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317 Gene, 144(1994)317-318
0 1994 Elsevier Science B.V. All rights reserved. 0378-l 119/94/$07.00
GENE 07999
Cloning and sequencing of the cDNA encoding a human testis phospholipid hydroperoxide glutathione peroxidase*
(Selenium-dependent glutathione peroxidase; homology; phosphorylation; opal codon suppression)
R. Steven Esworthy, Khiem Doan, James H. Doroshow and Fong-Fong Chu
Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, Duarte, CA 91010, USA
Received by J.L. Slightom: 15 October 1993; Revised/Accepted: 10 December/21 December 1993; Received at publishers: 28 March 1994
SUMMARY
A human cDNA that encodes a polypeptide that has 94% deduced amino-acid sequence identity to porcine phospholi- pid hydroperoxide glutathione peroxidase was cloned from a testis library. The sequence shows preservation of the UGA selenocysteine codon, putative active-site Trp and Glu residues and a Tyr residue that is phosphorylated in the porcine protein. The 3’-UTR shows some conservation of sequences implicated in the insertion of selenocysteine at an opal codon in human glutathione peroxidase-1.
Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is the most distinctive selenium-dependent glu- tathione peroxidase (SeGPx) isozyme. It was discovered as a 22-kDa glutathione-dependent protein in porcine liver that protected biomembranes from peroxidative de- gradation (Schuckelt et al., 1991). PHGPx differs from GPx-1 and the other two tetrameric SeGPx isozymes by a high level of activity toward phospholipid and choles- terol hydroperoxides (Thomas et al., 1990). As opposed to the other SeGPx isozymes, PHGPx is phosphorylated in vivo (Schuckelt et al., 1991). High levels of PHGPx activity and protein were found in rat testis where GPx-1
Correspondence to: Dr. R.S. Esworthy, Department of Medical
Oncology and Therapeutics Research, City of Hope National Medical
Center, 1500 E. Duarte Road, Duarte CA, 91010, USA. Tel. (1-818)
359-8111, ext. 2819; Fax (1-818) 301-8233.
*On request, the authors will supply detailed experimental evidence for
the conclusions reached in this Brief Note.
Abbreviations: aa, amino acid(s); bp, base pair(s); GPx, glutathione per-
oxidase; gpxl, gene encoding GPx-1; gpx4, gene encoding PHGPx; h,
human; kb, kilobase or 1000 bp; nt, nucleotide(s); p, porcine; PCR,
polymerase chain reaction; PHGPx, phospholipid hydroperoxide
GPx; SeC, seleno-Cys; SeGPx, selenium-dependent GPx; UTR, untranslated region.
levels are relatively low (Roveri et al., 1992). The situation is reversed in most other tissues.
The residual SeGPx activity detected in human tumor- derived cell lines devoid of GPx-1 was characterized as a putative hPHGPx (Maiorino et al., 1991). Confirmation of the production of hPHGPx was sought by isolation of a hgpx4 cDNA (gene synthesizing hPHGPx) with a sequence homologous to the cDNA encoding pPHGPx (Schuckelt et al., 1991).
