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Isolation of a New Peroxisome-Deficient CHO Cell Mutant Defective in Peroxisome Targeting Signal-1 Receptor
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
230, 402–406 (1997)
RC965971
Isolation of a New Peroxisome-Deficient CHO Cell Mutant Defective in Peroxisome Targeting Signal-1 Receptor Toshiro Tsukamoto,*,1 Akemi Bogaki,* Kanji Okumoto,*,† Keita Tateishi,*,† Yukio Fujiki,† Nobuyuki Shimozawa,‡ Yasuyuki Suzuki,‡ Naomi Kondo,‡ and Takashi Osumi* *Department of Life Science, Himeji Institute of Technology, Kamigori, Hyogo 678-12, Japan; †Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812-81, Japan; and ‡Department of Pediatrics, Gifu University School of Medicine, Gifu 500, Japan
Received December 6, 1996
For the study of mechanism of peroxisome biogenesis, we attempted to isolate CHO cell mutants deficient in peroxisome biogenesis. We used as the parent strain a stable CHO transformant of rat PEX2 (formerly named peroxisome assembly factor-1) cDNA, to avoid unusually frequent isolation of Pex2 mutants. Among the three peroxisome-deficient mutants obtained, ZP102 was a new CHO mutant of complementation group 2, and was restored for peroxisome assembly by the transfection of human PEX5 (formerly called PXR1 or PTS1R) cDNA. This approach would facilitate the isolation of new complementation gorups of peroxisome-deficient CHO mutants and the identification of essential genes for peroxisome biogenesis. q 1997 Academic Press
Peroxisome is a ubiquitous organelle present in a wide variety of eukaryotic cells from yeast to humans. This organelle contains at least one H2O2-producing oxidase and catalase, an H2O2-degrading enzyme. Genetic approach using peroxisome-deficient mutants of yeast and CHO cell revealed many protein factors essential for peroxisome biogenesis (1-3). Recently, the nomenclature for the genes of these peroxisome biogenesis factors was unified with the term, PEX (4). In humans, inherited peroxisome-deficient diseases, such as 1 Correspondence should be addressed to Dr. Toshiro Tsukamoto, Department of Life Science, Himeji Institute of Technology, Kamigori, Hyogo 678-12, Japan. Fax: 81-7915-8-0193. E-mail: tsukamot@ sci.himeji-tech.ac.jp. Abbreviations used: CHO, Chinese hamster ovary; DHAP-ATase, dihydroxyacetonephosphate acyltransferase; HAT, hypoxanthine/ aminopterin/thymidine; MNNG, N-methyl-N*-nitro-N-nitrosoguanidine; PTS, peroxisome targeting signal; P9OH/UV, 9-(1*-pyrene)nonanol/long wavelength ultraviolet light; P12/UV, 12-(1*-pyrene)dodecanoic acid/UV; RT-PCR, reverse transcription-polymerase chain reaction.
Zellweger syndrome and neonatal adrenoleukodystrophy, are known (5). Cell fusion studies on fibroblasts from these patients identified at least ten different complementation groups (6-8). Three pathogenic genes, rat and human PEX2 (2, 9), rat and human PEX6 (formerly named PAF-2 (10, 11) or PXAAA1 (12)) and PEX5 (formerly called PXR1 (13) or PTS1-receptor (14, 15)) have been identified for groups F, C and 2 of peroxisome deficiency, respectively. We previously isolated three distinct CHO cell mutants, Z24, Z65 and ZP92, defective in peroxisome assembly (16, 17). PEX2 and PEX6 genes were isolated by genetic complementation using Z65 and ZP92. Although this approach seemed useful to isolate other genes essential for peroxisome biogenesis, only CHO mutants belonging to complementation groups F, C and E have been successfully isolated to date, because of overwhelmingly frequent isolation of group F mutant (17). In this study, we used as a parent strain a stable transformant of the wildtype CHO cell with rat PEX2 cDNA, and isolated a new mutant belonging to human complementation group 2. MATERIALS AND METHODS Cell lines. Chinese Hamster Ovary (CHO) cells were cultured in Ham’s F12 medium supplemented with 10% fetal calf serum. Wildtype CHO cells were transfected with pcD65K (2), a rat PEX2 cDNA expression plasmid, by calcium phosphate co-precipitation method (18), and stable transformants were selected with 400 mg/ml G418 (GIBCO BRL). Three G418 resistant clones, named CHOTKa, CHOTKb and CHOTKc, were isolated. RT-PCR. Expression of transfected rat PEX2 cDNA was verified by RT-PCR. For this purpose, total RNA was isolated from CHOTKa, TKb and TKc cells by acid guanidine thiocyanate-phenol-chloroform extraction (19). First strand synthesis was performed for 60 min at 377C using rat PEX2 specific antisense primer T1 (5*-CAACTTGGTTTCTAAAGAGCATT-3*) and M-MLV reverse transcriptase (BRL) as recommended by manufacturer. PCR was done using Taq DNA polymerase (Takara Shuzo) and sense primer H1 (5*-AAAGACAGCATCAGAGAAGATATG-3*) and antisense primer T1, 40 cycles of 947C for 1 min, 607C for 2 min and 727C for 3 min, as described
