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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 31, Issue of August 4, pp.

23861–23868, 2000
© 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Promoter Sequences of the Putative Anopheles gambiae Apyrase


Confer Salivary Gland Expression in Drosophila melanogaster*
Received for publication, November 29, 1999, and in revised form, April 26, 2000
Published, JBC Papers in Press, May 5, 2000, DOI 10.1074/jbc.M909547199

Fabrizio Lombardo‡储§, Manlio Di Cristina¶, Lefteris Spanos储, Christos Louis储**, Mario Coluzzi‡,
and Bruno Arcà‡ ‡‡ §§
From the ‡Istituto di Parassitologia, Istituto Pasteur-Fondazione Cenci Bolognetti, Università di Roma “La Sapienza,”
00185 Roma, Italy, ¶Department of Biology, Imperial College of Science, Technology, and Medicine, London SW7 2AZ,
United Kingdom, 储Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, 71110
Heraklion, Crete, Greece, **Department of Biology, University of Crete, 71110 Heraklion, Crete, Greece, and
‡‡Dipartimento di Genetica, Biologia Generale e Molecolare, Università di Napoli Federico II, 80134 Napoli, Italy

The saliva of blood-feeding arthropods contains an ing blood feeding. Since hematophagy has arisen independ-
apyrase that facilitates hematophagy by inhibiting the ently several times in insects, even within the same order, a
ADP-induced aggregation of the host platelets. We re- large variety of different molecules have evolved to accomplish
port here the isolation of a salivary gland-specific cDNA the same or similar function (1– 4). Remarkably diverse sub-
encoding a secreted protein that likely represents the stances act as vasodilators in the saliva of distinct arthropod
Anopheles gambiae apyrase. We describe also two addi- species; they include prostaglandins in ticks, nitric oxide in the
tional members of the apyrase/5ⴕ-nucleotidase family. bugs Rhodnius prolixus and Cimex lectularius, peptides such

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The cDNA corresponding to the AgApyL1 gene encodes a as tachykinins or maxadilan in the mosquito Aedes aegypti and
secreted protein that is closely related in sequence to in the sandfly Lutzomya longipalpis, respectively, and the sal-
the apyrase of the yellow fever mosquito, Aedes aegypti,
ivary peroxidase/catechol oxidase in the mosquito Anopheles
and whose expression appears enriched in, but not re-
albimanus (4 – 6). Similarly, partly as a consequence of their
stricted to, female salivary glands. The AgApyL2 gene
was found searching an A. gambiae data base, and its different feeding habits, a multitude of different anticoagulants
expression is restricted to larval stages. We isolated the is found in different blood-eating species (7). In mosquitoes,
gene encoding the presumed A. gambiae apyrase thrombin and Factor Xa are the preferential targets within the
(AgApy) and we tested its putative promoter for the tis- blood coagulation cascade; anophelines produce anti-thrombin
sue-specific expression of the LacZ gene from Esche- activities, whereas culicines secrete Factor Xa-directed antico-
richia coli in transgenic Drosophila melanogaster. All agulants (8). In contrast, inhibition of platelet aggregation
the transgenic lines analyzed showed a weak but unam- seems to have been achieved in most hematophagous arthro-
biguous staining of the adult glands, indicating that pods by the salivary apyrase (ATP-diphosphohydrolase, E.C.
some of the salivary gland-specific transcriptional reg- 3.6.1.5) (2, 4). When vascular tissue is damaged, the disrupted
ulatory elements are conserved between the malaria cells release ATP and ADP at high concentrations into the
mosquito and the fruit fly. The availability of salivary extra cellular environment where ADP promotes platelet acti-
gland-specific promoters may be useful both for studies vation and aggregation. The activated platelets may in turn
on vector-parasite interactions and, potentially, for the release in the medium their ADP-containing granules, recruit-
targeted tissue-specific expression of anti-parasite ing additional platelets to the site of injury. The function of the
genes in the mosquito. apyrase, injected at the feeding site with the saliva, is to inhibit
the ADP-induced platelet recruitment and aggregation by hy-
drolyzing the ADP to AMP and inorganic phosphate.
The salivary glands of blood-sucking arthropods secrete a Molecular cloning and sequence analysis have revealed at
complex array of specific factors with vasodilatory, anti-clot- least three classes of apyrases of different evolutionary origin.
ting, and anti-platelet activities that assist the mosquito dur- They are represented by the apyrases of the yellow fever mos-
quito A. aegypti (9, 10), the intracellular parasite Toxoplasma
gondii (11), and the bedbug C. lectularius (12). The T. gondii
* This work was supported by European Union Grant ERBFM-
RXCT960017 (to M. C and C. L.) and by a grant from the United apyrase belongs to a large family of ecto-ATPases that are
Nations Developmental Program/World Bank/World Health Organiza- found in a wide variety of organisms and tissues ranging from
tion Special Program for Research and Training in Tropical Diseases plants (13) to humans (14), whose role is not yet well under-
(TDR) (to M. C.). The costs of publication of this article were defrayed in stood. The C. lectularius apyrase does not show sequence sim-
part by the payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 U.S.C. Section ilarity to any previously characterized nucleotide binding en-
1734 solely to indicate this fact. zyme and belongs to a novel type of ATPases (12). Finally the A.
The nucleotide sequence(s) reported in this paper has been submitted aegypti apyrase shows a high degree of sequence similarity to
to the GenBankTM/EBI Data Bank with accession number(s) AJ237704, 5⬘-nucleotidases from different organisms (9).
AJ237705, and AJ237706.
§ Supported by a training fellowship of the University of Rome “La
Using the signal sequence trap technique (15), we previously
Sapienza.” identified two cDNAs expressed in the salivary glands of the
§§ Supported by a postdoctoral fellowship of the Istituto Pasteur- malaria mosquito, Anopheles gambiae, showing similarity to
Fondazione Cenci Bolognetti and by European Union Return Grant the gene encoding the A. aegypti apyrase. Because of their
BIO4-CT98-5020. To whom correspondence should be addressed: Isti-
tuto di Parassitologia, Università di Roma “La Sapienza,” P. le Aldo
tissue-specific pattern of expression we suggested that they
Moro 5, Box 6, Roma 62, 00185 Roma, Italy. Tel.: 39-06-4991-4900; Fax: could be derived from malaria mosquito apyrase and 5⬘-nucle-
39-06-4991-4644; E-mail: b.Arca@Caspur.it. otidase genes. We report here the isolation of the corresponding

