J. Biochem. 112, 249-252 (1992)
Molecular Cloning of a cDNA Encoding a Member of a Novel Cytochrome P450 Family in the Mollusc Lymnaea stagnalis Yvonne Teunissen,' Wynand P.M. Geraerts,' Harm van Heerikhoizen,** Rudi J. Planta,** and Joos Joosse* 'Department of Biology, Free University, De Boelelaan 1087; and "Department of Biochemistry, Free University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands Received for publication, February 12, 1992
We isolated a cDNA encoding a cytochrome P450 from the mollusc Lymnaea stagnalis. The mRNA is 2.1 nucleotides long and contains an open reading frame encoding a protein of 546 amino acids. A conserved heme-binding domain, characteristic of cytochrome P460 proteins, is present in the deduced amino acid sequence. The Lymnaea cytochrome P450 protein shares less than 40% positional identity with any known member of the cytochrome P450 superfamily, and therefore, represents a separate family, which we propose to name CYP10. The CYP10 mRNA is shown to be uniquely and abundantly expressed in the female gonadotropic hormone producing dorsal bodies of L. stagnalis.
Cytochrome P450 enzymes are functionally related, ubiquitous enzymes, involved in the oxidative metabolism of endogenous compounds such as steroids, prostaglandins, vitamin D, and of numerous exogenous compounds (2). To date, 27 gene families, comprising 154 genes and 7 putative pseudogenes have been described, that together constitute the cytochrome P450 (CYP) superfamily (2). The majority of P450 enzymes is located in the endoplasmic reticulum. Only the CYP11A, CYP11B, and CYP27 gene families consist of enzymes that are known to be bound to mitochondrial membranes (2). Here, we report the sequence analysis of a cDNA encoding a novel cytochrome P450 in the mollusc, Lymnaea stagnalis. The Lymnaea cytochrome P450 gene represents a member of a distinct family of the P450 superfamily, which we propose to name the CYP10 family, following a widely recognized nomenclature for cytochrome P450s (2). The (CYP10) cytochrome P450 of Lymnaea shows the highest similarity with the mitochondrial P450 enzymes, known to be involved in the synthesis of hormones such as steroids (3) and vitamin D (4). The cDNA was isolated in the course of experiments designed to uncover the structural identity and biosynthetic route of the gonadotropic hormone of the dorsal bodies, endocrine organs involved in the control of vitellogenesis (5), and the female reproductive tract (6, 7) of Lymnaea. The present experiments show that the (CYP10) cytochrome P450 is uniquely and abundantly expressed in the dorsal bodies, indicating that it is involved in hormone synthesis. EXPERIMENTAL PROCEDURES Animals—Adult L. stagnalis, bred in the laboratory under standard conditions (8), at a photoperiod of 12 h per day, were used. Materials—A cDNA library in A.gtlO, specific for the Abbreviations: BSA, bovine serum albumin; CNS, central nervous system; CYP, cytochrome P450; SDS, sodium dodecyl sulfate. Vol. 112, No. 2, 1992
249
central nervous system (CNS) and the dorsal bodies of L. stagnalis, was used as described previously (9). Restriction enzymes, AMV reverse transcriptase and T4 DNA ligase were obtained from Bethesda Research Laboratories. T4 polynucleotide kinase and yeast tRNA were obtained from Boehringer. Enzymes were used according to Maniatis et aL {10). Nylon filters (Hybond N) for hybridization experiments and radioisotopes were purchased from Amersham. T7 DNA polymerase used for DNA sequencing was supplied by Pharmacia LKB Biotechnology. Oligonucleotides were synthesized on an Applied Biosystems' Model 381A DNA synthesizer. cDNA Screening—The cDNA library of the CNS and the dorsal bodies was subjected to the following differential screening procedure. About 100,000 clones were hybridized to radioactive cDNA (specific activity>2x 10' dpm/ fig RNA) synthesized from RNA extracted from the dorsal bodies (positive probe) and from the CNS without the cerebral ganglia and the dorsal bodies (negative probe). Total RNA was isolated according to Chirgwin et al. (11). Hybridization procedures were performed by standard methods (20). Dorsal body specific clones were identified and part of them were purified through a second round of screening. Inserts were subcloned in M13 vectors (12) by standard cloning procedures (10) and subjected to DNA sequence analysis. DNA Sequencing—Nucleotide sequence analysis was performed by the dideoxy-chain-termination technique (13) in both orientations, using a M13-specific primer or synthetic oligonucleotides prepared according to known sequences. Northern Blot Analysis—Total RNA isolation was carried out by the method of Chomczinsky and Sacchi (14). After denaturation in glyoxal/sulfoxide (15) and agarose gel electrophoresis, RNA was transferred to Hybond N in 20XSSC ( l x S S C : 0.15 M NaCl, 15 mM sodium citrate, pH 7.0). Hybridization was performed at 65'C in 6 X SSC, 0.5% SDS, 5xDenhardt's solution (0.1% w/v Ficoll, polyvinylpyrrolidone, and BSA each) and 0.02 mg/ml
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yeast tRNA. The membrane was washed three times in 2 X SSC, 0.1% SDS at 65"C. In Situ Hybridization—Methods used were basically according to Dirks et aL {16) as described by Bogerd et al (9).