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previously (3). These primers did not amplify CHO PEX2 cDNA under the same conditions. Isolation of peroxisome deficient mutants. Peroxisome deficient mutants were isolated as described (17), with several modifications for efficient screening of independent clones. CHOTKa cells (1 to 2 1 104 cells/well) were inoculated into a 24 well plate. Next day, cells were mutagenized with 0.2 mg/ml N-methyl-N*-nitro-N-nitrosoguanidine (MNNG) for 16 hr. The cells were then washed twice with complete medium, and cultured for three days. To assure isolation of independent mutant clones, cells in each well (one-tenth of each cell mass) were separately replated to a 100 mm dish, and treated as a separate pool in all procedures afterwards. After cultured for 2 to 3 days, about 5 1 105 cells from each dish were re-inoculated to a new 100 mm dish. Cells were then subjected to the 9-(1*-pyrene)nonanol/long wavelength ultraviolet light (P9OH/UV) selection procedure, which selectively kills peroxisome-containing wild-type cells (20), as described (17). P9OH/UV resistant colonies emerged in 7 days, when a single colony was picked from each dish. Cells from each colony were divided into two dishes, and one was subjected to immunocytochemical staining using rabbit anti-rat catalase antibody (10) to assess peroxisome-deficiency. Peroxisome-deficient mutants were further cloned from the other dish by limiting dilution method. Throughout the isolation procedure of mutants, 400 mg/ml G418 was added to the medium. Complementation analysis by cell fusion. Isolated mutants were fused using polyethylene glycol (21) with a derivative of wild-type CHO or Z24 mutant, both of which carried double resistance to 6thioguanine (TGr) and ouabain (Ouar). Hybrid cells were selected with HAT selection medium containing 1 mM ouabain. For complementation tests between CHO mutants and human peroxisome deficient disease, TGr clones of isolated CHO mutants were fused with patient’s fibroblasts and selected with HAT selection medium containing 400 mg/ml G418. Restoration of peroxisomes was determined by immunocytochemical staining of catalase. Complementation analysis by cDNA transfection. Transfection of rat PEX2 and rat PEX6 cDNA expression plasmids (2, 10) was also used for grouping of the mutants. Isolated CHO mutants were suspended in PBS(0) at 1 1 106 cells/ml. The cell suspension (400 ml) was mixed with 10 mg of cDNA expression plasmid and electroporated, as described (10). PCR cloning of human PEX5 cDNA. Human PEX5 cDNA was amplified by PCR using antisense oligonucleotide (5*-AAGATCGATCCCTCCAGGTGGACACTCAC-3*) and sense oligonucleotide (5*TGGTCTAGACCATGGCAATGCGGGAGCTG-3*), based on the published sequence (13). Human brain cDNA (Clontech) was used as a template. PCR conditions were: initial heating for 2 min at 947C, two cycles of 947C for 1 min, 627C for 2 min and 727C for 3 min, then 38 cycles of 947C for 1 min, 687C for 2 min and 727C for 3 min. A single fragment of about 1.8 kb was obtained. PCR product was digested with ClaI and XbaI and subcloned into pUcD2SRaMCS vector (10). Other methods. Continuous cell labeling with [35S]methionine and immunoprecipitation of acyl-CoA oxidase and peroxisomal 3-
FIG. 1. Expression of transfected rat PEX2 cDNA. RT-PCR was performed with rat PEX2 specific primers. Lanes 1, 2, and 3 are CHOTKa, CHOTKb, and CHOTKc, respectively. Arrowhead, PCR product.
FIG. 2. Morphological analysis of peroxisomes. (A)-(F): Immunofluorescence staining of peroxisomes. (A) Wild-type CHO cells. (B) CHOTKa. (C) ZP101. (D) ZP102. (E) ZP103. (F) ZP102 transfected with human PEX5 cDNA plasmid. Magnification, 1250. Bar, 50 mm.
ketoacyl-CoA thiolase were performed as described (10). Catalase latency, P9OH/UV and 12-(1*-pyrene)dodecanoic acid/UV (P12/UV) resistance (2) and peroxisomal DHAP-ATase activity (22) were monitored by published methods.
RESULTS Isolation of stable PEX2 transformant CHO cell lines. An expression plasmid of rat PEX2 cDNA, pcD65K, was transfected to wild-type CHO cells, and stably transfected cells were selected with G418. Three colonies were picked, and expression of transfected rat PEX2 gene was verified by RT-PCR. Under the conditions as described in Materials and Methods, only the rat PEX2 but not CHO PEX2 sequence was amplified by RT-PCR. Two clones, CHOTKa and CHOTKc, were revealed to express rat PEX2 mRNA (Fig. 1, arrowhead). CHOTKa was further used for isolation of peroxisome-deficient mutants. This cell line had morphologically normal peroxisomes (Fig. 2A and B). Isolation of new peroxisome-deficient CHO mutants. CHOTKa cells were mutagenized with MNNG and peroxisome-deficient mutants were isolated by the P9OH/ UV selection. This fatty alcohol analog is incorporated into plasmalogen molecules at an early step of synthesis, and produces active oxygen upon long wave UV irradiation (20) . Because this biosynthetic step occurs in peroxisomes, this procedure kills wild-type CHO cells, but not peroxisome-deficient cells. Cells in 48 wells were mutagenized and 39 wells gave one or more P9OH/UV resistant colonies. Out of them, three clones, named ZP101, ZP102 and ZP103 showed cytosolic localization of catalase upon immunofluorescence staining, indicating that they were deficient in peroxisomes (Fig. 2C, D and E).
