Preferential Expression of Biotransformation Enzymes in the Olfactory Organs of Drosophila melanogaster, the Antennae

Biotransformation enzymes have been found in the olfactory epithelium of vertebrates. We now show that inDrosophila melanogaster, a UDP-glycosyltransferase (UGT), as well as a short chain dehydrogenase/reductase and a cytochrome P450 are expressed specifically or preferentially in the olfactory organs, the antennae. The evolutionarily conserved expression of biotransformation enzymes in olfactory organs suggests that they play an important role in olfaction. In addition, we describe fiveDrosophila UGTs belonging to two families. All five UGTs contain a putative transmembrane domain at their C terminus as is the case for vertebrate UGTs where it is required for enzymatic activity. The primary sequence of the C terminus, including part of the transmembrane domain, differs between the two families but is highly conserved not only within each Drosophila family, but also between the members of one of the Drosophila families and vertebrate UGTs. The partial overlap of the conserved primary sequence with the transmembrane domain suggests that this part of the protein is involved in specific interactions occurring at the membrane surface. The presence of different C termini in the two Drosophilafamilies suggests that they interact with different targets, one of which is conserved between Drosophila and vertebrates. Biotransformation enzymes have been found in the olfactory epithelium of vertebrates. We now show that inDrosophila melanogaster, a UDP-glycosyltransferase (UGT), as well as a short chain dehydrogenase/reductase and a cytochrome P450 are expressed specifically or preferentially in the olfactory organs, the antennae. The evolutionarily conserved expression of biotransformation enzymes in olfactory organs suggests that they play an important role in olfaction. In addition, we describe fiveDrosophila UGTs belonging to two families. All five UGTs contain a putative transmembrane domain at their C terminus as is the case for vertebrate UGTs where it is required for enzymatic activity. The primary sequence of the C terminus, including part of the transmembrane domain, differs between the two families but is highly conserved not only within each Drosophila family, but also between the members of one of the Drosophila families and vertebrate UGTs. The partial overlap of the conserved primary sequence with the transmembrane domain suggests that this part of the protein is involved in specific interactions occurring at the membrane surface. The presence of different C termini in the two Drosophilafamilies suggests that they interact with different targets, one of which is conserved between Drosophila and vertebrates. short chain dehydrogenase/reductase UDP-glycosyltransferase open reading frame expressed sequence tag All organisms live in environments that contain potentially harmful chemicals, both natural and man-made. Extensive studies of detoxification in the vertebrate liver provide a framework to the study of detoxification mechanisms in other systems (1Tephly T.R. Burchell B. Trends Pharmacol. Sci. 1990; 11: 276-279Abstract Full Text PDF PubMed Scopus (247) Google Scholar, 2Brierley C.H. Burchell B. Bioessays. 1993; 15: 749-754Crossref PubMed Scopus (51) Google Scholar, 3Meyer U.A. J. Pharmacokinet. Biopharm. 1996; 24: 449-459Crossref PubMed Scopus (191) Google Scholar). Detoxification often occurs in two phases; in phase I, the initial compound is transformed into a more reactive species. A variety of different chemical transformations are involved, including redox reactions catalyzed by enzymes of the cytochrome P450 superfamily (4Nebert D.W. Gonzalez F.J. Annu. Rev. Biochem. 1987; 56: 945-993Crossref PubMed Scopus (1416) Google Scholar, 5Guengerich F.P. Sci. Am. 1993; 81: 440-447Google Scholar) and members of the short chain dehydrogenase/reductase (SDR)1 family (6Maser E. Opperman U.C.T. Eur. J. Biochem. 1997; 249: 365-369Crossref PubMed Scopus (58) Google Scholar). Phase II reactions consist in the addition, either to a product of a phase I reaction or directly to many toxic chemicals, of a highly polar group such as UDP-glucuronosyl (catalyzed by UDP-glucuronosyltransferases) (2Brierley C.H. Burchell B. Bioessays. 