A 8-35 is 1,870 nucleotides long and overlaps at the 3' end with the 1,306 nucleotide long clone A.8-8. Clone A 8-35 has E E
RESULTS AND DISCUSSION
Identification and Nucleotide Sequencing of cDNA Clones—Searching for dorsal body specific mRNA sequences we used a differential hybridization procedure to screen a L. stagnalis cDNA library of the CNS and the dorsal bodies with probes prepared from RNA isolated from dorsal bodies and from the CNS without the cerebral ganglia and the dorsal bodies. With this screening procedure, only abundantly expressed transcripts are identified. We identified 250 dorsal body specific cDNA clones out of 100,000 clones. Cross hybridization indicated that all 250 ,
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GATGATGTGGAGCCTATTCTAAACACCATGTTGACCCCTGACCGACCTGTCCGAATAGAGTTCAAACCAAGACAjtTAAtiACATAGCAATAGTAGAGAGGATGTTTTTAAAAAAAACCAAA D D V E P I L N T M L T P D R P V R I E F K P R Q -
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Fig. 2. Nucleotide sequence of ,18-38 and dedaced amino acid sequence. Nudeotides and amino adds are numbered at the right, starting with the initiator methionine codon (boxed) and residue as + 1 . A highly conserved region amongst cytochrome P450 enzymes containing the heme-binding cysteine residue (1, 17) is underlined. The termination codon and the putative polyadenylation signal are boxed. J. Biochan.
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Fig. 4. Comparison of the heme-blnding region of the Lymnaea P450 with other P450s. Amino acids shared between the L, stagnalis P450 (Lymnaea 10) and the other P450s are boxed. Sequences are taken from references as cited in Table I. Fig. 3. Northern blot analysis, indicating transcript length and tissue specificity of CYP10 mRNA. Total RNA (20 ^ g) isolated from (A) dorsal bodies, and (B) cerebral ganglia with dorsal bodies (lane 1); the other ganglia of the CNS (lane 2); penial complex (lane 3); prostate gland (lane 4); ovotestis and hepatopancreas (lane 5); vesiculae seminalis (lane 6); albumen gland (lane 7); vas deferens (lane 8); oothecal gland (lane 9); muciparua gland (lane 10) was electrophoresed in a 1.2% agarose gel, transferred to a nylon membrane and hybridized with a "P-labeled single stranded CYP10 cDNA. Siie markers are hybridization products from known sequences.