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Complementation Analysis by Cell Fusion (A) Cell fusion between CHO cells CHOTGrOuar
Z24TGrOuar
/ / /
0 / 0
ZP101 ZP102 ZP103
(B) Complementation analysis between ZP102 and human fibroblasts A
B
D
E
G
2
3
6
/
/
/
/
/
0
/
/
FIG. 3. Latency of catalase activity in CHOTKa, ZP101, and ZP102 cells. Circles, CHOTKa; Triangles, ZP101; Squares, ZP102. Relative free catalase activity is expressed as a percentage of the total activity measured in the presence of 1% Triton X-100 (16).
(A) Three peroxisome-deficient CHO mutants were fused with CHOTGrOuar or Z24TGrOuar. Fused cells were selected and immunocytochemically stained with rabbit anti-rat catalase antibody. / means peroxisomes were observed after cell fusion. 0 means no peroxisome-formation. (B) ZP102 was fused with fibroblasts derived from the patients belonging to one of eight different complementation groups. / and 0 signs are used as in (A). Designation of complementation groups is as described in refs. (6) (groups A to F), (8) (group G), (7) (groups 2, 3 and 6).
tion groups revealed that ZP102 belonged to group 2 (Table 1B). It was reported that PEX5, which encode peroxisome targeting signal-1 receptor, is responsible for this group of mutations. Transfection of PCR-amplified human PEX5 cDNA restored peroxisomes in ZP102 (Fig. 2F), strongly suggesting that peroxisome deficiency of ZP102 was due to the loss of Pex5p function.
ZP102
Complementation analysis was done by cell fusion and transfection of PEX2 or PEX6 cDNA. Cell fusion with wild-type CHO cells indicated that all mutants were recessive (Table 1A). ZP101 and ZP103 cells fused with a previously isolated mutant Z24 (complementation group E) were not restored for peroxisome assembly, indicating that they both belonged to group E (Table 1A). PEX2 and PEX6 cDNA transfection to ZP101 and 103 did not restore peroxisomes, confirming that they were not in group F or C (Table 2). In contrast, ZP102 was restored for peroxisomes upon cell fusion with Z24 (Table 1A), whereas no peroxisome was observed after the transfection of PEX2 and PEX6 cDNA (Table 2). These results clearly indicated that ZP102 was a new mutant belonging to a complementation group different from those of previously isolated CHO mutants, Z24, Z65 and ZP92. Cell fusion between ZP102 and human fibroblasts of several complementa-
TABLE 2
Complementation Analysis by DNA Transfection PEX2 cDNA
PEX6 cDNA
0 0 0
0 0 0
ZP101 ZP102 ZP103
Three peroxisome-deficient CHO mutants were transfected with expression vectors pcD65K (PEX2) or pUcD2r92A (PEX6) by electroporation. After 72 hr, cells were immunocytochemically stained with rabbit anti-rat catalase antibody. 0 means that no peroxisome was formed.
Characterization of ZP101 and ZP102. We further characterized ZP101 and ZP102 biochemically. ZP103 was omitted, because it was in the same complementation group as that of ZP101 and grew slower. In peroxisome-deficient cells, peroxisomal proteins are mislocalized to the cytosol, rapidly degraded or not converted to mature forms, despite a normal synthesis. Intracellular localization of catalase was determined by the titration with digitonin. In contrast to the case for wildtype cells, almost all catalase activity was released at the concentration as low as 100 mg/ml of digitonin for both mutants (Fig. 3), suggesting that catalase was present in the cytosol. The activity of the first enzyme of ether phospholipid synthesis, dihydroxyacetonephosphate acyltransferase (DHAP-ATase) was severely diminished in both mutant cells as compared to CHOTKa cells (Table 3). ZP101 and ZP102 were originally isolated as P9OH/UV resistant mutants; both were thus resistant to P9OH/UV treatment. Conversely, they were sensitive to P12/UV treatment which specifically kills peroxisome-deficient mutants, because there is no synthesis of plasmalogen, an oxygen radical scavenger (Table 3). Acyl-CoA oxidase, the first enzyme of the peroxisomal fatty acid b-oxidation system, is synthesized as a 75 kDa polypeptide (A component) and proteolytically converted into 53 kDa and 22 kDa polypeptides (B and C components, respectively) in peroxisomes. On continuous cell labeling, A and B components were observed in CHOTKa cells (Fig. 4, lane 1). On the other hand, only A component was seen in ZP101 or ZP102 mutant cells, in a much smaller amount probably due to rapid degradation (Fig. 4, lanes 2 and 3). Peroxisomal 3-ketoacyl-CoA thiolase,
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Properties of CHOTKa, ZP101, ZP102 and ZP103 Cells Cell
Peroxisome
DHAP-ATase (%)
P12/UV (%)
P9OH/UV (%)
CHOTKa ZP101 ZP102 ZP103
/ 0 0 0
100 4.2 4.4 6.4
66 0 0 0.01
0 97 96 100
DHAP-ATase activity was expressed as a percentage relative to that of wild-type CHOTKa cells (6.09 nmol 2 hr01 mg01). For determination of P12/UV and P9OH/UV resistance, 200 or 1 1 105 cells were inoculated into 60 mm dishes and selected. The numbers of colonies were counted and expressed as percentages of that of unselected control.
