Isolation and Identification of a Cytochrome P450 Sequence in an Australian Termite,Mastotermes darwiniensis

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.

241, 579–583 (1997)

RC977856

Isolation and Identification of a Cytochrome P450 Sequence in an Australian Termite, Mastotermes darwiniensis Patrick H. J. Falckh,1 Wendy Balcombe, Victoria S. Haritos, and Jorma T. Ahokas Key Centre for Applied and Nutritional Toxicology, RMIT-University, Melbourne, Australia, 3000

Received November 16, 1997

A partial cytochrome P450 sequence was RT/PCR amplified from total RNA isolated from the whole body of worker class termites (Mastotermes darwiniensis). The degenerate primers used were designed from conserved regions from 4 different species: rat, human, cockroach and drosophila. The sequence was defined by the presence of the typical P450 heme-binding region and invariant residues found in all P450 proteins. The deduced amino acid sequence is 67% identical to cockroach (Blaberus discoidalis) CYP4C1, with only 39% and 42% identity to either CYP4A1 or CYP4B1, respectively, and has been named CYP4C8. Similar low sequence homology was observed between the termite sequence and the mouse CYP3a16 (39%) and blackswallow butterfly CYP6B3 (41%) P450 proteins. The CYP4C8 sequence contains variations in the 13-residue sequence characteristic of family 4 members, distinct from those seen for CYP4D1 and CYP4F family members. M. darwiniensis has been proposed as the ‘‘missing link between cockroaches and termites,’’ with the genus Mastotermes dating back some 20–40 million years. The phylogenetic distance between B. discoidalis and M. darwiniensis would suggest that CYP4C8 represents the most ancient of the CYP4 family members. q 1997 Academic Press Key Words: cytochrome P450 monooxygenase; isoptera; termite; metabolism; CYP4.

One of the most important enzyme families used by living organisms is the cytochrome P450 superfamily that is involved in oxidation of a wide range of endogenous and exogenous (xenobiotic) compounds. The number and diversity of this superfamily is such that more than 480 P450 genes and 22 pseudogenes have been 1 Author for correspondence: Dr. Patrick H. J. Falckh, Key Centre for Applied & Nutritional Toxicology, GPO Box 2467V, Melbourne, Victoria, Australia 3001. Fax.: (/61-3) 9663 6087; E-mail: p.falckh@ rmit.edu.au.

described in 85 eukaryote and 20 prokaryote species (1). In phytophagous insects, the diversification of the cytochrome P450 gene families is thought to be driven by selective pressure of secondary plant metabolites (2), which until recently had little experimental evidence. In some cases, the expression of specific cytochrome P450 isozymes are only observed after a challenge, or exposure, to specific chemicals; for example CYP6B1, a P450 isolated from the caterpillar Papilio polyxenes (3), has been shown to be only detectable in selective Papilionidae that have consumed plant matter containing the secondary metabolite xanthotoxin and is not constitutively expressed. Moreover, CYP6 family members have only been found in insects and are generally associated with insecticide resistance. The CYP4 family is an enzyme group that has been found in more than 10 different species, from rats, fish (4) and humans to cockroaches, mosquitos and flies (1). This family of P450 enzymes is believed to have originated more than 700 million years ago, prior to the divergence of invertebrates and vertebrates (5) and appears to be a commonality that links all species. These metabolising enzyme sequences have been classed as CYP4 family members on the similarity of a 13-amino acid residue that demonstrates 92-100% homology between all identified CYP4 family members (6). The fact that all living organisms originated from some historic serendipitous event suggests that a common enzyme family, for example CYP4, may be constitutively expressed and fundamental to the normal function of all living organisms. The wood consuming insect, Mastotermes darwiniensis, is the most destructive termite found in Australia and is generally confined to the northern tropical region of that continent. M. darwiniensis is the sole surviving member of the ancient termite family Mastotermiditae, and is considered a ‘living fossil’ due to its retention of primitive cockroach characteristics (7). Present findings in our laboratory (8) have indicated that this termite species has multiple P450 isozymes,