1993; 15: 749-754Crossref PubMed Scopus (51) Google Scholar) or glutathione (catalyzed by glutathione S-transferases (7Shen H. Kauvar L. Tew K.D. Oncol. Res. 1997; 9: 295-302PubMed Google Scholar). Products of phase II reactions are hydrophilic; they can no longer cross membranes and are eliminated by secretion. In addition to the elimination of environmental toxins, phase I and II biotransformation enzymes participate in the removal of toxic side products of normal metabolism (e.g. bile acids), participate in drug clearance, and play an important role in the synthesis of hormones such as prostaglandins and some steroids (3Meyer U.A. J. Pharmacokinet. Biopharm. 1996; 24: 449-459Crossref PubMed Scopus (191) Google Scholar). Finally, the involvement of these enzymes in the production of carcinogens (8Nebert D.W. McKinnon R.A. Puga A. DNA Cell Biol. 1996; 15: 273-280Crossref PubMed Scopus (299) Google Scholar), drug clearance, and some hereditary diseases (9Owens I.S. Ritter J.K. Yeatman M.T. Chen F. J. Pharmacokinet. Biopharm. 1996; 24: 491-508Crossref PubMed Scopus (23) Google Scholar) makes an understanding of their function important for human health. Biotransformation enzymes related to those found in vertebrates have also been found in insects and are likely to play equally important roles. Cytochrome P450s and glutathione S-transferases in particular have been implicated in insect resistance to pesticides (10Feyereisen R. Toxicol. Lett. (Shannon). 1995; 82: 83-90Crossref PubMed Scopus (205) Google Scholar). UDP-glucuronosyltransferases are part of a superfamily of UDP-glycosyltransferases (UGTs) present in plants, animals, and bacteria (11Mackenzie P.I. Owens I.S. Burchell B. Bock K.W. Bairoch A. Belanger A. Fournet-Gigleux S. Green M. Hum D.W. Iyanagi T. Lancet D. Pharmacogenetics. 1997; 7: 255-269Crossref PubMed Scopus (1015) Google Scholar). These enzymes transfer the sugar moiety of UDP-glucose, UDP-glucuronic acid, UDP-galactose, or UDP-xylose to a variety of hydrophobic substrates (11Mackenzie P.I. Owens I.S. Burchell B. Bock K.W. Bairoch A. Belanger A. Fournet-Gigleux S. Green M. Hum D.W. Iyanagi T. Lancet D. Pharmacogenetics. 1997; 7: 255-269Crossref PubMed Scopus (1015) Google Scholar). Insects contain UGT activities that can use UDP-glucose but not UDP-glucuronic acid as a glycosyl donor (12Dutton G.J. Comp. Biochem. Physiol. 1962; 7: 39-46Crossref Scopus (15) Google Scholar, 13Ahmad S.A. Hopkins T.L. Arch. Insect Biochem. Physiol. 1992; 21: 207-224Crossref Scopus (30) Google Scholar, 14Rausel C. Llorca J. Real M.D. Arch. Insect Biochem. Physiol. 1997; 34: 347-358Crossref PubMed Scopus (20) Google Scholar). Although no molecular information on any insect UGT was available until this work, baculoviruses infecting several species of moths have been shown to encode ecdysteroid UDP-glucosyltransferases (15O'Reilly D.R. Miller L.K. Science. 1989; 245: 1110-1112Crossref PubMed Scopus (319) Google Scholar, 16Hu Z.H. Broer R. Westerlaken J. Martens J.W.M. Jin F. Jehle J.A. Wang L.M. Vlak J.M. Virus Res. 1997; 47: 91-97Crossref PubMed Scopus (32) Google Scholar). These viral enzymes specifically inactivate ecdysteroids, the molting hormones of the infected hosts, and thus prolong the larval stage permissive to viral replication. ecdysteroid UDP-glucosyltransferases lack a C-terminal transmembrane domain and are secreted in the hemolymph where ecdysone is present (15O'Reilly D.R. Miller L.K. Science. 1989; 245: 1110-1112Crossref PubMed Scopus (319) Google Scholar). Here we report that, in Drosophila, several phase I and II biotransformation enzymes are expressed preferentially in the olfactory organs, the antennae. This observation is reminiscent of the preferential or exclusive expression of a cytochrome P450, UGT, and glutathione S-transferase in the vertebrate olfactory epithelium (17Lazard D. Tal N. Rubinstein M. Khen M. Lancet D. Zupko K. Biochemistry. 1990; 29: 7433-7440Crossref PubMed Scopus (83) Google Scholar, 18Lazard D. Zupko K. Poria Y. Nef P. Lazarovits J. Horn S. Khen M. Lancet D. Nature. 