TABLE I. Sequence relatedness between Lymnaea P450 and various other cytochrome P460s. Comparisons between two proteins were made according to Needleman and Wunsch (19). Percentages of matched residues (% identity) are ratios of the numbers of matched sites to the sum of the numbers of matched, replaced, and unpaired sites. Percent similarity includes residues with single base substitutions. Sequences of human 11A1 (P450scc; 23), rabbit 27 (P450 26-ohp; 24), bovine 11B1 (P450 11£; 25), human 17 (P450cl7; 26), housefly 6 (P450 VLAI; 27), and Pseudomonas 101 (P450cam; 28) were taken from the references cited. Cytochrome P450 % similarity % identity Human 11A1 64.4 31.4 Rabbit 27 64.1 30.3 Bovine 11B1 53.8 28.5 Human 17 53.1 26.6 52.0 23.9 Housefly 6 Pseudomonas 101 47.6 18.9
an open reading frame of 1,638 nucleotides, starting with an ATG that is located 95 bases from the 5' end, and ending with a TAA stop codon. The 3' untranslated region contains a putative polyadenylation signal (AATAAA). Figure 2 shows the nucleotide sequence data and the deduced amino acid sequence. Northern blotting of dorsal body RNA revealed the presence of a single mRNA of about 2,100 nucleotides (Fig. 3A). Since the longest cDNA clone is only 1,870 nucleotides in length, we sequenced the mRNA directly with an oligonucleotide to obtain additional sequence information at the 5' end. This information, about 150 nucleotides (data not shown), did not lead to an extension of the open reading frame. The complete sequence thus contains an open reading frame encoding a protein of 545 amino acids with a predicted molecular weight of about 62 kDa. The sequence in Fig. 2 encodes a cytochrome P450 as evidenced by a conserved sequence near the carboxy-terminus, which contains the hemebinding cysteine, that has been found in all other P450 Vol. 112, No. 2, 1992
proteins (1, 17). Comparison with Other Cytochrome P450 Sequences— Data base searches of EMBL, GenBank, and VecBase, using the method of Pearson and Lipman (18), revealed the highest similarity of the Lymnaea P450 with mitochondrial P450s. We made pairwise alignments using the method of Needleman and Wunsch (19) of the Lymnaea P450 protein with a large number of other P450s encoded by different P450 families. The sequence relatedness with members of the mitochondrial P450 families (human 11A1, bovine 11B1, and rabbit 27) and of microsomal P450 families from a vertebrate (human 17), an invertebrate (housefly 6), and a bacterial sequence (Pseudomonas 101) are depicted in Table I. Comparison of the conserved heme-binding region of the Lymnaea P450 protein with members of these different P450 families is shown in Fig. 4. After optimal pairwise alignment, the Lymnaea P450 shows the highest percentage identity with the mitochondrial P450 proteins (31.4% with human 11A1,30.3% with rabbit 27, and 28.5% with bovine 11B1). As a "ground rule," P450 genes are placed in separate families when the corresponding proteins share less than 40% positional identity (2, 20). Thus, the Lymnaea cytochrome P450 gene has been classified as a member of a novel gene family, the CYP10 family, by the committee for the nomenclature of P450s (2), after having been provided with the Lymnaea P450 sequence by personal communication. Mitochondrial proteins are directed into the mitochondrion through an amino-terminal signal sequence, up to 70 residues long, which is cleaved off upon insertion (21, 22). Mitochondrial presequences have not been shown to contain a consensus sequence, but a general feature is that they contain positively charged residues spread along their entire length, with almost no negatively charged residues and with a high content of hydrophobic residues (22). The amino-terminal stretch of the first 40 amino acids of the CYP10 protein contains 2 arginine, 2 histidine, and 6 lysine residues and only one negatively charged residue (a glutamate), which is in accordance with this general feature, although overall the stretch is not hydrophobic. Evidence that the CYP10 protein contains a mitochondrial presequence and determination of the site of processing will have to await amino-terminal protein sequencing. Lymnaea (CYP10) Cytochrome P450 Is Specifically Expressed in the Dorsal Bodies—We confirmed the dorsal body specificity of the isolated cDNA encoding the cytochrome P450 by in situ hybridization and Northern blot analysis. The dorsal bodies show a strong signal in the in
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Y. Teunissen et al. Fig. 5. In situ hybridization of CYP10 cDN A to sections of the CNS of Lymnaea Btagnalis. Hybridization conditions were as described (9, 26). A "S-labeled single stranded CYP10 cDNA probe was used. The dorsal bodies show a clear labeling with the CYP10 cDNA clone, whereas the cerebral ganglia do not. Dorsal bodies (DB); cerebral ganglia (CG). Magnification, X45.
situ hybridization (Fig. 5), whereas the adjacent cerebral ganglia do not. Furthermore, Northern blot analysis of RNA isolated from the cerebral ganglia with the dorsal bodies, from the other ganglia of the CNS and from peripheral sources, confirms that the CYP10 mRNA is specifically and abundantly expressed in the dorsal bodies (Fig. 3B). Further extensive screening and analysis of cDNA clones did not result in the isolation of other dorsal body-specific cytochrome P450 sequences. This strongly suggests that the (CYP10) cytochrome P450 is involved in the synthesis of the hormone produced by the dorsal bodies. The fact that only one cytochrome P450 has been found makes steroid synthesis in the dorsal bodies unlikely, since this requires several cytochrome P450 enzymes belonging to distinct families (3).
