Figure 1.
Human PRDX5 gene organisation and protein forms.
(A) Human PRDX5 gene is located on chromosome 11q13 and is flanked by the HSPC152/TRMT112 gene, positioned in opposite direction. Human PRDX5 contains six exons (boxes) and five introns. 5′ and 3′ UTRs are indicated by open boxes. In human liver, three alternative transcription initiation sites were identified (black arrows) [11]. Exon I contains two in-frame ATG initiation codons. The regions encoding the mitochondrial targeting sequence (MTS) and peroxisomal targeting signal (PTS1) are in red and green, respectively. (B) The presence of alternative transcription and translation start sites in human PRDX5 gene results in the production of a long PRDX5 form (L-PRDX5) and a short PRDX5 form (S-PRDX5). L-PRDX5 contains a cleavable (scissors) N-terminal MTS (red) and is therefore imported into mitochondria. S-PRDX5 contains a weak C-terminal PTS1 (green) and is targeted to the cytosol and the peroxisomes. Peroxidatic (Cp) and resolving (Cr) Cys implicated in catalysis are indicated. Adapted from [6], [11].
Figure 2.
ClustalX 2.0.7 amino acid sequence alignment of human PRDX5 and animal orthologs.
Asterisks (*) indicate identities, dots (.) and double dots (:) indicate conservative and highly conservative substitutions, respectively. Methionines encoded by the two alternative translation initiation sites are indicated in bold type. Peroxidatic (Cp) and resolving (Cr) Cys implicated in catalysis are highlighted in yellow. The peroxisomal targeting signal (PTS1) is highlighted in green. Mitochondrial targeting sequence (MTS) predictions were performed with TargetP [22], [43] and Mitoprot [21]. MTS predicted by TargetP are highlighted in red. MitoProt and TargetP subcellular localization predictions for PRDX5 were similar, except for X. laevis PRDX5, which was predicted to be imported into mitochondria with high probabilities when using Mitoprot, while the protein was predicted to be secreted with TargetP. Taxonomic groups are indicated between brackets. GenBank accession numbers are NP_036226.1 (human, Homo sapiens); XP_533241.1 (domestic dog, Canis lupus familiaris); XP_003230134.1 (green anole, Anolis carolinensis); NP_001085580.1 (African clawed frog, Xenopus laevis); ADI78068.1 (gilthead seabream, Sparus aurata); AAM18076.1 (Japanese lancelet, Branchiostoma belcheri tsingtaunese); NP_650679.3 (fruitfly, Drosophila melanogaster); ADQ57291.1 (bay scallop, Argopecten irradians); AAY96293.1 (lugworm, Arenicola marina); XP_001629337.1 (Starlet sea anemone, Nematostella vectensis); XP_002108078.1 (Trichoplax adhaerens); XP_003384607.1 (Amphimedon queenslandica).
Figure 3.
Dog PRDX5 subcellular distribution.
(A) Intracellular localization of dog PRDX5 was assessed by liver subcellular fractionation. Post-nuclear (E), cytosolic (S), mitochondrial (Mito) and peroxisomal (Perox) fractions were analyzed for marker enzyme and PRDX5 content by Western blotting, using anti-Hsp90 (cytosolic), anti-COX4 (mitochondrial), anti-catalase (peroxisomal) and anti-PRDX5 antibodies. Fifteen µg of proteins from each fraction were loaded. PRDX5 subcellular localization was also assessed by immunofluorescence detection of endogenous PRDX5 in MDCK cells (B, E) with co-localization with MitoTracker Red (C) or with peroxisomal catalase (F). Nuclei were counterstained with DAPI. Hsp90, heat shock protein 90; COX4, cytochrome c oxidase subunit 4.
Figure 4.
Sequencing and alignment of the PRDX5 5′-region in Canidae, Ursidae and Phocidae.