the third enzyme of peroxisomal b-oxidation system, is synthesized as a larger precursor with an aminoterminal presequence which contains a peroxisomal targeting signal-2, and is converted to the mature form in peroxisomes. In CHOTKa cells, only the mature thiolase was detected (Fig. 4, lane 4), representing the rapid processing of the precursor form. In ZP101 or ZP102 cells, only the larger precursor was seen (Fig. 4, lane 5 and 6), implying the absence of processing activity. DISCUSSION We previously reported the isolation of two peroxisome deficient CHO mutants, Z24 and Z65, which were identified by the autoradiographic screening for mu-
FIG. 4. Biosynthesis of peroxisomal enzymes. Marks A, B and C indicate the positions of A, B and C components of acyl-CoA oxidase (AOx), respectively. P and M, a larger precursor and mature forms of 3-ketoacyl-CoA thiolase (TH), respectively. A faint band at the same position as that of AOx B component in lanes 2 and 3 is nonspecific, because a band of similar intensity is seen in lanes 4–6. C component of AOx is difficult to see in lane 1, because of the nonspecific band observed at the same mobility in all six lanes. Exposure, 43 days.
tants defective in peroxisomal DHAP-ATase activity (16) . In another work, we isolated eight clones of peroxisome-deficient mutants using P9OH/UV selection. One of them, ZP92, was the third mutant, but all other seven were of complementation group F, the same as that of Z65 (17) . Although the P9OH/UV method is effective to isolate peroxisome-deficient mutant, excessively frequent isolation of group F mutants made it difficult to isolate new mutants of different complementation groups. In this report, we overcame this difficulty by using CHOTKa cells that was transformed with rat PEX2 cDNA. By performing mutagenization with MNNG and P9OH/UV selection in the presence of G418, the ectopic PEX2 gene was stably retained, hence no group F mutant was obtained. Three mutants were isolated and two mutants, ZP101 and ZP103 were group E mutants, like previously isolated mutant Z24. ZP102 belonged to a complementation group different from those of previously isolated CHO mutants. Cell fusion analysis revealed that ZP102 was a group 2 mutant, and transfection of human PEX5 cDNA restored peroxisomes. At present, ten complementation groups are known in human peroxisome deficient disorders (68), and four CHO mutants corresponding to groups C, E, F and 2 have now been isolated. Group 2 peroxisome deficiency is caused by the mutation of PEX5 gene, which encode the receptor for PTS1 sequence at the C-termini of many peroxisomal proteins including acyl-CoA oxidase. Peroxisomal 3-ketoacyl-CoA thiolase have PTS2, another targeting signal for peroxisome, within its N-terminal presequence. Some patients of group 2 only show the defect of PTS1mediated import, but others are deficient in both PTS1and PTS2-mediated import (13) . ZP102 has a larger precursor form of thiolase and shows cytosolic distribution of green fluorescent protein fused to PTS2 (unpublished result), indicating that both import systems are affected in ZP102. The molecular basis of the effect of Pex5 mutation on the PTS2-dependent import should be investigated. CHO cell mutants grow faster than fibroblasts, and are immortal and feasible for the isolation of stable
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transformants. These characters are favorable for screening genes essential for peroxisome biogenesis by functional complementation, which was successfully applied for isolation of PEX2 (2) and PEX6 cDNA (10). Moreover, peroxisomal protein import was conveniently assayed with living (23) and semi-intact CHO cells (24). Thus, our approach will be effective to isolate new peroxisome deficient CHO mutants, and facilitate the identification of genes essential for peroxisome biogenesis and their functional characterization. ACKNOWLEDGMENTS This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan, The Sumitomo Foundation, and Uehara Memorial Foundation.