579

0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

AID

BBRC 7856

/

6942$$1361

12-02-97 03:55:53

bbrcg

AP: BBRC

Vol. 241, No. 2, 1997

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

although no genetic sequence data is available on any metabolising enzyme in termites. The fact that CYP4 sequences have been found in both insects and mammalian species, as well as regions of similarities between family 4 isozymes from cockroach (CYP4C1)(6), rat (CYP4A1)(9), human (CYP4B1) (10) and drosophila (CYP4D1 and CYP4D2)(11, 12), suggest that this class of P450 may also exist in the termite. Degenerate primers utilising the amino acid homology and uniqueness of this family of P450s have been designed which specifically amplify family 4 isozymes sequences using PCR methods. In this paper we present the isolation and sequence of a new cytochrome P450 cDNA from the termite, M. darwiniensis, using degenerate primers and RT/PCR. The similarities between this fragment and those on the genetic database, GenBank, defines this cDNA to belong to the cytochrome P450 IV gene family and has been designated CYP4C8. MATERIALS AND METHODS M. darwiniensis Froggatt (Isoptera: Mastoteritidae) were obtained from a colony in Northern Queensland and maintained at the CSIRO, Division of Forest Products, Clayton, Victoria, Australia. Only the worker class termites were used in this experiment, as they are the foragers and supplier of foodstuffs for the entire colony. Insects were frozen in liquid nitrogen , crushed and total RNA was isolated using the acid-guanidinium-phenol-chloroform (AGPC) method of Chomczynski and Sacchi (13). Total RNA was also extracted from the abdomen of a cockroach, Periplaneta americana, in a similar manner and used as a control tissue. Oligo(dT) primed total RNA was reverse-transcribed using MuLV reverse transcriptase and the resultant cDNA was PCR amplified with a set of degenerate primers. The forward primer targeted a conserved 13 amino acid region found in rat, human, cockroach and drosophila CYP4 sequences (accession numbers : M63798, X82029, M55719, X67645 and Z23005; respectively), upstream from the heme-binding cysteine (upstream primer: 5*-GGAAGTIGACACITTYATGTT-3*, where Y is T or C, and I is inosine). In contrast, the reverse primer was taken solely from a region of the cockroach CYP4C1, immediately downstream of the heme-binding cysteine (downstream primer : 5*-CGCAAGATGCTAGACAATAC-3*). The region downstream of the heme-binding region is exceptionally variant in the CY4 family which made the design of a degenerate reverse primer infeasible. cDNA was amplified in a reaction mix containing: 0.2 volumes of cDNA, 1.0 mM of each primer, 2.5U of Taq polymerase per 100 ml of reaction, 200 mM each dNTP and 2.0 mM MgCl2 in a final volume of either 20 or 50 ml. The PCR was performed for 30 cycles using an annealing temperature of 527C, which was earlier optimised for Mg2/ and temperature. PCR amplified cDNA was isolated directly from the PCR reaction by spin chromatography (ChromaSpin 200, Clontech) or from bands excised from agarose gels using the ‘freeze-squeeze method (14). Purified PCR products were directly sequenced using dye terminator cycle sequencing, performed on a GeneAmp 9600 thermal cycler (Perkin Elmer Corporation) with the class-specific CYP4 primers used to create the products and reagents from the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit. Northern blot analysis was performed as described in Feyereisen et al. (14) and probed with the reverse primer that was 5* end labelled with g-33P-dATP. Chromatograms of sequenced fragments were manually audited and the deduced amino acid sequence determined using computer

FIG. 1. PCR amplified products from the termite (T) and cockroach (C) separated on a 2% agarose gel stained with ethidium bromide. The expected product size is indicated (arrow) and a 100-bp ladder (L) was included for size determinations. M. darwiniensis (T) PCR product was of the expected size, however, P. americana (C) was approximately 200 bp smaller. The bands were excised from the gel and purified by spin chromatography for direct cycle sequencing.

programs on the Australian National Genomic Information System (ANGIS). The deduced sequence was BLASTP checked against the EMBL and GenBank databases and subsequent alignments with the deduced sequence performed using CLUSTALV (ANGIS).

