), which is similar to the peroxisomal targ eting signal 1 utilized for protein im. port into peroxisomes. HsPMP20 boun d directly to the human peroxisomal targeting signal 1 receptor, HsPEX5. Mu tagenesis analysis showed that the C-terminal tripeptide sequence, SQL, of HsPMP20 is necessary for its binding to HsPEX5. Subcellular fractionation o f HeLa cells, expressing epitope-tagged PMP20, revealed that HsPMP20 is loc alized in the cytoplasm and in a particulate fraction containing peroxisome s. Double-staining immunofluorescence studies showed colocalization of HsPM P20 and thiolase, a bona fide peroxisomal protein. The amino acid sequence alignment of HsPMP20, MmPMP20, CbPMP20A, and CbPMP20B displayed high simila rity to thiol-specific antioxidant proteins. HsPMP20 exerted an inhibitory effect on the inactivation of glutamine synthetase in the thiol metal-catal yzed oxidation system but not in the nonthiol metal-catalyzed oxidation sys tem, suggesting that HsPMP20 possesses thiol-specific antioxidant activity. in addition, HsPMP20 removed hydrogen peroxide by its thiol-peroxidase act ivity. These results indicate that HsPMP20 is imported into the peroxisomal matrix via PEX5p and may work to protect peroxisomal proteins against oxid ative stress. Because some portion of PMP20 might also be present in the cy tosol, HsPMP20 may also have a protective effect in the cytoplasm.