1991; 349: 790-793Crossref PubMed Scopus (202) Google Scholar, 19Dear T.N. Campbell K. Rabbitts T.H. Biochemistry. 1991; 30: 10376-10382Crossref PubMed Scopus (103) Google Scholar, 20Ben-Arie N. Khen M. Lancet D. Biochem. J. 1993; 292: 379-384Crossref PubMed Scopus (67) Google Scholar). The presence of these enzymes in the olfactory organs of such evolutionarily distant organisms supports the notion that they play an important role in olfaction. In addition, the availability of the first sequences of UGTs from insects sheds light on the structure and function of the C-terminal domain of vertebrate UGTs. Partial cDNA clones for AntP450,AntDH, and DmeUgt35b were initially found through random sequencing of clones in an antennae-minus-heads subtracted cDNA library that was described previously (21Pikielny C.W. Hasan G. Rouyer F. Rosbash M. Neuron. 1994; 12: 39-45Abstract Full Text PDF Scopus (311) Google Scholar). All cDNA sequences discussed in this paper were obtained from full-length cDNA clones isolated by using the partial clones as probes to screen an appendage cDNA library in Lambda-ZAP (Stratagene). Appendage RNA was generated from poly(A)+ RNA isolated from an appendage fraction (see below and Ref. 22Oliver D.V. Philips J.P. Drosophila Information Service. 1970; 45: 58Google Scholar). Total RNA for Northern blots was isolated either from hand-dissected antennae or legs or from mass-produced body fractions generated as follows(22). Frozen flies are vortexed, and the resulting body parts are then sieved to yield three fractions: appendages (antennae, legs, and wings), heads (without antennae), and bodies (abdomen and thorax, decapitated and without legs or wings). Because all the proteins under study belong to multigene families, the probes used were first tested on Southern blots under identical conditions to ensure that there was no detectable cross-reactivity to related genes (not shown). The probes used are indicated in the legend to Fig. 1. Sequences were assembled and analyzed using Wisconsin Package Version 9.1, Genetics Computer Group, Madison, WI. Data base searches were performed using BLAST (23Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60524) Google Scholar) both on the Berkeley Drosophila Genome Project and National Center for Biotechnology Information www servers (http://www.fruitfly.org/blast/andhttp://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast?Jform=0, respectively). The central portion of the DmeUgt35a cDNA is represented by nucleotides number 1–324 of P1 clone DS07339 that has been mapped by the BDGP to 86D5–10. Similarly, the 3′ end of DmeUgt35bis partially represented in preliminary sequence (nucleotides 1–326) of DS08785 mapped to 86D1-D2. AntP450 is a partial cDNA sequence identical to EST number GH06928 and with significant sequence similarity to the C terminus of the cyp6 family of cytochrome P450s (24Cohen M.B. Koener R. 1994; PubMed Scopus (51) Google Scholar). The three members of the family from of sequences from the The sequences the three open reading are present at nucleotides number to of P1 clone for of for and nucleotides to of for we a likely at In the case of of a in a of at in the of an with similarity to the (see The in the sequence by a sequencing or a resulting in a In either of the sequence a UGT with similarity to the other proteins open reading frame and likely a UGT, it no longer in present the of this we the to the that the of is not to the the domain structure of Drosophila UGTs that are discussed in the The sequences of the three discussed have been in the base and their are as and UGTs are to the by the UGT and are in the We have previously described a subtracted cDNA library in expressed specifically or preferentially in the antennae of Drosophila of a number of those to the of several putative proteins with expression on the of the a role for proteins in olfactory (21Pikielny C.W. Hasan G. Rouyer F. Rosbash M. Neuron. 