[1]
P450 GENE NOMENCLATURE
3
[1] P 4 5 0 G e n e N o m e n c l a t u r e B a s e d o n E v o l u t i o n B y DANIEL W. NEBERT and DAVID R. NELSON
Introduction P450 enzymes are important in the metabolism of numerous physiological substrates such as steroids, fatty acids, prostaglandins, cytokines, bile acids, and biogenic amines. ~-4 It has been postulated that these enzymes might play an important role in controlling the steady-state level of endogenous effectors of growth and differentiation. 5 Many of these enzymes also metabolize a wide range of foreign chemicals including environmental pollutants, drugs, and natural plant products. The metabolism of foreign chemicals can frequently produce toxic metabolites, some of which are believed to initiate carcinogenesis and tumor promotion. What had appeared 10-20 years ago to be a small number of P450 enzymes was simply a reflection of the limitations in biochemical methodology at the time) With the advent of recombinant DNA and ancillary techniques during the 1980s, we have come to appreciate the magnitude and complexity of the P450 gene superfamily. 6-13
Number of P450 Genes Existing in Any Given Species One approach to estimating the total number of P450 genes in any one species is to tally the number of genes reported at the end of each year in I A. H. Conney, Pharmacol. Rev. 19, 317 (1967). 2 A. Y. H. Lu and W. Levin, Biochim. Biophys. Acta 344, 205 (1974). 3 D. W. Nebert, Mol. Cell. Biochem. 27, 27 (1979). 4 R. E. White and M. J. Coon, Annu. Rev. Biochern. 49, 315 (1980). 5 D. W. Nebert, Nature (London) 347, 709 (1990). 6 D. W. Nebert, M. Adesnik, M. J. Coon, R. W. Estabrook, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, B. Kemper, W. Levin, I. R. Phillips, R. Sato, and M. R. Waterman, DNA 6, 1 (1987). 7 D. W. Nebert and F. J. Gonzalez, Annu. Rev. Biochem. 56, 945 (1987). 8 D. R. Nelson and H. W. Strobel, Mol. Biol. Eool. 4, 572 (1987). 9 F. J. Gonzalez, Pharmacol. Rev. 40, 243 (1988). 10 D. W. Nebert, D. R. Nelson, M. Adesnik, M. J. Coon, R. W. Estabrook, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, B. Kemper, W. Levin, I. R. Phillips, R. Sato, and M. R. Waterman, DNA 8, 1 (1989). i1 D. W. Nebert, D. R. Nelson, and R. Feyereisen, Xenobiotica 19, 1149 (1989). 12 F. J. Gonzalez and D. W. Nebert, Trends Genet. 6, 182 (1990).
METHODS IN ENZYMOLOGY, VOL, 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
4
MUTAGENESIS, MODIFICATION, PROTEIN STRUCTURE
[1]
40
30 0ul I-n0n LU r~
20 W
Z w o a.
10
FIG. 1. Rates at which P450 genes in three species have been discovered, or reported, at the end of 1986, 6 1988, l° and 1990. t3
which a P450 nomenclature update has been completed. Figure 1 shows that the total number of new human, rat, and mouse P450 genes appears to be increasing at least as rapidly between 1988 and 1990 as between 1986 and 1988, and we are currently approaching 30 distinct P450 genes in the human and 40 in the rat. Furthermore, most of the genes characterized so far represent those expressed in liver, lung, kidney, and endocrine tissues. Because many of these genes have been isolated from easily accessible tissues, represent (relatively) abundant and often inducible mRNA species, and exhibit tissue- and developmental-specific expression, we expect that many more P450 genes remain to be isolated--especially noninducible genes and those from tissues not mentioned above. Hence, we believe that the halfway point has not yet been reached and predict that each mammalian species might easily have at least 60 and perhaps more than 13 D. W. Nebert, D. R. Nelson, M. J. Coon, R. W. Estabrook, R. Feyereisen, Y. FujiiKuriyama, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, J. C. Loper, R. Sato, M. R. Waterman, and D. J. Waxman, DNA Cell Biol. 10, 1 (1991).
