Characterization of Human and Murine PMP20 Peroxisomal Proteins That Exhibit Antioxidant Activity in Vitro

Hiroshi Yamashita,Shalom Avraham,Shuxian Jiang,Roanna London,Paul P Van Veldhoven,Suresh Subramani,Rick A Rogers,Hava Avraham

doi:10.1074/jbc.274.42.29897

Abstract

We have isolated the cDNAs encoding human and mouse homologues of a yeast protein, termed peroxisomal membrane protein 20 (PMP20). Comparison of the amino acid sequences of human (HsPMP20) and mouse (MmPMP20) PMP20 proteins revealed a high degree of identity (93%), whereas resemblance to the yeast Candida boidinii PMP20A and PMP20B (CbPMP20A and CbPMP20B) was less (30% identity). Both HsPMP20 and MmPMP20 lack transmembrane regions, as do CbPMP20A and CbPMP20B. HsPMP20 mRNA expression was low in human fetal tissues, especially in the brain. In adult tissues, HsPMP20 mRNA was expressed in the majority of tissues tested. HsPMP20 and MmPMP20 contained the C-terminal tripeptide sequence Ser-Gln-Leu (SQL), which is similar to the peroxisomal targeting signal 1 utilized for protein import into peroxisomes. HsPMP20 bound directly to the human peroxisomal targeting signal 1 receptor, HsPEX5. Mutagenesis analysis showed that the C-terminal tripeptide sequence, SQL, of HsPMP20 is necessary for its binding to HsPEX5. Subcellular fractionation of HeLa cells, expressing epitope-tagged PMP20, revealed that HsPMP20 is localized in the cytoplasm and in a particulate fraction containing peroxisomes. Double-staining immunofluorescence studies showed colocalization of HsPMP20 and thiolase, a bona fide peroxisomal protein. The amino acid sequence alignment of HsPMP20, MmPMP20, CbPMP20A, and CbPMP20B displayed high similarity to thiol-specific antioxidant proteins. HsPMP20 exerted an inhibitory effect on the inactivation of glutamine synthetase in the thiol metal-catalyzed oxidation system but not in the nonthiol metal-catalyzed oxidation system, suggesting that HsPMP20 possesses thiol-specific antioxidant activity. In addition, HsPMP20 removed hydrogen peroxide by its thiol-peroxidase activity. These results indicate that HsPMP20 is imported into the peroxisomal matrix via PEX5p and may work to protect peroxisomal proteins against oxidative stress. Because some portion of PMP20 might also be present in the cytosol, HsPMP20 may also have a protective effect in the cytoplasm.

Highlights

This paper is dedicated to William Troy and Charlene Engelhard for their continuing friendship and support for our research program
Human peroxisomal membrane protein 20 (PMP20) Is Homologous to the Yeast PMP20 —Because mammalian cDNA for PMP20 had not been described, we used the EST179427 sequence to probe a human hippocampus cDNA library and obtained four clones ranging in length from 0.45 to 1 kilobase pair
The amount of GST fusion proteins used for the assay was similar (Fig. 6B, lower panel). These results indicate that HsPEX5 binds directly to the SQL sequence at the free C terminus of HsPMP20 in the same manner as HsPEX5 binds to the typical PTS1 sequence

Summary

Introduction

This paper is dedicated to William Troy and Charlene Engelhard for their continuing friendship and support for our research program. HsPMP20 and MmPMP20 contained the C-terminal tripeptide sequence Ser-Gln-Leu (SQL), which is similar to the peroxisomal targeting signal 1 utilized for protein import into peroxisomes. Subcellular fractionation of HeLa cells, expressing epitope-tagged PMP20, revealed that HsPMP20 is localized in the cytoplasm and in a particulate fraction containing peroxisomes.

Results

Conclusion