RESULTS AND DISCUSSION The degenerate primers, designed from conserved regions between cockroach, B. discoidalis, rat, human and Drosophila melangaster CYP4 sequences, amplified a product from M. darwiniensis cDNA of approximately 495 bp; consistent with that calculated from the cockroach sequence. Although there is no intron-exon mapping of the CYP4C1 sequence, data from CYP4D2 would suggest that the 2 primers are in different exons and hence PCR products obtained from the cDNA would be significantly smaller than products obtained from genomic DNA; by at least 55 bases. Interestingly, this primer pair was not able to amplify a product of the expected size from the cockroach species, Periplaneta americana. The fact that several CYP4 PCR products, of comparable size, were amplifiable from both P. americana and M. darwiniensis (data not shown), using reverse primers that were designed specifically from Drosophila CYP4D1 (11) and CYP4D2 (12) sequences or from the tobacco hornworm (accession numbers L38670 and L38671), strongly indicated that there is considerable inter-, as well as, intra-species differences within the CYP4 family and that there are additional CYP4 family members in both the termite and cockroach species studied (Fig. 1). A 432-bp sequence, encoding a deduced 144 amino acid peptide, was derived from the PCR product and is

580

AID

BBRC 7856

/

6942$$1361

12-02-97 03:55:53

bbrcg

AP: BBRC

Vol. 241, No. 2, 1997

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 2. The nucleotide and deduced amino acid (single-letter code) sequence derived from the open reading frame of the PCR generated P450 cDNA from the termite, M. darwiniensis.

shown in Fig. 2. The deduced sequence clearly demonstrated the P450 signature, FxxGxxCxG (1), indicating that the sequence encoded for a P450 isozyme, and the residues Gly-3, Glu-60, Pro-63, Pro-109, Phe-133, Gly136, Cys-140 and Gly-142 are invariant in all other aligned P450 proteins (15). The termite sequence data was compared to other sequences maintained on the GenBank data base, using BLASTP and FASTA which resulted in 828 sequences being considered to have some homology to the deduced amino acid sequence from the termite. The 100-most similar amino acid sequences, rated by the highest similarity scores, were all P450 sequences from either the CYP3A or CYP4 families, with the highest score associated with the sequences from the cockroach (6) which was used to design the primers. The alignment of the M. darwiniensis and B. discoidalis sequences (Fig. 3) demonstrated that 68% of the sequences were identical, with a further 18.8% representing conserved changes. Alignment of the termite sequence to other CYP4 sequences demonstrated lower identities of 39% and 42% to either CYP4A1 or CYP4B1, respectively, consistent with the low identity demonstrated between CYP4C1 and other CYP4 family

members (6). This data suggested that the termite sequence is a member of the CYP4 family, and possibly a subfamily of 4C. The sequence was forwarded to the P450 Nomenclature Committee, who confirmed (personal communication) that the sequence belonged to the CYP4C family of cytochrome P450 isozymes and phylogenetically was positioned between the sequences CYP4C1 and CYP4C6. The sequence has been given the name of CYP4C8 and has been entered into the GenBank data base (accession number U77126). The ancient CYP4 family of P450 isozymes has been considered to be closely related to the CYP3 and CYP6 families (16). An alignment of the 144 deduced amino acids of CYP4C8 with mouse CYP3a16 (17) and blackswallow butterfly CYP6B3 (accession number U25819) indicated homologies of 38% and 41%, respectively. The Northern blot of M. darwiniensis, utilising the endlabelled CYP4 primer as a probe, identified a single band at 1.37 kilobases, however, this size for the mRNA is not consistent with other CYP4 family members who have been shown to have larger mRNA transcripts greater than 1.48 kilobases. Bradfield et al. (6) has suggested that the strict conservation of a 13-amino acid peptide (EVDTFMFEG-