[1]
P450 GENE NOMENCLATURE
5
200 individual P450 genes. For prokaryotic and lower eukaryotic species, sufficient data are not yet available for estimating the total number of P450 genes. Each P450 gene almost always produces a single protein. To date, there appear to be two examples in which "functional" alternative splicing might occur, that is, differential processing of the P450 transcript such that entire exons or portions of exons are exchanged in order to produce an enzyme with a new catalytic activity.~3 Naming P450 Gene or Enzyme For naming a P450 gene it has been recommended ~3 that we include the italicized root symbol CYP (Cyp for the mouse), denoting cytochrome P450, an Arabic number designating the P450 family, a letter indicating the subfamily when two or more subfamilies are known to exist within that family, and an Arabic number representing the individual gene. With mouse genes, the final number is preceded by a hyphen. Arabic rather than Roman numerals should be used. It is also recommended that the same nomenclature for the gene be used for its transcript and product: for example, nonitalicized CYP1AI for the mRNA and protein in all species including mouse, and italicized CYP1A! (Cypla-! in mouse) for the gene and cDNA. On the contrary, one might continue using the trivial name for the enzyme and Roman numerals where they have already been established, for example, LM2 and rap-12 are encoded by the rabbit CYP2B4 and human CYP2C8 genes, respectively. When describing data about the gene and gene product, the authors are free to use, for example, CYP2B4 or C ¥P2C8 and CYP2B4 or CYP2C8 throughout the text. Another possibility for the protein might be P450 2B4 or P450 2C8, or simply 2B4 or 2C8. As a further possibility, the authors might use the trivial name LM2 or rap-12 in the text, as long as the assigned names of the genes and the species under study are provided in a footnote. Although consistent usage of the gene nomenclature is preferred, some authors may prefer to use trivial P450 enzyme names. However, for the names to be compatible with GenBank and other nucleic acid and protein databases, this means no hyphen in P450, no Greek letters, and no subscripts or superscripts. For example, P4507,~, P450scc, P45011js, P45017a, P450~rom, P450c21, and P450c27 should be referred to as P450c7, P450scc, P450cl 1, P450c17, P450arom (or P450c19), P450c21, and P450c27, respectively, or simply c7, scc, c l l , c17, arom (or c19), c21, and c27. If there seems to be no subfamily and no second functional gene in a particular family, one need not specify the subfamily and gene number. For example, the human (also cow, pig, and mouse) P450c21 is the sole
6
MUTAGENESIS, MODIFICATION, PROTEIN STRUCTURE
[1]
functional gene of the CYP21 family, and the gene and enzyme are named CYP21 (Cyp-21 in mouse) and CYP21, respectively. A P450 protein sequence from one gene family is defined as having at most 40% resemblance to that from any other family. Originally, this rule for a P450 gene family was arbitrary, but the definition has turned out to be very useful. In those instances that have been examined to date, genes within a defined subfamily have been found to be nonsegregating, that is, to lie within the same "gene cluster. ''m3 For example, the rat CYP2D1, CYP2D2, CYP2D3, CYP2D4, and CYP2D5 genes are located adjacent to one another on the same chromosome and form the CYP2D cluster) 4 The same appears to hold true for the human CYP2D6 gene and pseudogenes CYP2D7P and CYP2D8P) 5
The "40% Rule" Naming a P450 gene can be quite simple. The protein sequence is aligned with a representative sequence from each family and subfamily, and the percent identity is determined. This percentage only reflects comparisons of overlapping portions of the sequences; gaps and unmatched ends are not counted in the overall length. If the sequence is less than 40% identical to all other sequences, the new sequence constitutes the first member of a new family. If the new sequence is at least 40% identical to any other sequence, then the new sequence belongs in that family. Usually there is a clear affinity with only one family or subfamily, unless the sequence represents the first member of a new family. We have found no instance yet in which a sequence appears to belong to more than one group. Once the family or subfamily is identified, the new sequence is compared with that of all other members in the group. If the new sequence is only a few (-