(A) Phylogenetic relationships between the nine families composing the suborder Caniformia. The phylogeny of Caniformia is currently not completely established, e.g. concerning the position of Ailuridae. The presented phylogenetic tree is based on multiple nuclear gene sequences and is adapted from [44]. (B) Phylogeny of Canidae, based on ∼15 kb from 12 exons and 4 introns, resolved by [23]. Canidae family contains three major phylogenetic groups, which are indicated in pink for the fox-like canids, green for the South American canids and blue for the wolf-like canids. Additionally, several canids do not belong to any of the three main clades, such as the gray fox (yellow), which was shown to be more primitive. Adapted from [23]. (C) Schematic diagram of HSPC152/TRMT112 and PRDX5 genes and cloning strategy. Exons I from both genes are detailed. 5′ UTRs are indicated by open boxes. Gray box highlights the sequence encoding the potential PRDX5 MTS. Conserved translation start sites are indicated in black. The potential translation start site enabling the translation of L-PRDX5 is indicated in red. P1 and P2 indicate the positions to which the PCR primers (green arrows) map. The primers were designated to map on regions sharing 100% identity between giant panda and domestic dog sequences. (D) ClustalX 2.0.7. nucleotidic sequence alignment of PRDX5 5′ region from nine Caniformia. Asterisks (*) indicate identities. Sequences encoding a potential MTS are highlighted in gray. Translation start sites enabling the translation of L-PRDX5 and S-PRDX5 are highlighted in red and black respectively. STOP codons being in-frame with the ATG+1 codon are underlined. Gaps are boxed. Species names are written in brown for Phocidae, gray for Ursidae, yellow for gray fox, red for the fox-like canids, green for the South American canids and blue for the wolf-like canids. Only one of the two alleles (Allele 1) found for gray fox and American black bear PRDX5 are represented. GenBank accession numbers are the following:. Arctic wolf (Canis lupus arctos): JQ411232; American black bear (Ursus americanus) JQ411225; gray fox (Urocyon cinereoargenteus): JQ411227; maned wolf (Chrysocyon brachyurus): JQ411231; northern elephant seal (Mirounga angustirostris): JQ411224; raccoon dog (Nyctereutes procyonoides) JQ411229; red fox (Vulpes vulpes): JQ411230. The sequences of domestic dog and giant panda PRDX5 5′ region were downloaded from the Genbank references GeneID:476032 (domestic dog, Canis lupus familiaris) and GeneID:100473172 (giant panda, Ailuropoda melanoleuca).
Table 1.
Sequences of PRDX5 5′flanking region: Genbank accession numbers.
Figure 5.
Subcellular localization of giant panda (A–D) and northern elephant seal (E–H) PRDX5 MTS expressed in fusion with GFP in MDCK cells.
Structure of the constructs cloned into mammalian expression vector pcDNA3.1 and used for MDCK transfection (A and E). Both alternative ATG initiation codons are indicated (arrows). Transfected cells were examined for GFP fusion expression (B and F). Mitochondria were stained with Mitotracker Red (C and G) and nuclei were counterstained with DAPI. BGH polyA, bovine growth hormone polyadenylation signal; GFP, green fluorescent protein; MTS, mitochondrial targeting sequence; pCMV, promoter of cytomegalovirus.
Figure 6.
Overexpression of human PRDX5 in MDCK cells.
(A) Organization of the construct cloned into mammalian expression vector pEF-BOS. EF-1α prom, promoter region of human EF-1α chromosomal gene; G-CSF poly(A), polyadenylation signal from human granulocyte colony-stimulating factor. (B) Representation of the proteins encoded by the different constructs used for transfection. Mito-hum and Mito-hum-C47A correspond to the enzymatically active and inactive mitochondrial human PRDX5 with the cleavable (scissors) presequence (MTS), respectively. Cyto-hum and Cyto-hum-C47S correspond to enzymatically active and inactive cytosolic human PRDX5, respectively. PRDX5 content of each MDCK clone was verified by Western blotting (C) and total expression levels were quantified (D). PRDX5 protein levels were normalized with β-actin and were expressed in relative units of PRDX5 content in MDCK control cells. Values are means ± SEM of triplicates. The subcellular localization of PRDX5 in the MDCK clones was verified by immunofluorescence (E). Mitochondria were stained with MitoTracker Red prior to cell fixation. Cell nuclei were counterstained with DAPI. HsPRDX5: human PRDX5.
Figure 7.
Cytotoxicity induced by hydrogen peroxide (A–B) and t-BHP (C–D) in MDCK cells overexpressing human PRDX5.
After one hour exposure to high levels of peroxide, cell death was evaluated by LDH release. The total LDH release (100% cell death) was determined after lysis of the cells with 2% Triton X-100. Assays were performed three times for each clone and values are means ± SEM. Significance is designated as *p<0.05, **p<0.01, ***p<0.001. t-BHP, tert-butyl hydroperoxide.