581

AID

BBRC 7856

/

6942$$1361

12-02-97 03:55:53

bbrcg

AP: BBRC

Vol. 241, No. 2, 1997

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 3. The deduced single-letter amino acid sequence from M. darwiniensis is shown (accession number U77126), aligned with the CYP4C1 cockroach sequence from B. discoidalis (6). The termite sequence demonstrated 68% identical homology (*) with the cockroach sequence, which increased to 86.8% when conserved changes (r) were included. The required P450 signature (underlined) and residues invariant in all cytochrome P450 proteins (boxed) are illustrated (1). The deduced position of an intron is indicated (double underlined) and was determined from mosquito CYP4 sequences (23).

HDTT) found around position 315 of CYP4 family members 4A1, 4B1 and 4C1 defines the CYP4 family and may indicate that these enzymes act on closely related substrates. In contrast, CYP4C8 has 3 variations in this 13-amino acid peptide. Two conserved changes were found in the M. darwiniensis sequence, where Gln-4 replaces His-315, and Glu-5 replaces Asp-316, as defined in the B. discoidalis, as well as an outright replacement of Thr-317 with lysine. Two other CYP4 families also display variations in the 13-amino acid peptide; CYP4F, defined as leukotriene B4 v-hydroxylases (18), has asparagine replace the valine, and CYP4D1 which has the replacement of Glu-316 by lysine (12). The suggestion that this 13-amino acid peptide defines the substrate region of the CYP4 enzymes would appear to be incorrect. This is supported by examining the 4A and 4B subfamilies. CYP4A and CYP4B have identical 13-amino acid peptides, however, 4B enzymes (10, 19) are not able to catalyse the same reactions as 4A enzymes (20). As additional members of the CYP4 family are discovered, further variations in the 13-amino acid peptide may be demonstrated, however, this region still defines the CYP4 family as no other family appears to contain a similar configuration of residues at this position. While the function of the CYP4C8 enzyme is unknown, the relatively high homology between the cockroach and termite sequence would suggest that the role of the termite enzyme maybe similar to that suggested for CYP4C1; namely an enzyme associated with energy substrate mobilization (6). In particular, Bradfield et al. (6) suggests that CYP4C1 mediates the action of hypertrehalose hormone, a hormone that stimulates glycogenolysis and precursers for the synthesis of trehalose, in the fat body of insects. In termites, lipids, in the form of triglycerides, are the most important longterm energy reserve (21). M. darwiniensis is an ancient species and is more closely related to P. americana than B. discoidalis (22),

however, the similarities between CYP4C1 and CYP4C8 may reflect possible convergence of the CYP4C family in the two more distantly related species (M. darwiniensis and P. americana) as a CYP4C product of the expected size was not able to be amplified from P. americana. As such, the CYP4C8 sequence represents a more ancient sequence of the CYP4 family. Comparisons of sequences from present day M. darwiniensis, to that of DNA sequenced from fossilised specimens of M. electrodominicus, preserved in 25-30 million year old amber (7), would afford science an unique opportunity to directly analyse changes of detoxication enzymes, in the same genus. ACKNOWLEDGMENTS The authors would like to thank Dr. J. R. J. French for the collection and supply of termites, and Dr. David Nelson, University of Tennessee, Memphis, for naming the sequence. Dr. Falckh is supported by the RMIT postdoctoral research program. The support of the Faculty of Biomedical & Health Sciences, RMIT is gratefully acknowledged.