The nt sequence of the hgpx4 cDNA clone 5A2 is shown in Fig. 1. Using the hGPx-1 aa terminus for align- ment, the putative translation start codon in clone 5A2 is located at nt positions 161-163 (Mullenbach et al., 1987). The deduced peptide sequence from this reading frame shares 94% identity with the published pPHGPx sequence (Schuckelt et al., 1991). The peptide would be 19 kDa, which is close to the 18 f 0.7 kDa found for cyto- solic hPHGPx. PHGPx has also been detected in the mitochondria (Roveri et al., 1992). Utilization of an up- stream initiation codon (nt 80-83) could provide a mito- chondrial transfer leader sequence (Allison and Schatz, 1986). Encoded in this reading frame is an opal codon (nt positions 296-298) corresponding to the opal codon in the porcine nt sequence which is suppressed by inser- tion of the active site seleno-Cys. Sequences in the
a
318
GGGGAGGCG~GGAAAGCCG;CGCGCGTCC;TTGGTCGGC~GGACGAGGGdAGGAGCCGC;GGCTCCCAGSCCCGCCGC~GCCTC~CCCCCTTTG~CGCCTACTG~AGCCGGCGC~ 120
MSLGRLCRLLKPAL 14
PRI
GCTCTGTGGGCCTCTGGCCGCGCCTGGCCTGGCCGGGAC~TGCGCGTCCCGGGACGACTGGCGCTGTGCGCGCTCCATGCACGAGTTTTCCGCCAAGGACATCGACGGGCACATGG~ 240
LCGALAAPGLAGTMCASRDDWRCARSMHEFSAKD I D G H M V 54
TAACCTGGACAAGTACCGGGGCTTCGTGTGCATCGTCACCAACGTGGCCTCCCAGTGAGGCAAGACCGAAGTAAACTACACTCAGCTCGTCGACCTGCACGCCCGATACGCTGAGTGTGG 360
NLDKYRGFVC I V T N V A S Q SEC G KTEVNYTQLVDLHARYAECG 94
*** TTTGCGGATCCTGGCCTTCCCGTGTAACCAGTTCGGGAAGCAGGAGCCAGGGAGTAACGAAGAGATCAAAGAGTTCGCCGCGGGCTACAACGTCAAATTCGATATGTTCAGCAAGATCTG 480
LRILAFPCNQFGKQEPGSNEEIKEFAAGYNVKFDMFSKIC 134
PR2
CGTGAACGGGGACGACGCCCACCCGCTGTGGAAGTGGATGAAGATCCAACCCAAGGGCAAGGGCATCCTGGGAAATGCCATCAAGTGGAACTTCACCAAGTTCCTCATCGACAAGAACGG 600
VNGDDAHPLWKWMK IQPKGKG ILGNAIKWNFTKFL I D K N G 174
CTGCGTGGTGAAGCGCTACGGACCCATGGAGGAGCCCCTGGTGATAGAGAAGGACCTGCCCCACTATTTCTAGCTCCACAAGTGTGTGGCCCCGCCCGAGCCCCTGCCCACGCCCTTGGA 720
CV V K R Y G P ME E P L VIE K V L P H Y Fter
GCCTTCCACCGGCACTCATGACGGCCTGCCTGCAAACC7GCTGGTGGGGCAGACCCGAAAATCCAGCGTGCACCCCGCCGGAGGAAGGTCCCATGGCCTGCTGGGCTTGGCTCGGCGCCC 840
CCACCCCTGGCTACCTTGTGG~~~AGACAAATTAGAAAAAAAAAAAAAAA 895
Fig. 1. The nt sequence of the full-length gpx4 cDNA (clone 5A2) and the deduced aa sequence. The nt probe (underlined sequence) used to screen
a hgtll human testis cDNA library (Clontech) was prepared by PCR amplification of the whole library using primers (overlined sequences PRl and
PR2) based on the pPHGPx nt sequence presented in Schuckelt et al. (1991). Nineteen candidate gpx4 cDNA recombinants were selected from
30000 plaques. Seventeen were replated at lower density to isolate discrete recombinant clones. Ten of these clones had cDNA inserts that hybridized
on Southern blots with the 270-bp gpx4 cDNA probe. Clones A2 and 5A2 were selected for sequence analysis. Between A2 and 5A2, only one
significant difference was detected, an extra 17-bp sequence in 5A2 adjacent to the 5’ EcoRI cloning site. Two candidate start codons are boxed. The
opal codon suppressed by insertion of seleno-Cys is at nt 2966298. Sequences in italics in the 3’-UTR may be involved in the suppression of the opal
codon. The stop codon is at 671-673 (ter). The Tyr residue that is phosphorylated in pPHGPx is encoded by nt 4466448 (marked as ***). The boxed
sequence in the 3’-UTR is the putative poly(A)-addition signal. The 270-bp partial gpx4 cDNA has been used to determine that chromosome 19
harbors the hgpx4 gene (Chu, 1994). GenBank/EMBL accession No. X71973.
3’-UTR of gpxl are involved in the suppression of the opal codon (Shen et al., 1993). The hgpx4 3’-UTR dis- plays some of the opal codon suppression consensus 3’-UTR sequence. Active-site residues Trp163 and G1ulo8, which interact with seleno-Cys, are conserved in hPHGPx, as is the Tyr residue that is phosphorylated in pPHGPx (Schuckelt et al., 1991). The results of this study appear to confirm the production of a hPHGPx with homology to pPHGPx.
ACKNOWLEDGEMENTS
This work was supported by NIH grant DK 46921 and Cancer Center Core Grant CA33572.
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