Neth. J. ZooL 19, 131-139 9. Bogerd, J., Geraerts, W.P.M., van Heerikhuizen, H., Kerhoven, R.M., & Joosse, J. (1991) MoL Brain Res. 11, 47-54 10. Maniatis, T., Fritsch, E.T., & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York 11. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., & Rutter, W.J. (1979) Biochemistry 18, 5294-5299 12. Vieira, J. & Messing, J. (1982) Gene 19, 259-268 13. Sanger, F., Nicklen, S., & Coulsen, A. (1977) Proc. Natl. Acad ScL U.S.A. 74, 5463-5467 14. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156159 15. Thomas, P.S. (1980) Proc. NatL Acad Sci. USA 77, 5201-5205 16. Dirks, R.W., Raap, A.K., van Minnen, J., Vreugdenhil, E., Smit, A.B., & van der Ploeg, M. (1989) J. Histochem. Cytochem. 37, 714 17. Nebert, D.W. & Gonzalez, F.J. (1987) Annu. Rev. Biochem. 56, 945-993 We wish to thank Dr. A.B. Smit for performing initial experiments, 18. Pearson, W.R & Lipman, D.J. (1988) Proc Natl. Acad. Set. T. Bouwmeester, B. Blom, and P. Kooij for technical assistance, and USA 85, 2444-2448 M. Ramkema for performing the in situ hybridization. 19. Needleman, S.B. & Wunsch, CD. (1970) J. MoL BioL 48, 443453 20. Nebert, D.W., Nelson, D.R, Adesnik, M., Coon, M.J., EstaREFERENCES brook, R.W., Gonzalez, F.J., Guengerich, F.P., Gunsalus, I.C., 1. Ortiz de Montellano, P.R (1986) Cytochrome P450: Structure, Johnson, E.F., Kemper, B., Levin, W., Phillips, I.R, Sato, R, & Mechanism, and Biochemistry, Plenum, New York Waterman, M.R. (1989) DNA 8, 1-13 2. Nebert, D.W., Nelson, D.R, Coon, M.J., Estabrook, R.W., 21. Schatz, G. (1979) FEBS Lett. 103, 203-211 Feiereisen, R, Fujii-Kuriyama, Y., Gonzalez, F.J., Guengerich, 22. Roise, D. & Schatz, G. (1988) J. BioL Chem. 263, 4509-4511 F.P., Gunsalus, I.C., Johnson, E.F., Loper, J.C., Sato, R-, 23. Chung, B., Matteson, K.J., Voutilainen, R, Mohandas, T.K., & Waterman, M.R, & Waxman, D.J. (1991) DNA Cell BioL 10,1Miller, W.L. (1986) Proc Natl Acad. ScL USA 83, 8962-8966 14 24. Andersson, S., Davis, D.L., Dahlback, H., Joravall, H., &Russel, 3. Miller, W.L. (1988) Endocr. Rev. 9, 295-318 D.W. (1989) J. BioL Chem. 284, 8222-8229 4. Su, P., Rennert, H., Shayiq, RM., Yamamoto, R, Zheng, Y.M., 25. Chua, S.C., Szabo, P., Vitek, A., Grzeschik, K.H., John, M., & Addya, S., Strauss HI, J.F., & Avadhani, N.G. (1990) DNA Cell White, P.C. (1987) Proc NatL Acad. ScL USA 84, 7193-7197 BioL 9, 657-665 26. Chung, B., Picado-Leonard, J., Haniu, M., Bienkowski, M., Hall, 5. Geraerts, W.P.M. & Joosse, J. (1975) Gen. Comp. EndocrinoL P.F., Shively, J.E., & Miller, W.L. (1987) Proc. NatL Acad ScL 28, 350-375 USA 84, 407-411 6. Geraerts, W.P.M. & Algera, L.H. (1976) Gen. Comp. EndocrinoL 27. Feyereisen, R, Koener, J.F., Famsworth, D.E., & Nebert, D.W. 29, 109-118 (1989) Proc NatL Acad ScL USA 86, 1465-1469 7. Wijdenes, J., van Elk, R , & Joosse, J. (1983) Gen. Comp. 28. Unger, B.P., Gunsalus, I.C., & Sligar, S.G. (1986) J. BioL Chem. EndocrinoL 51, 263-271 261, 1158-1163 8. Steen, W.J. van der, Hoven, N.P. van den, & Jager, J.C. (1969)
