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

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 metalcatalyzed oxidation system but not in the nonthiol metalcatalyzed 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.

Identification of a New Type of Mammalian Peroxiredoxin That Forms an Intramolecular Disulfide as a Reaction Intermediate

“…However, the sequences surrounding Cys 73 and Cys 152 are not homologous to those surrounding the COOH-terminal conserved Cys residue of 2-Cys Prx enzymes, and the distances between Cys 48 and these other two Cys residues are substantially smaller than the 120 -123 residues that separate the two conserved Cys residues in typical 2-Cys Prx enzymes. PrxV shows 30% sequence identity to a Saccharomyces cerevisiae Prx known as type II Trx-dependent peroxidase (18) or peroxisomal membrane protein 20 (PMP20) (19) or alkyl hydroperoxide reductase (20); however, the yeast protein does not contain Cys residues corresponding to Cys 73 and Cys 152 of PrxV. Human PrxV was expressed in E. coli and purified to homogeneity.…”

The Trypanosoma cruzi Enzyme TcGPXI Is a Glycosomal Peroxidase and Can Be Linked to Trypanothione Reduction by Glutathione or Tryparedoxin

“…This dual localization may have functional significance and be related to the observation that TcGPXI can use both tryparedoxin and glutathione as electron donors. Studies on yeast and mammalian peroxiredoxin Vs have shown that these enzymes are distributed in the peroxisome and cytosol (39,(51)(52)(53)(54) and can also use both thioredoxin and glutathione as an electron donor (39,55). Because peroxisomes lack thioredoxin but do contain glutathione (41), it has been proposed that peroxiredoxin V functions as a thioredoxin-dependent enzyme within the cytosol, whereas within the peroxisome the glutathione-dependent activity predominates.…”

Peroxiredoxin, a Novel Family of Peroxidases

“…Although only the NH 2 -terminal Cys residue is conserved in Prx V, it is designated 2-Cys Prx enzyme because its function is dependent on two Cys residues. PMP20 (39) and AOEB166 (23), other terms for Prx V, were cloned as the result of a search for mammalian homologs of a yeast peroxisomal protein PMP20 and as the result of an attempt to characterize a 17-kDa bronchoalveolar protein, respectively. It was observed that the 162-residue mammalian PMP20 (Prx V) protein contained a peroxisomal targeting sequence (Ser-GlnLeu) at the COOH-terminus and that the AOEB166 cDNA contains two potential initiation sites in the same reading frame, the use of one of which would result in the production of a 162-residue protein identical to Prx V (PMP20), and the use of the other would generate a polypeptide of 214 residues.…”