This paper is available on line at http://www.jbc.org 23861


23862 5⬘-Nucleotidase Family Members from A. gambiae
full-length cDNAs and their developmental expression profiles. Oligonucleotide primers used for the construction of the clones de-
Expression of tagged recombinant proteins in COS-7 cells scribed above are available upon request.
Expression in COS-7 Cells and Western Blot Detection of Recombi-
shows that both proteins are secreted. Finally, we provide
nant Proteins—COS-7 cells were cultured in DMEM supplemented with
evidence that an 800 bp1 fragment located at the 5⬘-end of the 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and
putative A. gambiae apyrase-coding region is able to drive 100 ␮g/ml streptomycin (DMEM complete) in a humidified incubator at
specific expression of the Escherichia coli ␤-galactosidase re- 37 °C with 5% CO2. Cells were transfected using the LipofectAMINE
porter gene in the salivary glands of transgenic Drosophila reagent (Life Technologies, Inc.) according to the manufacturer’s in-
melanogaster. structions. 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
EXPERIMENTAL PROCEDURES using 2 ␮g of plasmid DNA. Twenty-four hours after transfection the
supernatant was removed, and the cells were carefully washed several
Mosquito Colony—The A. gambiae strain used in this study was the
times with fetal bovine serum-free DMEM to remove traces of serum
homokaryotypic GASUA reference strain (Xag, 2R, 2La, 3R, 3L). Indi-
and incubated in 2 ml of fetal bovine serum-free DMEM. After 48 to 72 h
viduals from different developmental stages were collected, frozen in
of incubation, the medium was removed, centrifuged twice at 1,000 ⫻ g
liquid nitrogen, and stored at ⫺80 °C before nucleic acid isolation.
for 10 min and then once at 14,000 ⫻ g for 5 min to remove detached
Libraries Screening and Sequence Analysis—If not otherwise speci-
cells and debris. After the addition of phenylmethylsulfonyl fluoride (40
fied, general nucleic acid manipulations were performed according to
standard procedures (16, 17). The A. gambiae thoracic cDNA library ␮g/ml), the supernatants were concentrated using Microcon YM-30
filter devices (Millipore) and stored at ⫺20 °C for Western blot analysis.
(15) and the ␭ genomic library (18) were screened by a gene amplifica-
Concentrated supernatants were subjected to SDS/polyacrylamide gel
tion-based method (19) using the following gene-specific oligonucleotide
electrophoresis and transferred onto nitrocellulose filters (Schleicher &
primers:
Schuell). myc-tagged recombinant proteins were stained with the
AgApy-F, 5⬘-AAAGTGCTGCTGCTAATC-3⬘;
mouse anti-c-myc-peroxidase monoclonal antibody (Roche Molecular
AgApy-R, 5⬘-AATACAGGTGTCACCTTCC-3⬘;
Biochemicals) and detected using the ECL-plus system (Amersham
AgApyL1-F, 5⬘-GGCAGAATGGCACTGGTACG-3⬘;
Pharmacia Biotech).
AgApyL1-R, 5⬘-CACTCTTCAGCTGCTTGATC-3⬘.
Drosophila Transformation and Histochemical Stainings—The oli-
Signal peptide prediction analysis was performed by using the SIG-
gonucleotide primers AgApyPr-5⬘EcoRI (5⬘-CTAGGAATTCGCTTG-
NALP program (20). Sequence comparison and data base searches were
TAGGTGACGCTGTG-3⬘) and AgApyPr-3⬘BamHI (5⬘-CTAGCCTAG-