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8. Poulos, A., Christodoulou, J., Chow, C. W., Goldblatt, J., Paton, B. C., Orii, T., Suzuki, Y., and Shimozawa, N. (1995) J. Pediatr. 127, 596–599. 9. Shimozawa, N., Tsukamoto, T., Suzuki, Y., Orii, T., Shirayoshi, Y., Mori, T., and Fujiki, Y. (1992) Science 255, 1132–1134. 10. Tsukamoto, T., Miura, S., Nakai, T., Yokota, S., Shimozawa, N., Suzuki, Y., Orii, T., Fujiki, Y., Sakai, F., Bogaki, A., Yasumo, H., and Osumi, T. (1995) Nature Genet. 11, 395–401. 11. Fukuda, S., Shimozawa, N., Suzuki, Y., Zhang, Z., Tomatsu, S., Tsukamoto, T., Hashiguchi, N., Osumi, T., Masuno, M., Imaizumi, K., Kuroki, Y., Fujiki, Y., Orii, T., and Kondo, N. (1996) Am. J. Hum. Genet. 59, in press. 12. Yahraus, T., Braverman, N., Dodt, G., Kalish, J. E., Morrell, J. C., Moser, H. W., Valle, D., and Gould, S. J. (1996) EMBO J. 15, 2914–2923. 13. Dodt, G., Braverman, N., Wong, C., Moser, A., Moser, H. W., Watkins, P., Valle, D., and Gould, S. J. (1995) Nature Genet. 9, 115–125. 14. Fransen, M., Brees, C., Baumgart, E., Vanhooren, J., Baes, M., Mannaerts, G. P., and Van, V. P. (1995) J. Biol. Chem. 270, 7731–7736. 15. Wiemer, E., Nuttley, W. M., Bertolaet, B. L., Li, X., Francke, U., Wheelock, M. J., Anne´, U. K., Johnson, K. R., and Subramani, S. (1995) J. Cell Biol. 130, 51–65. 16. Tsukamoto, T., Yokota, S., and Fujiki, Y. (1990) J. Cell Biol. 110, 651–660. 17. Shimozawa, N., Tsukamoto, T., Suzuki, Y., Orii, T., and Fujiki, Y. (1992) J. Clin. Invest. 90, 1864–1870. 18. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745–2752. 19. Chomczynski, P., and Sacchi, N. (1986) Anal. Biochem. 162, 156– 159. 20. Morand, O. H., Allen, L.-A. H., Zoeller, R. A., and Raetz, C. R. H. (1990) Biochim. Biophys. Acta 1034, 132–141. 21. Brul, S., Westerveld, A., Strijland, A., Wanders, R. J. A., Schram, A. W., Heymans, H. S. A., Schutgens, R. B. H., van den Bosch, H., and Tager, J. M. (1988) J. Clin. Invest. 81, 1710–1715. 22. Shimozawa, N., Suzuki, Y., Orii, T., Yokota, S., and Hashimoto, T. (1988) Pediatr. Res. 24, 723–727. 23. Tsukamoto, T., Hata, S., Yokota, S., Miura, S., Fujiki, Y., Hijikata, M., Miyazawa, S., Hashimoto, T., and Osumi, T. (1994) J. Biol. Chem. 269, 6001–6010. 24. Wendland, M., and Subramani, S. (1993) J. Cell Biol. 120, 675– 685.
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Isolation of a New Peroxisome-Deficient CHO Cell Mutant Defective in Peroxisome Targeting Signal-1 Receptor Toshiro Tsukamoto,*,1 Akemi Bogaki,* Kanji Okumoto,*,† Keita Tateishi,*,† Yukio Fujiki,† Nobuyuki Shimozawa,‡ Yasuyuki Suzuki,‡ Naomi Kondo,‡ and Takashi Osumi* *Department of Life Science, Himeji Institute of Technology, Kamigori, Hyogo 678-12, Japan; †Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812-81, Japan; and ‡Department of Pediatrics, Gifu University School of Medicine, Gifu 500, Japan
Received December 6, 1996
For the study of mechanism of peroxisome biogenesis, we attempted to isolate CHO cell mutants deficient in peroxisome biogenesis. We used as the parent strain a stable CHO transformant of rat PEX2 (formerly named peroxisome assembly factor-1) cDNA, to avoid unusually frequent isolation of Pex2 mutants. Among the three peroxisome-deficient mutants obtained, ZP102 was a new CHO mutant of complementation group 2, and was restored for peroxisome assembly by the transfection of human PEX5 (formerly called PXR1 or PTS1R) cDNA. This approach would facilitate the isolation of new complementation gorups of peroxisome-deficient CHO mutants and the identification of essential genes for peroxisome biogenesis. q 1997 Academic Press
Peroxisome is a ubiquitous organelle present in a wide variety of eukaryotic cells from yeast to humans. This organelle contains at least one H2O2-producing oxidase and catalase, an H2O2-degrading enzyme. Genetic approach using peroxisome-deficient mutants of yeast and CHO cell revealed many protein factors essential for peroxisome biogenesis (1-3). Recently, the nomenclature for the genes of these peroxisome biogenesis factors was unified with the term, PEX (4). In humans, inherited peroxisome-deficient diseases, such as 1 Correspondence should be addressed to Dr. Toshiro Tsukamoto, Department of Life Science, Himeji Institute of Technology, Kamigori, Hyogo 678-12, Japan. Fax: 81-7915-8-0193. E-mail: tsukamot@ sci.himeji-tech.ac.jp. Abbreviations used: CHO, Chinese hamster ovary; DHAP-ATase, dihydroxyacetonephosphate acyltransferase; HAT, hypoxanthine/ aminopterin/thymidine; MNNG, N-methyl-N*-nitro-N-nitrosoguanidine; PTS, peroxisome targeting signal; P9OH/UV, 9-(1*-pyrene)nonanol/long wavelength ultraviolet light; P12/UV, 12-(1*-pyrene)dodecanoic acid/UV; RT-PCR, reverse transcription-polymerase chain reaction.