1994; 12: 39-45Abstract Full Text PDF Scopus (311) Google Scholar). Here we report that, in addition to of library has to the of for a cytochrome P450 a UGT below for of the and a short chain dehydrogenase/reductase The proteins by these three are related to vertebrate enzymes involved in In addition to DmeUgt35b isolated from a cDNA was isolated from an appendage cDNA library by to DmeUgt35b (see of expression was performed by Northern blots using probes specific for and In case the probes were generated from sequences that show similarity with other genes of the family and the lack of was on Southern blots performed under identical conditions not shown). The Northern shown in from two of as indicated the parts were into three fractions (see appendages legs, and wings), heads (without and bodies and without to between different we and legs from of is to and within appendages it is in in AntP450 and DmeUgt35b are more they are in heads and bodies at in both genes are expressed at in the In to these three DmeUgt35a expression in the in Although involved in can found on the legs, and of Drosophila, the the of olfactory Cell Res. 1994; PubMed Scopus Google Scholar). The preferential expression of and AntP450 in the is of a role in olfaction. expression we used in on of heads and antennae the genes discussed expression was by this likely of expression not shown). with the Northern in is to the no expression is in the or in the (not shown). This observation supports a role for in the not contain Cell Res. 1994; PubMed Scopus Google Scholar). the expression and not in with several each of which is to a of with a on the (21Pikielny C.W. Hasan G. Rouyer F. Rosbash M. Neuron. 1994; 12: 39-45Abstract Full Text PDF Scopus (311) Google Scholar, P. J. Biol. 1994; Full Text PDF PubMed Google D. R.A. J. J. 1997; PubMed Google Scholar). S. A. G. P. G. A. and C. W. in The initial clone was used as a to a full-length cDNA clone from an appendage cDNA library (see A has sequence similarity to the members of a family of H. B. M. S. R. J. D. Biochemistry. 1995; 34: PubMed Scopus Google Scholar) found in organisms from and to H. B. M. S. R. J. D. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). The of sequence is as is in this family of proteins sequence we have found is from the in Fig. which has with has all the that have been to important for the function of In the to the terminus to a for either or In addition, the for can found at through and is the likely of the of N. S. J. R. R. Lett. 1997; PubMed Scopus Google Scholar). Although are some members of this family are or K. J. 1995; 9: PubMed Scopus Google Scholar). to the case of the in Fig. there is no sequence in the Because the short sequence the in clone not contain any we the that we are some sequences that for a the presence of an at the identical as it is found in many suggests that we have the terminus and that is a the appendage library with partial clone we found two of clones that at different Southern and sequence that these to two different each by a which we DmeUgt35a and (see the under for a of this We have mapped both sequences by in to in the Drosophila that these two genes have their in a the Berkeley and have parts of the to and for the a and We have also found that several other likely sequences are present in the Drosophila These three DNA sequences full-length that three members of a family of Drosophila and (see below for an of the other likely are represented by partial cDNA sequences or and The first are sequences for termini the one is a 3′ sequence for a C terminus In we describe or putative Drosophila UGTs each of the has only been from one identical to one of the other The two as well as the three sequences to they with and end with at that those for this family of genes (see for of DNA sequence In to the use of for the of as is the case of the (2Brierley C.H. Burchell B. Bioessays. 