Promoter Sequences of the Putative Anopheles gambiae Apyrase Confer Salivary Gland Expression in Drosophila melanogaster
2000; Elsevier BV; Volume: 275; Issue: 31 Linguagem: Inglês
10.1074/jbc.m909547199
ISSN1083-351X
AutoresFabrizio Lombardo, Manlio Di Cristina, Lefteris Spanos, Christos Louis, M. Cóluzzi, Bruno Arcà,
Tópico(s)Insect symbiosis and bacterial influences
ResumoThe saliva of blood-feeding arthropods contains an apyrase that facilitates hematophagy by inhibiting the ADP-induced aggregation of the host platelets. We report here the isolation of a salivary gland-specific cDNA encoding a secreted protein that likely represents the Anopheles gambiae apyrase. We describe also two additional members of the apyrase/5′-nucleotidase family. The cDNA corresponding to the AgApyL1 gene encodes a secreted protein that is closely related in sequence to the apyrase of the yellow fever mosquito, Aedes aegypti, and whose expression appears enriched in, but not restricted to, female salivary glands. The AgApyL2 gene was found searching anA. gambiae data base, and its expression is restricted to larval stages. We isolated the gene encoding the presumed A. gambiae apyrase (AgApy) and we tested its putative promoter for the tissue-specific expression of the LacZgene from Escherichia coli in transgenic Drosophila melanogaster. All the transgenic lines analyzed showed a weak but unambiguous staining of the adult glands, indicating that some of the salivary gland-specific transcriptional regulatory elements are conserved between the malaria mosquito and the fruit fly. The availability of salivary gland-specific promoters may be useful both for studies on vector-parasite interactions and, potentially, for the targeted tissue-specific expression of anti-parasite genes in the mosquito. The saliva of blood-feeding arthropods contains an apyrase that facilitates hematophagy by inhibiting the ADP-induced aggregation of the host platelets. We report here the isolation of a salivary gland-specific cDNA encoding a secreted protein that likely represents the Anopheles gambiae apyrase. We describe also two additional members of the apyrase/5′-nucleotidase family. The cDNA corresponding to the AgApyL1 gene encodes a secreted protein that is closely related in sequence to the apyrase of the yellow fever mosquito, Aedes aegypti, and whose expression appears enriched in, but not restricted to, female salivary glands. The AgApyL2 gene was found searching anA. gambiae data base, and its expression is restricted to larval stages. We isolated the gene encoding the presumed A. gambiae apyrase (AgApy) and we tested its putative promoter for the tissue-specific expression of the LacZgene from Escherichia coli in transgenic Drosophila melanogaster. All the transgenic lines analyzed showed a weak but unambiguous staining of the adult glands, indicating that some of the salivary gland-specific transcriptional regulatory elements are conserved between the malaria mosquito and the fruit fly. The availability of salivary gland-specific promoters may be useful both for studies on vector-parasite interactions and, potentially, for the targeted tissue-specific expression of anti-parasite genes in the mosquito. base pair(s) reverse transcription polymerase chain reaction Dulbecco's modified Eagle's medium 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside untranslated region group of overlapping clones The salivary glands of blood-sucking arthropods secrete a complex array of specific factors with vasodilatory, anti-clotting, and anti-platelet activities that assist the mosquito during blood feeding. Since hematophagy has arisen independently several times in insects, even within the same order, a large variety of different molecules have evolved to accomplish the same or similar function (1Ribeiro J.M.C. Annu. Rev. Entomol. 1987; 32: 463-478Crossref PubMed Scopus (448) Google Scholar, 2James A.A. Bull. Inst. Pasteur. 1994; 92: 133-150Google Scholar, 3Law J.H. Ribeiro J.M.C. Wells M.A. Annu. Rev. Biochem. 