J. Biochem.
Molecular Cloning of a cDNA Encoding a Member of a Novel Cytochrome P450 Family in the Mollusc Lymnaea stagnalis Yvonne Teunissen,' Wynand P.M. Geraerts,' Harm van Heerikhoizen,** Rudi J. Planta,** and Joos Joosse* 'Department of Biology, Free University, De Boelelaan 1087; and "Department of Biochemistry, Free University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands Received for publication, February 12, 1992
We isolated a cDNA encoding a cytochrome P450 from the mollusc Lymnaea stagnalis. The mRNA is 2.1 nucleotides long and contains an open reading frame encoding a protein of 546 amino acids. A conserved heme-binding domain, characteristic of cytochrome P460 proteins, is present in the deduced amino acid sequence. The Lymnaea cytochrome P450 protein shares less than 40% positional identity with any known member of the cytochrome P450 superfamily, and therefore, represents a separate family, which we propose to name CYP10. The CYP10 mRNA is shown to be uniquely and abundantly expressed in the female gonadotropic hormone producing dorsal bodies of L. stagnalis.
Cytochrome P450 enzymes are functionally related, ubiquitous enzymes, involved in the oxidative metabolism of endogenous compounds such as steroids, prostaglandins, vitamin D, and of numerous exogenous compounds (2). To date, 27 gene families, comprising 154 genes and 7 putative pseudogenes have been described, that together constitute the cytochrome P450 (CYP) superfamily (2). The majority of P450 enzymes is located in the endoplasmic reticulum. Only the CYP11A, CYP11B, and CYP27 gene families consist of enzymes that are known to be bound to mitochondrial membranes (2). Here, we report the sequence analysis of a cDNA encoding a novel cytochrome P450 in the mollusc, Lymnaea stagnalis. The Lymnaea cytochrome P450 gene represents a member of a distinct family of the P450 superfamily, which we propose to name the CYP10 family, following a widely recognized nomenclature for cytochrome P450s (2). The (CYP10) cytochrome P450 of Lymnaea shows the highest similarity with the mitochondrial P450 enzymes, known to be involved in the synthesis of hormones such as steroids (3) and vitamin D (4). The cDNA was isolated in the course of experiments designed to uncover the structural identity and biosynthetic route of the gonadotropic hormone of the dorsal bodies, endocrine organs involved in the control of vitellogenesis (5), and the female reproductive tract (6, 7) of Lymnaea. The present experiments show that the (CYP10) cytochrome P450 is uniquely and abundantly expressed in the dorsal bodies, indicating that it is involved in hormone synthesis. EXPERIMENTAL PROCEDURES Animals—Adult L. stagnalis, bred in the laboratory under standard conditions (8), at a photoperiod of 12 h per day, were used. Materials—A cDNA library in A.gtlO, specific for the Abbreviations: BSA, bovine serum albumin; CNS, central nervous system; CYP, cytochrome P450; SDS, sodium dodecyl sulfate. Vol. 112, No. 2, 1992
249
central nervous system (CNS) and the dorsal bodies of L. stagnalis, was used as described previously (9). Restriction enzymes, AMV reverse transcriptase and T4 DNA ligase were obtained from Bethesda Research Laboratories. T4 polynucleotide kinase and yeast tRNA were obtained from Boehringer. Enzymes were used according to Maniatis et aL {10). Nylon filters (Hybond N) for hybridization experiments and radioisotopes were purchased from Amersham. T7 DNA polymerase used for DNA sequencing was supplied by Pharmacia LKB Biotechnology. Oligonucleotides were synthesized on an Applied Biosystems' Model 381A DNA synthesizer. cDNA Screening—The cDNA library of the CNS and the dorsal bodies was subjected to the following differential screening procedure. About 100,000 clones were hybridized to radioactive cDNA (specific activity>2x 10' dpm/ fig RNA) synthesized from RNA extracted from the dorsal bodies (positive probe) and from the CNS without the cerebral ganglia and the dorsal bodies (negative probe). Total RNA was isolated according to Chirgwin et al. (11). Hybridization procedures were performed by standard methods (20). Dorsal body specific clones were identified and part of them were purified through a second round of screening. Inserts were subcloned in M13 vectors (12) by standard cloning procedures (10) and subjected to DNA sequence analysis. DNA Sequencing—Nucleotide sequence analysis was performed by the dideoxy-chain-termination technique (13) in both orientations, using a M13-specific primer or synthetic oligonucleotides prepared according to known sequences. Northern Blot Analysis—Total RNA isolation was carried out by the method of Chomczinsky and Sacchi (14). After denaturation in glyoxal/sulfoxide (15) and agarose gel electrophoresis, RNA was transferred to Hybond N in 20XSSC ( l x S S C : 0.15 M NaCl, 15 mM sodium citrate, pH 7.0). Hybridization was performed at 65'C in 6 X SSC, 0.5% SDS, 5xDenhardt's solution (0.1% w/v Ficoll, polyvinylpyrrolidone, and BSA each) and 0.02 mg/ml
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yeast tRNA. The membrane was washed three times in 2 X SSC, 0.1% SDS at 65"C. In Situ Hybridization—Methods used were basically according to Dirks et aL {16) as described by Bogerd et al (9).