P450 GENE NOMENCLATURE
3
[1] P 4 5 0 G e n e N o m e n c l a t u r e B a s e d o n E v o l u t i o n B y DANIEL W. NEBERT and DAVID R. NELSON
Introduction P450 enzymes are important in the metabolism of numerous physiological substrates such as steroids, fatty acids, prostaglandins, cytokines, bile acids, and biogenic amines. ~-4 It has been postulated that these enzymes might play an important role in controlling the steady-state level of endogenous effectors of growth and differentiation. 5 Many of these enzymes also metabolize a wide range of foreign chemicals including environmental pollutants, drugs, and natural plant products. The metabolism of foreign chemicals can frequently produce toxic metabolites, some of which are believed to initiate carcinogenesis and tumor promotion. What had appeared 10-20 years ago to be a small number of P450 enzymes was simply a reflection of the limitations in biochemical methodology at the time) With the advent of recombinant DNA and ancillary techniques during the 1980s, we have come to appreciate the magnitude and complexity of the P450 gene superfamily. 6-13
Number of P450 Genes Existing in Any Given Species One approach to estimating the total number of P450 genes in any one species is to tally the number of genes reported at the end of each year in I A. H. Conney, Pharmacol. Rev. 19, 317 (1967). 2 A. Y. H. Lu and W. Levin, Biochim. Biophys. Acta 344, 205 (1974). 3 D. W. Nebert, Mol. Cell. Biochem. 27, 27 (1979). 4 R. E. White and M. J. Coon, Annu. Rev. Biochern. 49, 315 (1980). 5 D. W. Nebert, Nature (London) 347, 709 (1990). 6 D. W. Nebert, M. Adesnik, M. J. Coon, R. W. Estabrook, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, B. Kemper, W. Levin, I. R. Phillips, R. Sato, and M. R. Waterman, DNA 6, 1 (1987). 7 D. W. Nebert and F. J. Gonzalez, Annu. Rev. Biochem. 56, 945 (1987). 8 D. R. Nelson and H. W. Strobel, Mol. Biol. Eool. 4, 572 (1987). 9 F. J. Gonzalez, Pharmacol. Rev. 40, 243 (1988). 10 D. W. Nebert, D. R. Nelson, M. Adesnik, M. J. Coon, R. W. Estabrook, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, B. Kemper, W. Levin, I. R. Phillips, R. Sato, and M. R. Waterman, DNA 8, 1 (1989). i1 D. W. Nebert, D. R. Nelson, and R. Feyereisen, Xenobiotica 19, 1149 (1989). 12 F. J. Gonzalez and D. W. Nebert, Trends Genet. 6, 182 (1990).
METHODS IN ENZYMOLOGY, VOL, 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
4
MUTAGENESIS, MODIFICATION, PROTEIN STRUCTURE
[1]
40
30 0ul I-n0n LU r~
20 W
Z w o a.
10
FIG. 1. Rates at which P450 genes in three species have been discovered, or reported, at the end of 1986, 6 1988, l° and 1990. t3
which a P450 nomenclature update has been completed. Figure 1 shows that the total number of new human, rat, and mouse P450 genes appears to be increasing at least as rapidly between 1988 and 1990 as between 1986 and 1988, and we are currently approaching 30 distinct P450 genes in the human and 40 in the rat. Furthermore, most of the genes characterized so far represent those expressed in liver, lung, kidney, and endocrine tissues. Because many of these genes have been isolated from easily accessible tissues, represent (relatively) abundant and often inducible mRNA species, and exhibit tissue- and developmental-specific expression, we expect that many more P450 genes remain to be isolated--especially noninducible genes and those from tissues not mentioned above. Hence, we believe that the halfway point has not yet been reached and predict that each mammalian species might easily have at least 60 and perhaps more than 13 D. W. Nebert, D. R. Nelson, M. J. Coon, R. W. Estabrook, R. Feyereisen, Y. FujiiKuriyama, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, J. C. Loper, R. Sato, M. R. Waterman, and D. J. Waxman, DNA Cell Biol. 10, 1 (1991).