1992; 61: 87-112Crossref PubMed Scopus (149) Google Scholar, 4Ribeiro J.M.C. Infect. Agents Dis. 1995; 4: 143-152PubMed Google Scholar). Remarkably diverse substances act as vasodilators in the saliva of distinct arthropod species; they include prostaglandins in ticks, nitric oxide in the bugs Rhodnius prolixus and Cimex lectularius, peptides such as tachykinins or maxadilan in the mosquito Aedes aegypti and in the sandfly Lutzomya longipalpis, respectively, and the salivary peroxidase/catechol oxidase in the mosquito Anopheles albimanus (4Ribeiro J.M.C. Infect. Agents Dis. 1995; 4: 143-152PubMed Google Scholar, 5Champagne D.E. Parasitol. Today. 1994; 10: 430-433Abstract Full Text PDF PubMed Scopus (53) Google Scholar, 6Ribeiro J.M.C. Valenzuela J.G. J. Exp. Biol. 1999; 202: 809-816Crossref PubMed Google Scholar). Similarly, partly as a consequence of their different feeding habits, a multitude of different anticoagulants is found in different blood-eating species (7Stark K.R. James A.A. Parasitol. Today. 1996; 12: 430-437Abstract Full Text PDF PubMed Scopus (45) Google Scholar). In mosquitoes, thrombin and Factor Xa are the preferential targets within the blood coagulation cascade; anophelines produce anti-thrombin activities, whereas culicines secrete Factor Xa-directed anticoagulants (8Stark K.R. James A.A. J. Med. Entomol. 1996; 33: 645-650Crossref PubMed Scopus (54) Google Scholar). In contrast, inhibition of platelet aggregation seems to have been achieved in most hematophagous arthropods by the salivary apyrase (ATP-diphosphohydrolase, E.C. 3.6.1.5) (2James A.A. Bull. Inst. Pasteur. 1994; 92: 133-150Google Scholar, 4Ribeiro J.M.C. Infect. Agents Dis. 1995; 4: 143-152PubMed Google Scholar). When vascular tissue is damaged, the disrupted cells release ATP and ADP at high concentrations into the extra cellular environment where ADP promotes platelet activation and aggregation. The activated platelets may in turn release in the medium their ADP-containing granules, recruiting additional platelets to the site of injury. The function of the apyrase, injected at the feeding site with the saliva, is to inhibit the ADP-induced platelet recruitment and aggregation by hydrolyzing the ADP to AMP and inorganic phosphate. Molecular cloning and sequence analysis have revealed at least three classes of apyrases of different evolutionary origin. They are represented by the apyrases of the yellow fever mosquito A. aegypti (9Champagne D.E. Smartt C.T. Ribeiro J.M.C. James A.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 694-698Crossref PubMed Scopus (218) Google Scholar, 10Smartt C.T. Kim A.P. Grossman G.L. James A.A. Exp. Parasitol. 1995; 81: 239-248Crossref PubMed Scopus (55) Google Scholar), the intracellular parasite Toxoplasma gondii (11Asai T. Miura S. Sibley L.D. Okabayashi H. Takeuchi T. J. Biol. Chem. 1995; 270: 11391-11397Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), and the bedbug C. lectularius (12Valenzuela J.G. Charlab R. Galperin M.Y. Ribeiro J.M.C. J. Biol. Chem. 1998; 273: 30583-30590Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The T. gondii apyrase belongs to a large family of ecto-ATPases that are found in a wide variety of organisms and tissues ranging from plants (13Handa M. Guidotti G. Biochem. Biophys. Res. Commun. 1996; 218: 916-923Crossref PubMed Scopus (272) Google Scholar) to humans (14Kaczmarek E. Koziak K. Sévigny J. Siegel J.B. Anrather J. Beaudoin A.R. Bach F.H. Robson S.C. J. Biol. Chem. 1996; 271: 33116-33122Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar), whose role is not yet well understood. The C. lectularius apyrase does not show sequence similarity to any previously characterized nucleotide binding enzyme and belongs to a novel type of ATPases (12Valenzuela J.G. Charlab R. Galperin M.Y. Ribeiro J.M.C. J. Biol. Chem. 1998; 273: 30583-30590Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Finally the A. aegypti apyrase shows a high degree of sequence similarity to 5′-nucleotidases from different organisms (9Champagne D.E. Smartt C.T. Ribeiro J.M.C. James A.