A 8-35 is 1,870 nucleotides long and overlaps at the 3' end with the 1,306 nucleotide long clone A.8-8. Clone A 8-35 has E E
RESULTS AND DISCUSSION
Identification and Nucleotide Sequencing of cDNA Clones—Searching for dorsal body specific mRNA sequences we used a differential hybridization procedure to screen a L. stagnalis cDNA library of the CNS and the dorsal bodies with probes prepared from RNA isolated from dorsal bodies and from the CNS without the cerebral ganglia and the dorsal bodies. With this screening procedure, only abundantly expressed transcripts are identified. We identified 250 dorsal body specific cDNA clones out of 100,000 clones. Cross hybridization indicated that all 250 ,
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clones were homologous. Two clones, A. 8-8 and A 8-35, were selected for further analysis. The restriction maps and sequencing strategy are shown in Fig. 1. The insert of clone
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GATGATGTGGAGCCTATTCTAAACACCATGTTGACCCCTGACCGACCTGTCCGAATAGAGTTCAAACCAAGACAjtTAAtiACATAGCAATAGTAGAGAGGATGTTTTTAAAAAAAACCAAA D D V E P I L N T M L T P D R P V R I E F K P R Q -
1680 545
AAAACCAACCAATCTTTTGGCAAGATTGGTTATAGTATAGGGACTATGTGGAAAACAACTATTGTATACTGCTGTCTGACJ^ATAAJI^CTAACTTC
1776
Fig. 2. Nucleotide sequence of ,18-38 and dedaced amino acid sequence. Nudeotides and amino adds are numbered at the right, starting with the initiator methionine codon (boxed) and residue as + 1 . A highly conserved region amongst cytochrome P450 enzymes containing the heme-binding cysteine residue (1, 17) is underlined. The termination codon and the putative polyadenylation signal are boxed. J. Biochan.
Lymnaea stagnate Cytochrome P450 1
1200—1
2
3
4
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6
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8
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Pseudomonas 101 F
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3
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Q\E| L F L 1 M[gjF S H L
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Fig. 4. Comparison of the heme-blnding region of the Lymnaea P450 with other P450s. Amino acids shared between the L, stagnalis P450 (Lymnaea 10) and the other P450s are boxed. Sequences are taken from references as cited in Table I. Fig. 3. Northern blot analysis, indicating transcript length and tissue specificity of CYP10 mRNA. Total RNA (20 ^ g) isolated from (A) dorsal bodies, and (B) cerebral ganglia with dorsal bodies (lane 1); the other ganglia of the CNS (lane 2); penial complex (lane 3); prostate gland (lane 4); ovotestis and hepatopancreas (lane 5); vesiculae seminalis (lane 6); albumen gland (lane 7); vas deferens (lane 8); oothecal gland (lane 9); muciparua gland (lane 10) was electrophoresed in a 1.2% agarose gel, transferred to a nylon membrane and hybridized with a "P-labeled single stranded CYP10 cDNA. Siie markers are hybridization products from known sequences.