Zellweger syndrome and neonatal adrenoleukodystrophy, are known (5). Cell fusion studies on fibroblasts from these patients identified at least ten different complementation groups (6-8). Three pathogenic genes, rat and human PEX2 (2, 9), rat and human PEX6 (formerly named PAF-2 (10, 11) or PXAAA1 (12)) and PEX5 (formerly called PXR1 (13) or PTS1-receptor (14, 15)) have been identified for groups F, C and 2 of peroxisome deficiency, respectively. We previously isolated three distinct CHO cell mutants, Z24, Z65 and ZP92, defective in peroxisome assembly (16, 17). PEX2 and PEX6 genes were isolated by genetic complementation using Z65 and ZP92. Although this approach seemed useful to isolate other genes essential for peroxisome biogenesis, only CHO mutants belonging to complementation groups F, C and E have been successfully isolated to date, because of overwhelmingly frequent isolation of group F mutant (17). In this study, we used as a parent strain a stable transformant of the wildtype CHO cell with rat PEX2 cDNA, and isolated a new mutant belonging to human complementation group 2. MATERIALS AND METHODS Cell lines. Chinese Hamster Ovary (CHO) cells were cultured in Ham’s F12 medium supplemented with 10% fetal calf serum. Wildtype CHO cells were transfected with pcD65K (2), a rat PEX2 cDNA expression plasmid, by calcium phosphate co-precipitation method (18), and stable transformants were selected with 400 mg/ml G418 (GIBCO BRL). Three G418 resistant clones, named CHOTKa, CHOTKb and CHOTKc, were isolated. RT-PCR. Expression of transfected rat PEX2 cDNA was verified by RT-PCR. For this purpose, total RNA was isolated from CHOTKa, TKb and TKc cells by acid guanidine thiocyanate-phenol-chloroform extraction (19). First strand synthesis was performed for 60 min at 377C using rat PEX2 specific antisense primer T1 (5*-CAACTTGGTTTCTAAAGAGCATT-3*) and M-MLV reverse transcriptase (BRL) as recommended by manufacturer. PCR was done using Taq DNA polymerase (Takara Shuzo) and sense primer H1 (5*-AAAGACAGCATCAGAGAAGATATG-3*) and antisense primer T1, 40 cycles of 947C for 1 min, 607C for 2 min and 727C for 3 min, as described
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previously (3). These primers did not amplify CHO PEX2 cDNA under the same conditions. Isolation of peroxisome deficient mutants. Peroxisome deficient mutants were isolated as described (17), with several modifications for efficient screening of independent clones. CHOTKa cells (1 to 2 1 104 cells/well) were inoculated into a 24 well plate. Next day, cells were mutagenized with 0.2 mg/ml N-methyl-N*-nitro-N-nitrosoguanidine (MNNG) for 16 hr. The cells were then washed twice with complete medium, and cultured for three days. To assure isolation of independent mutant clones, cells in each well (one-tenth of each cell mass) were separately replated to a 100 mm dish, and treated as a separate pool in all procedures afterwards. After cultured for 2 to 3 days, about 5 1 105 cells from each dish were re-inoculated to a new 100 mm dish. Cells were then subjected to the 9-(1*-pyrene)nonanol/long wavelength ultraviolet light (P9OH/UV) selection procedure, which selectively kills peroxisome-containing wild-type cells (20), as described (17). P9OH/UV resistant colonies emerged in 7 days, when a single colony was picked from each dish. Cells from each colony were divided into two dishes, and one was subjected to immunocytochemical staining using rabbit anti-rat catalase antibody (10) to assess peroxisome-deficiency. Peroxisome-deficient mutants were further cloned from the other dish by limiting dilution method. Throughout the isolation procedure of mutants, 400 mg/ml G418 was added to the medium. Complementation analysis by cell fusion. Isolated mutants were fused using polyethylene glycol (21) with a derivative of wild-type CHO or Z24 mutant, both of which carried double resistance to 6thioguanine (TGr) and ouabain (Ouar). Hybrid cells were selected with HAT selection medium containing 1 mM ouabain. For complementation tests between CHO mutants and human peroxisome deficient disease, TGr clones of isolated CHO mutants were fused with patient’s fibroblasts and selected with HAT selection medium containing 400 mg/ml G418. Restoration of peroxisomes was determined by immunocytochemical staining of catalase. Complementation analysis by cDNA transfection. Transfection of rat PEX2 and rat PEX6 cDNA expression plasmids (2, 10) was also used for grouping of the mutants. Isolated CHO mutants were suspended in PBS(0) at 1 1 106 cells/ml. The cell suspension (400 ml) was mixed with 10 mg of cDNA expression plasmid and electroporated, as described (10). PCR cloning of human PEX5 cDNA. Human PEX5 cDNA was amplified by PCR using antisense oligonucleotide (5*-AAGATCGATCCCTCCAGGTGGACACTCAC-3*) and sense oligonucleotide (5*TGGTCTAGACCATGGCAATGCGGGAGCTG-3*), based on the published sequence (13). Human brain cDNA (Clontech) was used as a template. PCR conditions were: initial heating for 2 min at 947C, two cycles of 947C for 1 min, 627C for 2 min and 727C for 3 min, then 38 cycles of 947C for 1 min, 687C for 2 min and 727C for 3 min. A single fragment of about 1.8 kb was obtained. PCR product was digested with ClaI and XbaI and subcloned into pUcD2SRaMCS vector (10). Other methods. Continuous cell labeling with [35S]methionine and immunoprecipitation of acyl-CoA oxidase and peroxisomal 3-
FIG. 1. Expression of transfected rat PEX2 cDNA. RT-PCR was performed with rat PEX2 specific primers. Lanes 1, 2, and 3 are CHOTKa, CHOTKb, and CHOTKc, respectively. Arrowhead, PCR product.
FIG. 2. Morphological analysis of peroxisomes. (A)-(F): Immunofluorescence staining of peroxisomes. (A) Wild-type CHO cells. (B) CHOTKa. (C) ZP101. (D) ZP102. (E) ZP103. (F) ZP102 transfected with human PEX5 cDNA plasmid. Magnification, 1250. Bar, 50 mm.
ketoacyl-CoA thiolase were performed as described (10). Catalase latency, P9OH/UV and 12-(1*-pyrene)dodecanoic acid/UV (P12/UV) resistance (2) and peroxisomal DHAP-ATase activity (22) were monitored by published methods.