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 694-698Crossref PubMed Scopus (218) Google Scholar). Using the signal sequence trap technique (15Arcà B. Lombardo F. Capurro M. della Torre A. Dimopoulos G. James A.A. Coluzzi M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1516-1521Crossref PubMed Scopus (127) Google Scholar), we previously identified two cDNAs expressed in the salivary glands of the malaria mosquito,Anopheles gambiae, showing similarity to the gene encoding the A. aegypti apyrase. Because of their tissue-specific pattern of expression we suggested that they could be derived from malaria mosquito apyrase and 5′-nucleotidase genes. We report here the isolation of the corresponding full-length cDNAs and their developmental expression profiles. Expression of tagged recombinant proteins in COS-7 cells shows that both proteins are secreted. Finally, we provide evidence that an 800 bp1 fragment located at the 5′-end of the putative A. gambiae apyrase-coding region is able to drive specific expression of the Escherichia coliβ-galactosidase reporter gene in the salivary glands of transgenicDrosophila melanogaster. The A. gambiae strain used in this study was the homokaryotypic GASUA reference strain (Xag, 2R, 2La, 3R, 3L). Individuals from different developmental stages were collected, frozen in liquid nitrogen, and stored at −80 °C before nucleic acid isolation. If not otherwise specified, general nucleic acid manipulations were performed according to standard procedures (16Ausubel F.M. Brent R. Kingston R.F. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1991Google Scholar, 17Sambrook J. Fritsh E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The A. gambiae thoracic cDNA library (15Arcà B. Lombardo F. Capurro M. della Torre A. Dimopoulos G. James A.A. Coluzzi M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1516-1521Crossref PubMed Scopus (127) Google Scholar) and the λ genomic library (18Müller H.M. Crampton J.M. della Torre A. Sinden R. Crisanti A. EMBO J. 1993; 12: 2891-2900Crossref PubMed Scopus (153) Google Scholar) were screened by a gene amplification-based method (19Israel D.I. Nuclic Acids Res. 1993; 21: 2627-2631Crossref PubMed Scopus (143) Google Scholar) using the following gene-specific oligonucleotide primers: AgApy-F, 5′-AAAGTGCTGCTGCTAATC-3′; AgApy-R, 5′-AATACAGGTGTCACCTTCC-3′; AgApyL1-F, 5′-GGCAGAATGGCACTGGTACG-3′; AgApyL1-R, 5′-CACTCTTCAGCTGCTTGATC-3′. Signal peptide prediction analysis was performed by using the SIGNALP program (20Nielsen H. Engelbrecht J. Brunak S. von Heijne G. Protein Eng. 1997; 10: 1-6Crossref PubMed Scopus (4911) Google Scholar). Sequence comparison and data base searches were done by using the Wisconsin Package Version 9.1 (Genetics Computer Group, Madison, WI) and the BLAST program (21Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (58771) Google Scholar). Multiple alignments were obtained using the CLUSTAL W program (22Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (54908) Google Scholar) and the Java Multiple Alignment editor at the World Wide Web server of the European Bioinformatics Institute. The phenogram was obtained using the Neighbor joining option in PAUP* 4.0b2 (23Swofford D.L. PAUP: Phylogenetic Analysis Using Parsimony (and Other Methods) , Version 4.0b2. Sinauer Associates, Sunderland, MA1999Google Scholar) using the E. coli5′-nucleotidase sequence as the out-group to root the tree. Tree topology was statistically tested by bootstrap analysis (2000 replicates). Five micrograms of total RNA was used for Northern analysis (16Ausubel F.M. Brent R. Kingston R.F. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1991Google Scholar). The full-length cDNAs encoded by AgApy and AgApyL1 and a 590-bp fragment from the 3′-UTR of the A. gambiae actin gene (U02964: nucleotides 1707–2297) were used as probes (24Salazar C.E. Hamm D.M. Wesson D.M. Beard C.B. Kumar V. Collins F.H. Insect Mol. Biol. 1994; 3: 1-13Crossref PubMed Scopus (47) Google Scholar). Approximately 100 ng of DNase-treated total RNA (RNase-free DNaseI, Roche Molecular Biochemicals) were used for the RT-PCR amplifications (25Arcà B. Savakis C. The Molecular Biology of Insect Disease Vectors: A Methods Manual. Chapman & Hall, London1997: 244-260Crossref Google Scholar) with the SuperScript One-Step RT-PCR System (Life Technologies, Inc.) using the following gene-specific primers: AgApy-F4, 5′-CAACAGTGTGCCGCAAAGTC-3′; AgApy-R4, 5′-TAGCTTACACCATCGTTCAG-3′; AgApyL1-F, 5′-GGCAGAATGGCACTGGTACG-3′; AgApyL1-R, 5′-CACTCTTCAGCTGCTTGATC- 3′; AgApyL2-F1, 5′-GCCATAATAGCGAGCGAAG-3′; AgApyL2-R1, 