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done by using the Wisconsin Package Version 9.1 (Genetics Computer
GCACGCTTCGCAGATATTAC-3⬘), containing the EcoRI and BamHI
Group, Madison, WI) and the BLAST program (21). Multiple align-
restriction sites at their ends respectively, were used to amplify the
ments were obtained using the CLUSTAL W program (22) and the Java
800-bp segment upstream of the AgApy gene. This segment was direc-
Multiple Alignment editor at the World Wide Web server of the Euro-
tionally cloned into the expression vector pCaSpeR-AUG-␤gal (27). The
pean Bioinformatics Institute. The phenogram was obtained using the
resulting pCaSpeR-Apy-␤gal was microinjected into yw D. melano-
Neighbor joining option in PAUP* 4.0b2 (23) using the E. coli 5⬘-
gaster embryos (carrying a mutation in the yellow and white genes)
nucleotidase sequence as the out-group to root the tree. Tree topology
along with an integration-defective helper plasmid as the source of P
was statistically tested by bootstrap analysis (2000 replicates).
transposase. Several transformed individuals were obtained, and three
RNA Purification and Expression Analysis—Five micrograms of total
RNA was used for Northern analysis (16). The full-length cDNAs en- independent homozygous lines were established through crosses with
coded by AgApy and AgApyL1 and a 590-bp fragment from the 3⬘-UTR strains carrying appropriate balancer chromosomes. The lines, desig-
of the A. gambiae actin gene (U02964: nucleotides 1707–2297) were nated Apy5, Apy9, and Apy13, were analyzed by Southern blot hybrid-
used as probes (24). ization and assayed for ␤-galactosidase activity. Apy5 and Apy13 con-
Approximately 100 ng of DNase-treated total RNA (RNase-free DNa- tained a single insertion, whereas the Apy9 line carried a double
seI, Roche Molecular Biochemicals) were used for the RT-PCR amplifi- insertion of the transposon.
cations (25) with the SuperScript One-Step RT-PCR System (Life Tech- Small openings were made in otherwise intact adult flies to allow the
nologies, Inc.) using the following gene-specific primers: staining solution to enter the body cavity. Flies were assayed individ-
AgApy-F4, 5⬘-CAACAGTGTGCCGCAAAGTC-3⬘; ually in 96-well plates and incubated at 37 °C in 100 ␮l of staining
AgApy-R4, 5⬘-TAGCTTACACCATCGTTCAG-3⬘; solution (50 mM sodium phosphate, pH 8.0, 2 mM potassium ferrocya-
AgApyL1-F, 5⬘-GGCAGAATGGCACTGGTACG-3⬘; nide, 2 mM potassium ferricyanide, 0.3% X-gal, 15% Ficoll-400). Several
individuals of both sexes were analyzed for each line. Typically, stain-
AgApyL1-R, 5⬘-CACTCTTCAGCTGCTTGATC- 3⬘;
ing started to appear after 5 to 6 h, but intense staining of the glands
AgApyL2-F1, 5⬘-GCCATAATAGCGAGCGAAG-3⬘;
was only observed after overnight incubations. After incubation, flies
AgApyL2-R1, 5⬘-CAATAGCATCGAGTACAGCC-3⬘;
were dissected in phosphate-buffered saline to extract the salivary
act-F1, 5⬘-ACCCCATCTCACACACTTC-3⬘;
glands. The staining pattern of each line was compared with that of the
act-R1, 5⬘-ATGTCTTTCATTGCCGCC-3⬘.
recipient strain yw and of the Lys␤-gal stock. The latter carries a P
Briefly, after the reverse transcription step (50 °C, 30 min) and heat
element insertion with ␤-galactosidase expression under the control of
inactivation of reverse transcriptase (94 °C, 2 min), 35 cycles of ampli-
the salivary gland-specific lysozyme P promoter of D. melanogaster (28)
fication (94 °C, 30 s; 55 °C, 30 s; 72 °C, 45 s) were employed for the
and typically exhibits a strong staining in salivary glands after 1–2 h of
detection of the apyrase and apyrase-like mRNAs; 25 cycles were used
incubation at 37 °C.2
for the actin control amplification in order to keep the reaction below
saturation levels. RESULTS
Construction of myc-tagged Recombinant Clones—The myc epitope,
EQKLISEEDL, was used to replace the peptides of identical length, The Full-length cDNA Corresponding to the cF3 Fragment
SERSSKCKAA (amino acids 55– 64) and NQKSSTCTNS (amino acids May Encode the A. gambiae Apyrase—In a previous study, we
52– 61), respectively, in the AgApy and AgApyL1 proteins. The AgApy- identified two short cDNA fragments, cF3 and iC6, whose con-
myc and the AgApyL1-myc were obtained by the overlap PCR amplifi- ceptually translated proteins showed similarity to the A. ae-