RESULTS Isolation of stable PEX2 transformant CHO cell lines. An expression plasmid of rat PEX2 cDNA, pcD65K, was transfected to wild-type CHO cells, and stably transfected cells were selected with G418. Three colonies were picked, and expression of transfected rat PEX2 gene was verified by RT-PCR. Under the conditions as described in Materials and Methods, only the rat PEX2 but not CHO PEX2 sequence was amplified by RT-PCR. Two clones, CHOTKa and CHOTKc, were revealed to express rat PEX2 mRNA (Fig. 1, arrowhead). CHOTKa was further used for isolation of peroxisome-deficient mutants. This cell line had morphologically normal peroxisomes (Fig. 2A and B). Isolation of new peroxisome-deficient CHO mutants. CHOTKa cells were mutagenized with MNNG and peroxisome-deficient mutants were isolated by the P9OH/ UV selection. This fatty alcohol analog is incorporated into plasmalogen molecules at an early step of synthesis, and produces active oxygen upon long wave UV irradiation (20) . Because this biosynthetic step occurs in peroxisomes, this procedure kills wild-type CHO cells, but not peroxisome-deficient cells. Cells in 48 wells were mutagenized and 39 wells gave one or more P9OH/UV resistant colonies. Out of them, three clones, named ZP101, ZP102 and ZP103 showed cytosolic localization of catalase upon immunofluorescence staining, indicating that they were deficient in peroxisomes (Fig. 2C, D and E).
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Complementation Analysis by Cell Fusion (A) Cell fusion between CHO cells CHOTGrOuar
Z24TGrOuar
/ / /
0 / 0
ZP101 ZP102 ZP103
(B) Complementation analysis between ZP102 and human fibroblasts A
B
D
E
G
2
3
6
/
/
/
/
/
0
/
/
FIG. 3. Latency of catalase activity in CHOTKa, ZP101, and ZP102 cells. Circles, CHOTKa; Triangles, ZP101; Squares, ZP102. Relative free catalase activity is expressed as a percentage of the total activity measured in the presence of 1% Triton X-100 (16).
(A) Three peroxisome-deficient CHO mutants were fused with CHOTGrOuar or Z24TGrOuar. Fused cells were selected and immunocytochemically stained with rabbit anti-rat catalase antibody. / means peroxisomes were observed after cell fusion. 0 means no peroxisome-formation. (B) ZP102 was fused with fibroblasts derived from the patients belonging to one of eight different complementation groups. / and 0 signs are used as in (A). Designation of complementation groups is as described in refs. (6) (groups A to F), (8) (group G), (7) (groups 2, 3 and 6).
tion groups revealed that ZP102 belonged to group 2 (Table 1B). It was reported that PEX5, which encode peroxisome targeting signal-1 receptor, is responsible for this group of mutations. Transfection of PCR-amplified human PEX5 cDNA restored peroxisomes in ZP102 (Fig. 2F), strongly suggesting that peroxisome deficiency of ZP102 was due to the loss of Pex5p function.
ZP102
Complementation analysis was done by cell fusion and transfection of PEX2 or PEX6 cDNA. Cell fusion with wild-type CHO cells indicated that all mutants were recessive (Table 1A). ZP101 and ZP103 cells fused with a previously isolated mutant Z24 (complementation group E) were not restored for peroxisome assembly, indicating that they both belonged to group E (Table 1A). PEX2 and PEX6 cDNA transfection to ZP101 and 103 did not restore peroxisomes, confirming that they were not in group F or C (Table 2). In contrast, ZP102 was restored for peroxisomes upon cell fusion with Z24 (Table 1A), whereas no peroxisome was observed after the transfection of PEX2 and PEX6 cDNA (Table 2). These results clearly indicated that ZP102 was a new mutant belonging to a complementation group different from those of previously isolated CHO mutants, Z24, Z65 and ZP92. Cell fusion between ZP102 and human fibroblasts of several complementa-
TABLE 2
Complementation Analysis by DNA Transfection PEX2 cDNA
PEX6 cDNA
0 0 0
0 0 0
ZP101 ZP102 ZP103
Three peroxisome-deficient CHO mutants were transfected with expression vectors pcD65K (PEX2) or pUcD2r92A (PEX6) by electroporation. After 72 hr, cells were immunocytochemically stained with rabbit anti-rat catalase antibody. 0 means that no peroxisome was formed.