TABLE I. Sequence relatedness between Lymnaea P450 and various other cytochrome P460s. Comparisons between two proteins were made according to Needleman and Wunsch (19). Percentages of matched residues (% identity) are ratios of the numbers of matched sites to the sum of the numbers of matched, replaced, and unpaired sites. Percent similarity includes residues with single base substitutions. Sequences of human 11A1 (P450scc; 23), rabbit 27 (P450 26-ohp; 24), bovine 11B1 (P450 11£; 25), human 17 (P450cl7; 26), housefly 6 (P450 VLAI; 27), and Pseudomonas 101 (P450cam; 28) were taken from the references cited. Cytochrome P450 % similarity % identity Human 11A1 64.4 31.4 Rabbit 27 64.1 30.3 Bovine 11B1 53.8 28.5 Human 17 53.1 26.6 52.0 23.9 Housefly 6 Pseudomonas 101 47.6 18.9
an open reading frame of 1,638 nucleotides, starting with an ATG that is located 95 bases from the 5' end, and ending with a TAA stop codon. The 3' untranslated region contains a putative polyadenylation signal (AATAAA). Figure 2 shows the nucleotide sequence data and the deduced amino acid sequence. Northern blotting of dorsal body RNA revealed the presence of a single mRNA of about 2,100 nucleotides (Fig. 3A). Since the longest cDNA clone is only 1,870 nucleotides in length, we sequenced the mRNA directly with an oligonucleotide to obtain additional sequence information at the 5' end. This information, about 150 nucleotides (data not shown), did not lead to an extension of the open reading frame. The complete sequence thus contains an open reading frame encoding a protein of 545 amino acids with a predicted molecular weight of about 62 kDa. The sequence in Fig. 2 encodes a cytochrome P450 as evidenced by a conserved sequence near the carboxy-terminus, which contains the hemebinding cysteine, that has been found in all other P450 Vol. 112, No. 2, 1992
proteins (1, 17). Comparison with Other Cytochrome P450 Sequences— Data base searches of EMBL, GenBank, and VecBase, using the method of Pearson and Lipman (18), revealed the highest similarity of the Lymnaea P450 with mitochondrial P450s. We made pairwise alignments using the method of Needleman and Wunsch (19) of the Lymnaea P450 protein with a large number of other P450s encoded by different P450 families. The sequence relatedness with members of the mitochondrial P450 families (human 11A1, bovine 11B1, and rabbit 27) and of microsomal P450 families from a vertebrate (human 17), an invertebrate (housefly 6), and a bacterial sequence (Pseudomonas 101) are depicted in Table I. Comparison of the conserved heme-binding region of the Lymnaea P450 protein with members of these different P450 families is shown in Fig. 4. After optimal pairwise alignment, the Lymnaea P450 shows the highest percentage identity with the mitochondrial P450 proteins (31.4% with human 11A1,30.3% with rabbit 27, and 28.5% with bovine 11B1). As a "ground rule," P450 genes are placed in separate families when the corresponding proteins share less than 40% positional identity (2, 20). Thus, the Lymnaea cytochrome P450 gene has been classified as a member of a novel gene family, the CYP10 family, by the committee for the nomenclature of P450s (2), after having been provided with the Lymnaea P450 sequence by personal communication. Mitochondrial proteins are directed into the mitochondrion through an amino-terminal signal sequence, up to 70 residues long, which is cleaved off upon insertion (21, 22). Mitochondrial presequences have not been shown to contain a consensus sequence, but a general feature is that they contain positively charged residues spread along their entire length, with almost no negatively charged residues and with a high content of hydrophobic residues (22). The amino-terminal stretch of the first 40 amino acids of the CYP10 protein contains 2 arginine, 2 histidine, and 6 lysine residues and only one negatively charged residue (a glutamate), which is in accordance with this general feature, although overall the stretch is not hydrophobic. Evidence that the CYP10 protein contains a mitochondrial presequence and determination of the site of processing will have to await amino-terminal protein sequencing. Lymnaea (CYP10) Cytochrome P450 Is Specifically Expressed in the Dorsal Bodies—We confirmed the dorsal body specificity of the isolated cDNA encoding the cytochrome P450 by in situ hybridization and Northern blot analysis. The dorsal bodies show a strong signal in the in
252
Y. Teunissen et al. Fig. 5. In situ hybridization of CYP10 cDN A to sections of the CNS of Lymnaea Btagnalis. Hybridization conditions were as described (9, 26). A "S-labeled single stranded CYP10 cDNA probe was used. The dorsal bodies show a clear labeling with the CYP10 cDNA clone, whereas the cerebral ganglia do not. Dorsal bodies (DB); cerebral ganglia (CG). Magnification, X45.
situ hybridization (Fig. 5), whereas the adjacent cerebral ganglia do not. Furthermore, Northern blot analysis of RNA isolated from the cerebral ganglia with the dorsal bodies, from the other ganglia of the CNS and from peripheral sources, confirms that the CYP10 mRNA is specifically and abundantly expressed in the dorsal bodies (Fig. 3B). Further extensive screening and analysis of cDNA clones did not result in the isolation of other dorsal body-specific cytochrome P450 sequences. This strongly suggests that the (CYP10) cytochrome P450 is involved in the synthesis of the hormone produced by the dorsal bodies. The fact that only one cytochrome P450 has been found makes steroid synthesis in the dorsal bodies unlikely, since this requires several cytochrome P450 enzymes belonging to distinct families (3).