cation technique (26). The final amplification products were cloned into
gypti apyrase and to several members of the 5⬘-nucleotidase
the pBKCMV vector (Stratagene), modified according to manufacturer’s
instructions to allow for higher expression levels in eukaryotic cells. family (15). cF3 expression was found to be restricted to female
The AgApyL1-myc-Crat construct contains the carboxyl terminus of the salivary glands, with the corresponding transcripts mainly lo-
rat 5⬘-nucleotidase (amino acids 548 –576) in place of the corresponding calized in the distal-lateral lobes. In contrast, iC6 expression,
AgApyL1 end (amino acids 549 –570). It was constructed from AgA- which was clearly enriched in female glands, could also be
pyL1-myc by substitution of a BglII-XhoI restriction fragment with a detected at a lower level in other tissues. These results were
PCR fragment containing the carboxyl-terminal domain of rat 5⬘-nucle-
compatible with cF3 representing the A. gambiae salivary
otidase. All of the constructs were verified by sequencing before use.
apyrase and iC6, a 5⬘-nucleotidase. To confirm these observa-
tions, we screened a thoracic cDNA library from A. gambiae
1
The abbreviations used are: bp, base pair(s); RT, reverse transcrip-
tion; PCR, polymerase chain reaction; DMEM, Dulbecco’s modified Ea-
gle’s medium; X-gal, 5-bromo-4-chloro-3-indolyl ␤-D-galactopyranoside; 2
G. Valianatos, I. Sidén-Kiamos, and C. Louis, unpublished
UTR, untranslated region; contig, group of overlapping clones. information.
5⬘-Nucleotidase Family Members from A. gambiae 23863
adult females and isolated the corresponding full-length biae apyrase-like 1). The AgApyL1 cDNA is ⬃1.8 kilobases in
cDNAs. In the remaining part of the text we will keep the length, with an open reading frame potentially encoding a
designation cF3 and iC6 to indicate the partial clones previ- protein of 570 amino acids and containing an amino-terminal
ously isolated by the signal sequence trap technique and we signal peptide. Sequence comparison showed that the putative
will refer to the corresponding full-length cDNAs as AgApy and protein shared higher similarity with apyrases than with 5⬘-
AgApyL1, respectively. nucleotidases, and surprisingly, the degree of similarity was
Using cF3-specific oligonucleotide primers for a PCR-based significantly higher to the apyrase of A. aegypti than to the
screening we isolated a cDNA that was 2253 bp in length. putative A. gambiae apyrase (Table I). The conceptual trans-
Sequence analysis showed that it lacked 19 nucleotides at the lation products of AgApy and AgApyL1 can be easily aligned to
5⬘-end that were present in the cF3 clone (15). The 2272- several other members of the apyrase/5⬘-nucleotidase family
nucleotide-long mRNA that can be reconstructed from this (not shown). All these proteins show a common general struc-
two-clone contig is likely to represent the full-length transcrip- ture, with an amino-terminal signal peptide of variable length
tion product and contains a 1671-nucleotide-long open reading and a high degree of conservation in the seven domains known
frame and a 584-base 3⬘-UTR (Fig. 1A). The putative protein to characterize enzymes having apyrase or 5⬘-nucleotidase ac-
encoded by this mRNA is similar in size (557 amino acids), tivity (9, 30). The six-amino acid sequence GKYVGR previously
molecular mass (61.7 kDa), and isoelectric point (8.83) to the A. identified in the sixth domain of the A. aegypti apyrase as the
aegypti apyrase (9, 10) and contains five potential N-linked putative nucleotide-binding site (9) is perfectly conserved in
glycosylation sites. Prediction analysis showed the presence at both the presumed A. gambiae apyrase and the apyrase-like-1
the amino terminus of a cleavable signal peptide, whereas no proteins. Fig. 2 shows the alignments of the carboxyl-terminal
regions with transmembrane properties could be detected, sug- domains of the conceptually translated proteins AgApy and
gesting that this mRNA encodes a secreted protein. Sequence AgApyL1, the A. aegypti apyrase (9), the 5⬘-nucleotidases from
comparison of the conceptually translated protein to the A. rat, human, and from the cattle tick Boophilus microplus (29,
aegypti apyrase showed an overall identity of 50.8% and a 31, 32), and two additional dipteran members of the family,