Characterization of ZP101 and ZP102. We further characterized ZP101 and ZP102 biochemically. ZP103 was omitted, because it was in the same complementation group as that of ZP101 and grew slower. In peroxisome-deficient cells, peroxisomal proteins are mislocalized to the cytosol, rapidly degraded or not converted to mature forms, despite a normal synthesis. Intracellular localization of catalase was determined by the titration with digitonin. In contrast to the case for wildtype cells, almost all catalase activity was released at the concentration as low as 100 mg/ml of digitonin for both mutants (Fig. 3), suggesting that catalase was present in the cytosol. The activity of the first enzyme of ether phospholipid synthesis, dihydroxyacetonephosphate acyltransferase (DHAP-ATase) was severely diminished in both mutant cells as compared to CHOTKa cells (Table 3). ZP101 and ZP102 were originally isolated as P9OH/UV resistant mutants; both were thus resistant to P9OH/UV treatment. Conversely, they were sensitive to P12/UV treatment which specifically kills peroxisome-deficient mutants, because there is no synthesis of plasmalogen, an oxygen radical scavenger (Table 3). Acyl-CoA oxidase, the first enzyme of the peroxisomal fatty acid b-oxidation system, is synthesized as a 75 kDa polypeptide (A component) and proteolytically converted into 53 kDa and 22 kDa polypeptides (B and C components, respectively) in peroxisomes. On continuous cell labeling, A and B components were observed in CHOTKa cells (Fig. 4, lane 1). On the other hand, only A component was seen in ZP101 or ZP102 mutant cells, in a much smaller amount probably due to rapid degradation (Fig. 4, lanes 2 and 3). Peroxisomal 3-ketoacyl-CoA thiolase,
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 3
Properties of CHOTKa, ZP101, ZP102 and ZP103 Cells Cell
Peroxisome
DHAP-ATase (%)
P12/UV (%)
P9OH/UV (%)
CHOTKa ZP101 ZP102 ZP103
/ 0 0 0
100 4.2 4.4 6.4
66 0 0 0.01
0 97 96 100
DHAP-ATase activity was expressed as a percentage relative to that of wild-type CHOTKa cells (6.09 nmol 2 hr01 mg01). For determination of P12/UV and P9OH/UV resistance, 200 or 1 1 105 cells were inoculated into 60 mm dishes and selected. The numbers of colonies were counted and expressed as percentages of that of unselected control.
the third enzyme of peroxisomal b-oxidation system, is synthesized as a larger precursor with an aminoterminal presequence which contains a peroxisomal targeting signal-2, and is converted to the mature form in peroxisomes. In CHOTKa cells, only the mature thiolase was detected (Fig. 4, lane 4), representing the rapid processing of the precursor form. In ZP101 or ZP102 cells, only the larger precursor was seen (Fig. 4, lane 5 and 6), implying the absence of processing activity. DISCUSSION We previously reported the isolation of two peroxisome deficient CHO mutants, Z24 and Z65, which were identified by the autoradiographic screening for mu-
FIG. 4. Biosynthesis of peroxisomal enzymes. Marks A, B and C indicate the positions of A, B and C components of acyl-CoA oxidase (AOx), respectively. P and M, a larger precursor and mature forms of 3-ketoacyl-CoA thiolase (TH), respectively. A faint band at the same position as that of AOx B component in lanes 2 and 3 is nonspecific, because a band of similar intensity is seen in lanes 4–6. C component of AOx is difficult to see in lane 1, because of the nonspecific band observed at the same mobility in all six lanes. Exposure, 43 days.
tants defective in peroxisomal DHAP-ATase activity (16) . In another work, we isolated eight clones of peroxisome-deficient mutants using P9OH/UV selection. One of them, ZP92, was the third mutant, but all other seven were of complementation group F, the same as that of Z65 (17) . Although the P9OH/UV method is effective to isolate peroxisome-deficient mutant, excessively frequent isolation of group F mutants made it difficult to isolate new mutants of different complementation groups. In this report, we overcame this difficulty by using CHOTKa cells that was transformed with rat PEX2 cDNA. By performing mutagenization with MNNG and P9OH/UV selection in the presence of G418, the ectopic PEX2 gene was stably retained, hence no group F mutant was obtained. Three mutants were isolated and two mutants, ZP101 and ZP103 were group E mutants, like previously isolated mutant Z24. ZP102 belonged to a complementation group different from those of previously isolated CHO mutants. Cell fusion analysis revealed that ZP102 was a group 2 mutant, and transfection of human PEX5 cDNA restored peroxisomes. At present, ten complementation groups are known in human peroxisome deficient disorders (68), and four CHO mutants corresponding to groups C, E, F and 2 have now been isolated. Group 2 peroxisome deficiency is caused by the mutation of PEX5 gene, which encode the receptor for PTS1 sequence at the C-termini of many peroxisomal proteins including acyl-CoA oxidase. Peroxisomal 3-ketoacyl-CoA thiolase have PTS2, another targeting signal for peroxisome, within its N-terminal presequence. Some patients of group 2 only show the defect of PTS1mediated import, but others are deficient in both PTS1and PTS2-mediated import (13) . ZP102 has a larger precursor form of thiolase and shows cytosolic distribution of green fluorescent protein fused to PTS2 (unpublished result), indicating that both import systems are affected in ZP102. The molecular basis of the effect of Pex5 mutation on the PTS2-dependent import should be investigated. CHO cell mutants grow faster than fibroblasts, and are immortal and feasible for the isolation of stable
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transformants. These characters are favorable for screening genes essential for peroxisome biogenesis by functional complementation, which was successfully applied for isolation of PEX2 (2) and PEX6 cDNA (10). Moreover, peroxisomal protein import was conveniently assayed with living (23) and semi-intact CHO cells (24). Thus, our approach will be effective to isolate new peroxisome deficient CHO mutants, and facilitate the identification of genes essential for peroxisome biogenesis and their functional characterization. ACKNOWLEDGMENTS This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan, The Sumitomo Foundation, and Uehara Memorial Foundation.
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