Neth. J. ZooL 19, 131-139 9. Bogerd, J., Geraerts, W.P.M., van Heerikhuizen, H., Kerhoven, R.M., & Joosse, J. (1991) MoL Brain Res. 11, 47-54 10. Maniatis, T., Fritsch, E.T., & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York 11. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., & Rutter, W.J. (1979) Biochemistry 18, 5294-5299 12. Vieira, J. & Messing, J. (1982) Gene 19, 259-268 13. Sanger, F., Nicklen, S., & Coulsen, A. (1977) Proc. Natl. Acad ScL U.S.A. 74, 5463-5467 14. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156159 15. Thomas, P.S. (1980) Proc. NatL Acad Sci. USA 77, 5201-5205 16. Dirks, R.W., Raap, A.K., van Minnen, J., Vreugdenhil, E., Smit, A.B., & van der Ploeg, M. (1989) J. Histochem. Cytochem. 37, 714 17. Nebert, D.W. & Gonzalez, F.J. (1987) Annu. Rev. Biochem. 56, 945-993 We wish to thank Dr. A.B. Smit for performing initial experiments, 18. Pearson, W.R & Lipman, D.J. (1988) Proc Natl. Acad. Set. T. Bouwmeester, B. Blom, and P. Kooij for technical assistance, and USA 85, 2444-2448 M. Ramkema for performing the in situ hybridization. 19. Needleman, S.B. & Wunsch, CD. (1970) J. MoL BioL 48, 443453 20. Nebert, D.W., Nelson, D.R, Adesnik, M., Coon, M.J., EstaREFERENCES brook, R.W., Gonzalez, F.J., Guengerich, F.P., Gunsalus, I.C., 1. Ortiz de Montellano, P.R (1986) Cytochrome P450: Structure, Johnson, E.F., Kemper, B., Levin, W., Phillips, I.R, Sato, R, & Mechanism, and Biochemistry, Plenum, New York Waterman, M.R. (1989) DNA 8, 1-13 2. Nebert, D.W., Nelson, D.R, Coon, M.J., Estabrook, R.W., 21. Schatz, G. (1979) FEBS Lett. 103, 203-211 Feiereisen, R, Fujii-Kuriyama, Y., Gonzalez, F.J., Guengerich, 22. Roise, D. & Schatz, G. (1988) J. BioL Chem. 263, 4509-4511 F.P., Gunsalus, I.C., Johnson, E.F., Loper, J.C., Sato, R-, 23. Chung, B., Matteson, K.J., Voutilainen, R, Mohandas, T.K., & Waterman, M.R, & Waxman, D.J. (1991) DNA Cell BioL 10,1Miller, W.L. (1986) Proc Natl Acad. ScL USA 83, 8962-8966 14 24. Andersson, S., Davis, D.L., Dahlback, H., Joravall, H., &Russel, 3. Miller, W.L. (1988) Endocr. Rev. 9, 295-318 D.W. (1989) J. BioL Chem. 284, 8222-8229 4. Su, P., Rennert, H., Shayiq, RM., Yamamoto, R, Zheng, Y.M., 25. Chua, S.C., Szabo, P., Vitek, A., Grzeschik, K.H., John, M., & Addya, S., Strauss HI, J.F., & Avadhani, N.G. (1990) DNA Cell White, P.C. (1987) Proc NatL Acad. ScL USA 84, 7193-7197 BioL 9, 657-665 26. Chung, B., Picado-Leonard, J., Haniu, M., Bienkowski, M., Hall, 5. Geraerts, W.P.M. & Joosse, J. (1975) Gen. Comp. EndocrinoL P.F., Shively, J.E., & Miller, W.L. (1987) Proc. NatL Acad ScL 28, 350-375 USA 84, 407-411 6. Geraerts, W.P.M. & Algera, L.H. (1976) Gen. Comp. EndocrinoL 27. Feyereisen, R, Koener, J.F., Famsworth, D.E., & Nebert, D.W. 29, 109-118 (1989) Proc NatL Acad ScL USA 86, 1465-1469 7. Wijdenes, J., van Elk, R , & Joosse, J. (1983) Gen. Comp. 28. Unger, B.P., Gunsalus, I.C., & Sligar, S.G. (1986) J. BioL Chem. EndocrinoL 51, 263-271 261, 1158-1163 8. Steen, W.J. van der, Hoven, N.P. van den, & Jager, J.C. (1969)
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