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similarity of 60.8%, whereas identity and similarity to different recently submitted to data bases: the 5⬘-nucleotidase from the
members of the 5⬘-nucleotidase family were significantly lower sandfly L. longipalpis and the chrysoptin from Chrysops sp.
(Table I). These observations along with the previously deter- The comparison of the carboxyl-terminal portions of these pro-
mined female salivary gland-specific expression provide fur- teins shows that the 5⬘-nucleotidases from rat, human, and B.
ther support for the notion that this mRNA could code for the microplus contain an additional terminal domain that is highly
A. gambiae salivary apyrase. However, we should point out hydrophobic and which is known to function as the signal for
here that we were unable to prove the apyrase activity of the glycosylphosphatidylinositol anchoring to plasma membranes
AgApy gene product by using a myc-tagged recombinant ver- (29, 32). This terminal portion is not present in AgApy, in the
sion of the protein (see also “Discussion”). A. aegypti apyrase, and in the chrysoptin, whereas the L. lon-
Because of our interest in the potential applications of up- gipalpis 5⬘-nucleotidase and the AgApyL1 protein contain
stream regulatory sequences determining salivary gland-spe- shorter carboxyl-terminal regions of 5 and 13 amino acids,
cific gene expression in A. gambiae, we screened a genomic respectively. However, these regions do not show the charac-
library and isolated a clone containing the entire region encod- teristic hydrophobic profile exhibited by 5⬘-nucleotidases (not
ing this gene. The primary transcript and 800 bp of 5⬘ end shown), suggesting that these proteins as well as the two mos-
sequences as well as ⬃150 bp to the 3⬘ end of the polyadenyl- quito apyrases and the chrysoptin also may be secreted. The
ation site are shown in Fig. 1A. The putative A. gambiae relationships among these different members of the family are
apyrase gene (AgApy) contains six exons separated by five more strikingly represented in Fig. 3, where the tree obtained
small introns and, as outlined in Fig. 1B, it is comparable in its from the alignment of the entire peptide sequences is shown.
general organization with the A. aegypti apyrase. The position Interestingly two different clusters can be clearly recognized;
of introns I to IV is perfectly conserved in the two mosquito the first includes the A. aegypti apyrase, AgApy, AgApyL1, and
species, whereas the intron V of the A. gambiae gene clearly the chrysoptin, whereas the remaining 5⬘-nucleotidases form a
corresponds to intron VI of the A. aegypti apyrase. The other second group.
introns present in the A. aegypti gene are not found in A. An additional member of this family of apyrase/5⬘-nucleotid-
gambiae. An additional difference involves the 3⬘-UTR, which ase-like proteins was found by searching a data base that
in A. gambiae is longer by almost 600 nucleotides, compared includes sequences from the ends of genomic clones of an A.
with the only 30-base-long 3⬘-UTR found in the A. aegypti gambiae bacterial artificial chromosome library. One of these
apyrase gene. clones, AK0AA011L15B1, contains sequences that showed sim-
Two Additional Apyrase-like Genes in A. gambiae—As pre- ilarity to apyrases and 5⬘-nucleotidases. The entry could poten-
viously reported, a second cDNA, iC6, showing similarity to the tially include an exon that is flanked by sequences matching
A. aegypti apyrase was isolated in the signal sequence trap the consensus for donor and acceptor splice sites and that has
screen (15). However, because its expression was not restricted the potential to encode a 277-amino acid-long polypeptide. This
to the salivary glands, it was thought to represent an A. gam- putative partial protein showed similarity to the region en-
biae 5⬘-nucleotidase. Champagne et al. (9) suggest that the A. coded by the exons 3 to 5 of the AgApy cDNA, and it is most
aegypti apyrase evolved from a 5⬘-nucleotidase family member likely another representative of the apyrase/5⬘-nucleotidase
by gene duplication and divergent evolution. Because ecto-5⬘- family. We will refer to this additional member of the family as
nucleotidases are attached to the plasma membrane by glyco- AgApyL2 (A. gambiae apyrase-like 2).
sylphosphatidylinositol anchors, the evolution of the secreted Developmental Expression of the Putative Apyrase and
apyrase proteins, adapted to blood-feeding, may have involved Apyrase-like Genes—We had previously shown that the AgApy
the loss of the hydrophobic carboxyl-terminal domain that in- gene is specifically expressed in the salivary glands of adult
cludes this structure (29). With the aim of better understand- females, whereas AgApyL1 also was found to be expressed at
ing of the evolutionary relationship between these two pro- lower levels in other female tissues and in males (15). This was
teins, we isolated the full-length cDNA corresponding to iC6, confirmed by Northern analysis on total RNA from adult male
and we designated the corresponding gene AgApyL1 (A. gam- and female mosquitoes (Fig. 4A), the only difference being that
23864 5⬘-Nucleotidase Family Members from A. gambiae