5′-CAATAGCATCGAGTACAGCC-3′; act-F1, 5′-ACCCCATCTCACACACTTC-3′; act-R1, 5′-ATGTCTTTCATTGCCGCC-3′. Briefly, after the reverse transcription step (50 °C, 30 min) and heat inactivation of reverse transcriptase (94 °C, 2 min), 35 cycles of amplification (94 °C, 30 s; 55 °C, 30 s; 72 °C, 45 s) were employed for the detection of the apyrase and apyrase-like mRNAs; 25 cycles were used for the actin control amplification in order to keep the reaction below saturation levels. The myc epitope, EQKLISEEDL, was used to replace the peptides of identical length, SERSSKCKAA (amino acids 55–64) and NQKSSTCTNS (amino acids 52–61), respectively, in the AgApy and AgApyL1 proteins. The AgApy-myc and the AgApyL1-myc were obtained by the overlap PCR amplification technique (26Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene. 1989; 77: 61-68Crossref PubMed Scopus (2614) Google Scholar). The final amplification products were cloned into the pBKCMV vector (Stratagene), modified according to manufacturer's instructions to allow for higher expression levels in eukaryotic cells. The AgApyL1-myc-Crat construct contains the carboxyl terminus of the rat 5′-nucleotidase (amino acids 548–576) in place of the corresponding AgApyL1 end (amino acids 549–570). It was constructed from AgApyL1-myc by substitution of a BglII-XhoI restriction fragment with a PCR fragment containing the carboxyl-terminal domain of rat 5′-nucleotidase. All of the constructs were verified by sequencing before use. Oligonucleotide primers used for the construction of the clones described above are available upon request. COS-7 cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (DMEM complete) in a humidified incubator at 37 °C with 5% CO2. Cells were transfected using the LipofectAMINE reagent (Life Technologies, Inc.) according to the manufacturer's instructions. Briefly, 1–3 × 105 cells were seeded in 2 ml of DMEM complete in 35-mm wells and, after 24–36 h, transfected for 6 h at 37 °C using 2 μg of plasmid DNA. Twenty-four hours after transfection the supernatant was removed, and the cells were carefully washed several times with fetal bovine serum-free DMEM to remove traces of serum and incubated in 2 ml of fetal bovine serum-free DMEM. After 48 to 72 h of incubation, the medium was removed, centrifuged twice at 1,000 × g for 10 min and then once at 14,000 ×g for 5 min to remove detached cells and debris. After the addition of phenylmethylsulfonyl fluoride (40 μg/ml), the supernatants were concentrated using Microcon YM-30 filter devices (Millipore) and stored at −20 °C for Western blot analysis. Concentrated supernatants were subjected to SDS/polyacrylamide gel electrophoresis and transferred onto nitrocellulose filters (Schleicher & Schuell). myc-tagged recombinant proteins were stained with the mouse anti-c-myc-peroxidase monoclonal antibody (Roche Molecular Biochemicals) and detected using the ECL-plus system (Amersham Pharmacia Biotech). The oligonucleotide primers AgApyPr-5′EcoRI (5′-CTAGGAATTCGCTTGTAGGTGACGCTGTG-3′) and AgApyPr-3′BamHI (5′-CTAGCCTAGGCACGCTTCGCAGATATTAC-3′), containing the EcoRI and BamHI restriction sites at their ends respectively, were used to amplify the 800-bp segment upstream of the AgApygene. This segment was directionally cloned into the expression vector pCaSpeR-AUG-βgal (27Thummel C.S. Boulet A.M. Lipshitz H.D. Gene. 1988; 74: 445-456Crossref PubMed Scopus (472) Google Scholar). The resulting pCaSpeR-Apy-βgal was microinjected into yw D. melanogaster embryos (carrying a mutation in the yellow and whitegenes) along with an integration-defective helper plasmid as the source of P transposase. Several transformed individuals were obtained, and three independent homozygous lines were established through crosses with strains carrying appropriate balancer chromosomes. The lines, designated Apy5, Apy9, and Apy13, were analyzed by Southern blot hybridization and assayed for β-galactosidase activity. Apy5 and Apy13 contained a single insertion, whereas the Apy9 line carried a double insertion of the transposon. Small openings were made in otherwise intact adult flies to allow the staining solution to enter the body cavity. Flies were assayed individually in 96-well plates and incubated at 37 °C in 100 μl of staining solution (50 mm sodium phosphate, pH 8.0, 2 mm potassium ferrocyanide, 2 mm potassium ferricyanide, 0.3% X-gal, 15% Ficoll-400). Several individuals of both sexes were analyzed for each line. Typically, staining started to appear after 5 to 6 h, but intense staining of the glands was only observed after overnight incubations. After incubation, flies were dissected in phosphate-buffered saline to extract the salivary glands. The staining pattern of each line was compared with that of the recipient strain yw and of the Lysβ-gal stock. The latter carries a P element insertion with β-galactosidase expression under the control of the salivary gland-specific lysozyme P promoter of D. melanogaster (28Kylsten P. Kimbrell D.A. Daffre S. Samaklovis C. Hultmark D. Mol. Gen. Genet. 1992; 232: 335-343Crossref PubMed Scopus (77) Google Scholar) and typically exhibits a strong staining in salivary glands after 1–2 h of incubation at 37 °C. 2G. Valianatos, I. Sidén-Kiamos, and C. Louis, unpublished information. In a previous study, we identified two short cDNA fragments, cF3 and iC6, whose conceptually translated proteins showed similarity to the A. aegyptiapyrase and to several members of the 5′-nucleotidase family (15Arcà B. Lombardo F. Capurro M. della Torre A. Dimopoulos G. James A.A. Coluzzi M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1516-1521Crossref PubMed Scopus (127) Google Scholar). cF3 expression was found to be restricted to female salivary glands, with the corresponding transcripts mainly localized in the distal-lateral lobes. In contrast, iC6 expression, which was clearly enriched in female glands, could also be detected at a lower level in other tissues. These results were compatible with cF3 representing the A. gambiae salivary apyrase and iC6, a 5′-nucleotidase. To confirm these observations, we screened a thoracic cDNA library from A. gambiae adult females and isolated the corresponding full-length cDNAs. In the remaining part of the text we will keep the designation cF3 and iC6 to indicate the partial clones previously isolated by the signal sequence trap technique and we will refer to the corresponding full-length cDNAs as AgApy and AgApyL1, respectively. Using cF3-specific oligonucleotide primers for a PCR-based screening we isolated a cDNA that was 2253 bp in length. Sequence analysis showed that it lacked 19 nucleotides at the 5′-end that were present in the cF3 clone (15Arcà B. Lombardo F. Capurro M. della Torre A. Dimopoulos G. James A.A. Coluzzi M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1516-1521Crossref PubMed Scopus (127) Google Scholar). The 2272-nucleotide-long mRNA that can be reconstructed from this two-clone contig is likely to represent the full-length transcription product and contains a 1671-nucleotide-long open reading frame and a 584-base 3′-UTR (Fig.1 A). The putative protein encoded by this mRNA is similar in size (557 amino acids), molecular mass (61.7 kDa), and isoelectric point (8.83) to the A. aegypti apyrase (9Champagne D.E. Smartt C.T. Ribeiro J.M.C. James A.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 694-698Crossref PubMed Scopus (218) Google Scholar, 10Smartt C.T. Kim A.P. Grossman G.L. James A.A. Exp. Parasitol. 1995; 81: 239-248Crossref PubMed Scopus (55) Google Scholar) and contains five potentialN-linked glycosylation sites. Prediction analysis showed the presence at the amino terminus of a cleavable signal peptide, whereas no regions with transmembrane properties could be detected, suggesting that this mRNA encodes a secreted protein. Sequence comparison of the conceptually translated protein to the A. aegyptiapyrase showed an overall identity of 50.8% and a similarity of 60.8%, whereas identity and similarity to different members of the 5′-nucleotidase family were significantly lower (TableI). These observations along with the previously determined female salivary gland-specific expression provide further support for the notion that this mRNA could code for the A. gambiae salivary apyrase. However, we should point out here that we were unable to prove the apyrase activity of the AgApy gene product by using a myc-tagged recombinant version of the protein (see also “Discussion”).Table