1993; 15: 749-754Crossref PubMed Scopus (51) Google Scholar), we have found no of and an is present in only one of the three sequences (see and have C-terminal to those of vertebrate UGTs but different from those and UGTs and can with all fiveDrosophila UGTs by to a where the sequences of the two families The three were generated using but the sequences shown are and their within the has been and not to similarity in five of three of and two of three for the and The transmembrane domain indicated for members of the family and vertebrate is the one for vertebrate P.I. J. Biol. Full Text PDF PubMed Google Scholar). for members of families are with the that the transmembrane the sequence not shown). was by the and using the structure of Genetics Computer the legend to Fig. for UGT and of the similarity between the five is found a sequence present in all UGTs and by the in which all that can at a are (11Mackenzie P.I. Owens I.S. Burchell B. Bock K.W. Bairoch A. Belanger A. Fournet-Gigleux S. Green M. Hum D.W. Iyanagi T. Lancet D. Pharmacogenetics. 1997; 7: 255-269Crossref PubMed Scopus (1015) Google Scholar). The presence of this sequence supports the of these five proteins as UGTs In addition, the five contain C-terminal hydrophobic by several (see below and Fig. In the case of vertebrate sequences have been as a transmembrane domain and a sequence that in are for the of the protein to the membrane (1Tephly T.R. Burchell B. Trends Pharmacol. Sci. 1990; 11: 276-279Abstract Full Text PDF PubMed Scopus (247) Google Scholar) and are for enzymatic R. G. P.I. DNA Cell Biol. 1996; 15: PubMed Scopus (32) Google Scholar). ecdysteroid which are and secreted in the hemolymph of the lack such C-terminal transmembrane (15O'Reilly D.R. Miller L.K. Science. 1989; 245: 1110-1112Crossref PubMed Scopus (319) Google Scholar). Finally, at of the putative Drosophila UGTs a of similarity at their terminus, the G. J. Biol. PubMed Scopus Google Scholar, G. J. Biol. PubMed Scopus Google Scholar) In the case of the putative only the C-terminal sequence is In vertebrate UGTs this of the is involved in the of R. P.I. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), which the of the In at one case a has enzymatic activities that from those of either S. Y. Iyanagi T. Biochemistry. 1997; PubMed Scopus Google Scholar), that the of different into provide In with the UGT (11Mackenzie P.I. Owens I.S. Burchell B. Bock K.W. Bairoch A. Belanger A. Fournet-Gigleux S. Green M. Hum D.W. Iyanagi T. Lancet D. Pharmacogenetics. 1997; 7: 255-269Crossref PubMed Scopus (1015) Google Scholar), we have the two we have to a family, The other three full-length sequences found through the into a families the for protein families within a family and between different families, not shown). Finally, the C-terminal sequence of suggests that it is a of the family the of the five UGTs only as to one of these two families, or their acid present in all Drosophila proteins in the first as well as in the C-terminal of the the sequence the first the of the proteins are highly (not as is the case for vertebrate to different the C-terminal of all proteins are there is a of this similarity at their C a highly conserved the sequences of the two families each a different C-terminal domain putative transmembrane and sequences each family, there is a of sequence the of sequence similarity with the likely transmembrane for members of the P.I. J. Biol. Full Text PDF PubMed Google Scholar) as well as families of the first in the putative transmembrane domain are identical and human and of is for the This of suggests specific and different for the C-terminal of the two Drosophila families. The that, as in the olfactory epithelium of vertebrates (17Lazard D. Tal N. Rubinstein M. Khen M. Lancet D. Zupko K. Biochemistry. 1990; 29: 7433-7440Crossref PubMed Scopus (83) Google Scholar, 18Lazard D. Zupko K. Poria Y. Nef P. Lazarovits J. Horn S. Khen M. Lancet D. Nature. 1991; 349: 790-793Crossref PubMed Scopus (202) Google Scholar, 19Dear T.N. Campbell K. Rabbitts T.H. Biochemistry. 