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FIG. 1. A, sequence of a genomic fragment containing the AgApy gene (AJ237705). The nucleotide coding sequence is shown above the conceptual
translation product of the corresponding cDNA. The putative TATA and CAAT boxes, the translation initiation (ATG), the putative signal peptide,
and the polyadenylation signal are underlined. The arrow indicates the probable transcription start site. Introns are shown in lowercase letters,
and the two invariable nucleotides of the donor and acceptor splice sites are in bold characters. Circles highlight potential N-linked glycosylation
5⬘-Nucleotidase Family Members from A. gambiae 23865
TABLE I
Similarity among selected members of the apyrase/5⬘-nucleotidase family
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. longipalpis 5⬘-nucleotidase (AF131933); chrysoptin, Chrysops sp.
chrysoptin precursor (AF169229); Bm5N, B. microplus 5⬘-nucleotidase (P52307); Rat5N, Rattus norvegicus 5⬘-nucleotidase (P21588); Hum5N,
Homo sapiens 5⬘-nucleotidase (P21589).
AgApy AgApyL1 AaApy L15N Chrysoptin Bm5N Rat5N Hum5N

AgApy 47.6 (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)
AgApyL1 58.5 (67.4) 36.5 (46.7) 38.2 (51.1) 29.7 (40.7) 32.6 (42.9) 34.0 (44.5)
AaApy 34.8 (48.1) 38.0 (50.9) 32.0 (42.8) 32.8 (43.3) 33.5 (45.2)
Ll5N 37.4 (50.1) 41.0 (51.2) 45.6 (53.6) 44.4 (52.2)
Chrysoptin 35.8 (48.7) 37.9 (48.4) 37.9 (47.7)
Bm5N 41.0 (50.1) 40.1 (49.1)
Rat5N 87.6 (90.7)
Hum5N