ISimilarity among selected members of the apyrase/5′-nucleotidase familyAgApyAgApyL1AaApyL15NChrysoptinBm5NRat5NHum5NAgApy47.6 (58.2)50.8 (60.8)34.8 (44.0)39.6 (51.4)34.2 (43.5)36.5 (45.1)38.0 (46.9)AgApyL158.5 (67.4)36.5 (46.7)38.2 (51.1)29.7 (40.7)32.6 (42.9)34.0 (44.5)AaApy34.8 (48.1)38.0 (50.9)32.0 (42.8)32.8 (43.3)33.5 (45.2)Ll5N37.4 (50.1)41.0 (51.2)45.6 (53.6)44.4 (52.2)Chrysoptin35.8 (48.7)37.9 (48.4)37.9 (47.7)Bm5N41.0 (50.1)40.1 (49.1)Rat5N87.6 (90.7)Hum5NPercentages of identity and similarity (in parenthesis) are shown. AgApy, putative A. gambiae apyrase (AJ237704); AgApyL1,A. gambiae apyrase-like 1 (AJ237706); AaApy, A. aegypti apyrase (P50635); Ll5N, L. longipalpis5′-nucleotidase (AF131933); chrysoptin, Chrysops sp. chrysoptin precursor (AF169229); Bm5N, B. microplus5′-nucleotidase (P52307); Rat5N, Rattus norvegicus5′-nucleotidase (P21588); Hum5N, Homo sapiens5′-nucleotidase (P21589). Open table in a new tab Percentages of identity and similarity (in parenthesis) are shown. AgApy, putative A. gambiae apyrase (AJ237704); AgApyL1,A. gambiae apyrase-like 1 (AJ237706); AaApy, A. aegypti apyrase (P50635); Ll5N, L. longipalpis5′-nucleotidase (AF131933); chrysoptin, Chrysops sp. chrysoptin precursor (AF169229); Bm5N, B. microplus5′-nucleotidase (P52307); Rat5N, Rattus norvegicus5′-nucleotidase (P21588); Hum5N, Homo sapiens5′-nucleotidase (P21589). Because of our interest in the potential applications of upstream regulatory sequences determining salivary gland-specific gene expression in A. gambiae, we screened a genomic library and isolated a clone containing the entire region encoding this gene. The primary transcript and 800 bp of 5′ end sequences as well as ∼150 bp to the 3′ end of the polyadenylation site are shown in Fig.1 A. The putative A. gambiae apyrase gene (AgApy) contains six exons separated by five small introns and, as outlined in Fig. 1 B, it is comparable in its general organization with the A. aegypti apyrase. The position of introns I to IV is perfectly conserved in the two mosquito species, whereas the intron V of the A. gambiae gene clearly corresponds to intron VI of the A. aegypti apyrase. The other introns present in the A. aegypti gene are not found in A. gambiae. An additional difference involves the 3′-UTR, which in A. gambiae is longer by almost 600 nucleotides, compared with the only 30-base-long 3′-UTR found in the A. aegypti apyrase gene. As previously reported, a second cDNA, iC6, showing similarity to the A. aegypti apyrase was isolated in the signal sequence trap screen (15Arcà B. Lombardo F. Capurro M. della Torre A. Dimopoulos G. James A.A. Coluzzi M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1516-1521Crossref PubMed Scopus (127) Google Scholar). However, because its expression was not restricted to the salivary glands, it was thought to represent an A. gambiae5′-nucleotidase. Champagne et al. (9Champagne D.E. Smartt C.T. Ribeiro J.M.C. James A.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 694-698Crossref PubMed Scopus (218) Google Scholar) suggest that the A. aegypti apyrase evolved from a 5′-nucleotidase family member by gene duplication and divergent evolution. Because ecto-5′-nucleotidases are attached to the plasma membrane by glycosylphosphatidylinositol anchors, the evolution of the secreted apyrase proteins, adapted to blood-feeding, may have involved the loss of the hydrophobic carboxyl-terminal domain that includes this structure (29Misumi Y. Ogata S. Ohkubo K. Hirose S. Ikehara Y. Eur. J. Biochem. 1990; 191: 563-569Crossref PubMed Scopus (117) Google Scholar). With the aim of better understanding of the evolutionary relationship between these two proteins, we isolated the full-length cDNA corresponding to iC6, and we designated the corresponding gene AgApyL1 (A. gambiaeapyrase-like 1). The AgApyL1 cDNA is ∼1.8 kilobases in length, with an open reading frame potentially encoding a protein of 570 amino acids and containing an amino-terminal signal peptide. Sequence comparison showed that the putative protein shared higher similarity with apyrases than with 5′-nucleotidases, and surprisingly, the degree of similarity was significantly higher to the apyrase of A. aegypti than to the putative A. gambiae apyrase (TableI). The conceptual translation products of AgApy and AgApyL1 can be easily aligned to several other members of the apyrase/5′-nucleotidase family (not shown). All these proteins show a common general structure, with an amino-terminal signal peptide of variable length and a high degree of conservation in the seven domains known to characterize enzymes having apyrase or 5′-nucleotidase activity (9Champagne D.E. Smartt C.T. Ribeiro J.M.C. James A.