1991; 30: 10376-10382Crossref PubMed Scopus (103) Google Scholar, 20Ben-Arie N. Khen M. Lancet D. Biochem. J. 1993; 292: 379-384Crossref PubMed Scopus (67) Google Scholar), several enzymes are expressed specifically or at in the antennae This conserved expression of enzymes in olfactory organs for an important function in olfaction. a role is also with the presence in the antennae of an involved in cytochrome P450 P450 F. J. 1997; PubMed Scopus Google Scholar). In a highly case of olfactory moths are to many by between two of a and from side to side as as the is no longer J. Biol. Google Scholar). The to without a that the of the olfactory organs short to the of the on these have for and found enzymes that can specifically in the antennae of several species of moths L.M. Sci. S. A. 82: PubMed Scopus Google Scholar, R. J. Biol. 1990; Full Text PDF PubMed Google Scholar). In vertebrate a role has been to biotransformation enzymes that are for their role in detoxification in the The sequence of that toxic involved in of olfactory with this an UGT, more liver UGTs D. Zupko K. Poria Y. Nef P. Lazarovits J. Horn S. Khen M. Lancet D. Nature. 1991; 349: 790-793Crossref PubMed Scopus (202) Google Scholar). A function for these enzymes in the of olfactory organs from environmental to which they by preferentially Although and are not exclusive and any protein involved in the expression of each the to either Because detoxification occurs in many organs, proteins expression is highly specific to the such as and are likely to involved in the other proteins that have more expression such as participate in We report that the Drosophila at different UGTs. The five genes for which sequences are available contain the of this superfamily and thus the first UGTs from any insect other ecdysteroid UDP-glucosyltransferases from baculoviruses (11Mackenzie P.I. Owens I.S. Burchell B. Bock K.W. Bairoch A. Belanger A. Fournet-Gigleux S. Green M. Hum D.W. Iyanagi T. Lancet D. Pharmacogenetics. 1997; 7: 255-269Crossref PubMed Scopus (1015) Google Scholar). In addition to the similarity to UGTs in a variety of and vertebrate proteins have at their C terminus a transmembrane domain by a sequence of several (1Tephly T.R. Burchell B. Trends Pharmacol. Sci. 1990; 11: 276-279Abstract Full Text PDF PubMed Scopus (247) Google Scholar, P.I. J. Biol. Full Text PDF PubMed Google Scholar). Although this domain is from ecdysteroid UDP-glucose and some UGTs (11Mackenzie P.I. Owens I.S. Burchell B. Bock K.W. Bairoch A. Belanger A. Fournet-Gigleux S. Green M. Hum D.W. Iyanagi T. Lancet D. Pharmacogenetics. 1997; 7: 255-269Crossref PubMed Scopus (1015) Google Scholar), in either the transmembrane domain or the sequence of vertebrate and R. G. P.I. DNA Cell Biol. 1996; 15: PubMed Scopus (32) Google Scholar). is the of primary sequence between the Drosophila members of the family and vertebrate sequences in a to and partially with the putative transmembrane domain and to the human a acid for the a and respectively). In addition, the sequences of members of the family are different from those of the family in this they are also highly conserved within this family This of primary sequences suggests that the C-terminal of UGTs are involved in specific interactions at the membrane that between and the vertebrate enzymes on one and the members of the family on the these the of the two of proteins have some In all for the two at and the of sequence are which are by a of hydrophobic likely to part of the transmembrane domain P.I. J. Biol. Full Text PDF PubMed Google and Fig. In the case of a three acid to and and the putative transmembrane domain at These that the sequences the two different of C-terminal have and interact with related is the function of these C-terminal The primary sequence within each family suggests it involved in an with protein that occurs at in part within the The enzymatic reactions catalyzed by UGTs in the of the and are on specific that of nucleotides into this N. Biochem. J. 1997; PubMed Scopus Google Scholar). is that the C-terminal domain of UGTs in interactions with specific to the of these enzymes for different glycosyl of membranes with not to proteins with in the C-terminal domain R. G. P.I. DNA Cell Biol. 1996; 15: PubMed Scopus (32) Google Scholar), only Although of the have not any UGT from any with a C-terminal domain to that of the family, the sequence of the human such this domain the of the of insects and it an domain and a for The availability of the genes for all these enzymes inDrosophila the of their involvement in as well as a of the function of the UGT C-terminal using both and We and for on the and also to Rosbash in this was