REFERENCES 1. Nelson, D. R., Koymanis, L., Kamataki, T., Stegeman, J. J., Feyereisen, R., Waxman, D. J., Waterman, M. R., Gotoh, O., Coon, M. J., Estabrook, R. W., Gunsalus, I. C., and Nebert, D. W. (1996) Pharmacogenetics 6, 1–42. 2. Tomita, T., and Scott, J. G. (1995) Insect Biochem. Molec. Biol. 25, 275–283. 3. Cohen, M. B., Schuler, M. A., and Berenbaum, M. R. (1992) Proc. Natl. Acad. Sci. USA 89, 10920–10924. 4. Falckh, P. H. J., Wu, Q. K., and Ahokas, J. P. (1997) Biochem. Biophys. Res Comm. 236, 302–305. 5. Nelson, D. R., and Strobel, H. W. (1987) Mol. Biol. Evol. 4, 572– 593. 6. Bradfield, J. Y., Lee, Y.-H., and Keeley, L. L. (1991) Proc. Natl. Acad. Sci. USA 88, 4558–4562. 7. DeSalle, R., Gatesy, J., Wheeler, W., and Grimaldi, D. (1992) Science 257, 1933–1936.

582

AID

BBRC 7856

/

6942$$1361

12-02-97 03:55:53

bbrcg

AP: BBRC

Vol. 241, No. 2, 1997

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

8. Haritos, V. S., French, J. R. J., and Ahokas, J. T. (1994) Insect Biochem. Mol. Biol. 24, 929–935. 9. Hardwick, J. P., Song, B.-J., Huberman, E., and Gonzalez, F. J. (1987) J. Biol. Chem. 262, 801–810. 10. Nhamburo, P. T., Gonzalez, F. J., McBride, W., Gelboin, H. V., and Kimura, S. (1989) Biochemistry 28, 8060–8066. 11. Gandhi, R., Varak, E., and Goldberg, M. L. (1992) DNA Cell Biol. 11, 397–404. 12. Frolov, M. V., and Alatortsev, V. E. (1994) DNA Cell Biol. 1313, 663–668. 13. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156– 159. 14. Thuring, R. W. J., Sanders, J. P. M., and Borst, P. (1975) Anal. Biochem. 66, 213–220. 15. Feyereisen, R., Koener, J. F., Farnsworth, D. E., and Nebert, D. W. (1989) Proc. Natl. Acad. Sci. USA 86, 1465–1469. 16. Nebert, D. W., Nelson, D. R., Coon, M. J., Estabrook, R. W., Feyereisen, R., Fujii-Kuriyama, Y., Gonzalez, F. J., Guengerich,

17. 18. 19. 20.

21.

22. 23.

F. P., Gunsalus, I. C., Johnson, E. F., Loper, J. C., Sato, R., Waterman, M. R., and Waxman, D. J. (1991) DNA Cell Biol. 10, 1– 14. Itoh, S., Satoh, M., Abe, Y., Hashimoto, H., Yanagimoto, T., and Kamataki, T. (1994) Eur. J. Biochem. 226, 877–882. Kikuta, Y., Kusunose, M., Kondo, T., Yamamoto, S., Kinoshita, H., and Kusunose, M. (1994) FEBS Lett. 348, 70–74. Gasser, R., and Philpot, R. M. (1989) Mol. Pharmacol. 35, 617– 625. Yokotani, N., Bernhardt, R., Sogawa, K., Kusunose, E., Kusunose, M., and Fujii-Kuriyama, Y. (1989) J. Biol. Chem. 264, 21665–21669. LaFage, J. P., and Nutting, W. L. (1978) in Production Ecology of Ants and Termites (Brian, M. V., Ed.), Cambridge Univ. Press, Cambridge, UK. Kambhampati, S. (1995) Proc. Natl. Acad. Sci. USA 92, 2017– 2020. Scott, J. A., Collins, F. H., and Feyereisen, R. (1994) Biochem. Biophys. Res. Comm. 205, 1452–1459.

583

AID

BBRC 7856

/

6942$$1361

12-02-97 03:55:53

bbrcg

AP: BBRC