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 694-698Crossref PubMed Scopus (218) Google Scholar, 30Volknandt W. Vogel M. Pevsner J. Misumi Y. Ikehara Y. Zimmermann H. Eur. J. Biochem. 1991; 202: 855-861Crossref PubMed Scopus (45) Google Scholar). The six-amino acid sequence GKYVGR previously identified in the sixth domain of the A. aegypti apyrase as the putative nucleotide-binding site (9Champagne D.E. Smartt C.T. Ribeiro J.M.C. James A.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 694-698Crossref PubMed Scopus (218) Google Scholar) is perfectly conserved in both the presumed A. gambiae apyrase and the apyrase-like-1 proteins. Fig. 2 shows the alignments of the carboxyl-terminal domains of the conceptually translated proteins AgApy and AgApyL1, the A. aegypti apyrase (9Champagne D.E. Smartt C.T. Ribeiro J.M.C. James A.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 694-698Crossref PubMed Scopus (218) Google Scholar), the 5′-nucleotidases from rat, human, and from the cattle tickBoophilus microplus (29Misumi Y. Ogata S. Ohkubo K. Hirose S. Ikehara Y. Eur. J. Biochem. 1990; 191: 563-569Crossref PubMed Scopus (117) Google Scholar, 31Misumi Y. Ogata S. Hirose S. Ikehara Y. J. Biol. Chem. 1990; 265: 2178-2183Abstract Full Text PDF PubMed Google Scholar, 32Liyou N. Hamilton S. Elvin C. Willadsen P. Insect Mol. Biol. 1999; 8: 257-266Crossref PubMed Scopus (28) Google Scholar), and two additional dipteran members of the family, recently submitted to data bases: the 5′-nucleotidase from the sandfly L. longipalpis and the chrysoptin from Chrysops sp. The comparison of the carboxyl-terminal portions of these proteins shows that the 5′-nucleotidases from rat, human, and B. microplus contain an additional terminal domain that is highly hydrophobic and which is known to function as the signal for glycosylphosphatidylinositol anchoring to plasma membranes (29Misumi Y. Ogata S. Ohkubo K. Hirose S. Ikehara Y. Eur. J. Biochem. 1990; 191: 563-569Crossref PubMed Scopus (117) Google Scholar, 32Liyou N. Hamilton S. Elvin C. Willadsen P. Insect Mol. Biol. 1999; 8: 257-266Crossref PubMed Scopus (28) Google Scholar). This terminal portion is not present in AgApy, in the A. aegypti apyrase, and in the chrysoptin, whereas the L. longipalpis 5′-nucleotidase and the AgApyL1 protein contain shorter carboxyl-terminal regions of 5 and 13 amino acids, respectively. However, these regions do not show the characteristic hydrophobic profile exhibited by 5′-nucleotidases (not shown), suggesting that these proteins as well as the two mosquito apyrases and the chrysoptin also may be secreted. The relationships among these different members of the family are more strikingly represented in Fig. 3, where the tree obtained from the alignment of the entire peptide sequences is shown. Interestingly two different clusters can be clearly recognized; the first includes the A. aegypti apyrase, AgApy, AgApyL1, and the chrysoptin, whereas the remaining 5′-nucleotidases form a second group.Figure 3Neighbor joining tree showing the relationships among members of the apyrase/5′-nucleotidase family.The E. coli 5′-nucleotidase (Ec5N, P07024) was used as an out-group. Numbers indicate bootstrap values (2000 replicates). For the other abbreviations, see the legend to TableI. Rat5N, R. norvegicus 5′-nucleotidase;Ll5N, L. longipalpis 5′-nucleotidase;Bm5N, B. microplus 5′-nucleotidase;Hum5N, H. sapiens human 5′-nucleotidase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) An additional member of this family of apyrase/5′-nucleotidase-like proteins was found by searching a data base that includes sequences from the ends of genomic clones of an A. gambiae bacterial artificial chromosome library. One of
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