[1]
P450 GENE NOMENCLATURE
5
200 individual P450 genes. For prokaryotic and lower eukaryotic species, sufficient data are not yet available for estimating the total number of P450 genes. Each P450 gene almost always produces a single protein. To date, there appear to be two examples in which "functional" alternative splicing might occur, that is, differential processing of the P450 transcript such that entire exons or portions of exons are exchanged in order to produce an enzyme with a new catalytic activity.~3 Naming P450 Gene or Enzyme For naming a P450 gene it has been recommended ~3 that we include the italicized root symbol CYP (Cyp for the mouse), denoting cytochrome P450, an Arabic number designating the P450 family, a letter indicating the subfamily when two or more subfamilies are known to exist within that family, and an Arabic number representing the individual gene. With mouse genes, the final number is preceded by a hyphen. Arabic rather than Roman numerals should be used. It is also recommended that the same nomenclature for the gene be used for its transcript and product: for example, nonitalicized CYP1AI for the mRNA and protein in all species including mouse, and italicized CYP1A! (Cypla-! in mouse) for the gene and cDNA. On the contrary, one might continue using the trivial name for the enzyme and Roman numerals where they have already been established, for example, LM2 and rap-12 are encoded by the rabbit CYP2B4 and human CYP2C8 genes, respectively. When describing data about the gene and gene product, the authors are free to use, for example, CYP2B4 or C ¥P2C8 and CYP2B4 or CYP2C8 throughout the text. Another possibility for the protein might be P450 2B4 or P450 2C8, or simply 2B4 or 2C8. As a further possibility, the authors might use the trivial name LM2 or rap-12 in the text, as long as the assigned names of the genes and the species under study are provided in a footnote. Although consistent usage of the gene nomenclature is preferred, some authors may prefer to use trivial P450 enzyme names. However, for the names to be compatible with GenBank and other nucleic acid and protein databases, this means no hyphen in P450, no Greek letters, and no subscripts or superscripts. For example, P4507,~, P450scc, P45011js, P45017a, P450~rom, P450c21, and P450c27 should be referred to as P450c7, P450scc, P450cl 1, P450c17, P450arom (or P450c19), P450c21, and P450c27, respectively, or simply c7, scc, c l l , c17, arom (or c19), c21, and c27. If there seems to be no subfamily and no second functional gene in a particular family, one need not specify the subfamily and gene number. For example, the human (also cow, pig, and mouse) P450c21 is the sole
6
MUTAGENESIS, MODIFICATION, PROTEIN STRUCTURE
[1]
functional gene of the CYP21 family, and the gene and enzyme are named CYP21 (Cyp-21 in mouse) and CYP21, respectively. A P450 protein sequence from one gene family is defined as having at most 40% resemblance to that from any other family. Originally, this rule for a P450 gene family was arbitrary, but the definition has turned out to be very useful. In those instances that have been examined to date, genes within a defined subfamily have been found to be nonsegregating, that is, to lie within the same "gene cluster. ''m3 For example, the rat CYP2D1, CYP2D2, CYP2D3, CYP2D4, and CYP2D5 genes are located adjacent to one another on the same chromosome and form the CYP2D cluster) 4 The same appears to hold true for the human CYP2D6 gene and pseudogenes CYP2D7P and CYP2D8P) 5
The "40% Rule" Naming a P450 gene can be quite simple. The protein sequence is aligned with a representative sequence from each family and subfamily, and the percent identity is determined. This percentage only reflects comparisons of overlapping portions of the sequences; gaps and unmatched ends are not counted in the overall length. If the sequence is less than 40% identical to all other sequences, the new sequence constitutes the first member of a new family. If the new sequence is at least 40% identical to any other sequence, then the new sequence belongs in that family. Usually there is a clear affinity with only one family or subfamily, unless the sequence represents the first member of a new family. We have found no instance yet in which a sequence appears to belong to more than one group. Once the family or subfamily is identified, the new sequence is compared with that of all other members in the group. If the new sequence is only a few (-