FIG. 2. Alignment of the carboxyl-terminal regions of different members of the apyrase/5ⴕ-nucleotidase family. Abbreviations are as
listed in Table I. Identities in at least four of the aligned sequences are shaded. Hydrophobic carboxyl-terminal domains that are replaced by the
glycosylphosphatidylinositol anchor are shown in dark gray. The boxed peptide sequence of AgApyL1 is the one substituted by the boxed peptide
sequence of the rat 5⬘-nucleotidase (Rat5N) in the AgapyL1-myc-Crat construct. LI5N, L. longipalpis 5⬘-nucleotidase; Bm5N, B. microplus

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5⬘-nucleotidase; Hum5N, Homo sapiens 5⬘-nucleotidase.

FIG. 3. Neighbor joining tree showing the relationships among


members of the apyrase/5ⴕ-nucleotidase family. The E. coli 5⬘-
nucleotidase (Ec5N, P07024) was used as an out-group. Numbers indi- FIG. 4. A, Northern blot analysis on total RNA from adult female (f)
cate bootstrap values (2000 replicates). For the other abbreviations, see and male mosquitoes (m). Probes used for the hybridizations are indi-
the legend to Table I. Rat5N, R. norvegicus 5⬘-nucleotidase; Ll5N, L. cated on the right side. B, developmental expression of the putative A.
longipalpis 5⬘-nucleotidase; Bm5N, B. microplus 5⬘-nucleotidase; gambiae apyrase and of the two apyrase-like genes obtained by reverse
Hum5N, H. sapiens human 5⬘-nucleotidase. transcription-PCR. The PCR amplification of the actin mRNA is shown
as control. ⫺, no template; E1, 0 –24 h embryos; E2, 24 – 48 h embryos;
the AgApyL1 transcript, which is highly abundant in females, L1, 1st instar larvae; L2–L3, 2nd and 3rd instar larvae; L4, 4th instar
larvae; ep, early pupae; lp, late pupae; f0, f1, and f2, adult females 0, 1,
is not detectable in adult males. This is presumably the result or 2 days-old; m0 and m2, adult males 0 or 2 days old, respectively; bf0,
of the lower sensitivity of this technique as compared with bf24, and bf72, adult females 0, 24, or 72 h after blood-feeding,
RT-PCR. respectively.
Using RT-PCR with gene-specific primers, we also analyzed
the developmental expression profiles of the presumed A. gam- is expressed only in adult females, and no transcript is detected
biae apyrase and apyrase-like genes (Fig. 4B). The AgApy gene at any other developmental stage; moreover, the transcript

sites. The asterisk shows the stop codon. The boxed nucleotide at position 3402 marks the polyadenylation site. B, structural comparison of the
putative apyrase genes from A. gambiae and A. aegypti. Shaded boxes represent exons, and roman numerals refer to introns. Numbers express
lengths in nucleotides. The transcription start point is shown by an arrow. Untranslated regions are represented by black boxes. The polyadenyl-
ation site at the end of the transcript is indicated by a dot on a vertical line.
23866 5⬘-Nucleotidase Family Members from A. gambiae
cells (lane 4) or in cells transfected with AgApyL1-myc-Crat
(lane 3). The 13-amino acid difference in length between
AgApy-myc and AgApyL1-myc can only partially account for
the observed different relative mobilities of the two recombi-
nant proteins, which may be due to post-translational modifi-
cations. Altogether, these results strongly suggest that both
AgApy and AgApyL1 have the properties of secretory proteins.
Transformation of D. melanogaster with the AgApy Promot-
er—Because of our interest in salivary gland-specific promot-
ers, which may be of use in future vector control campaign, we
decided to test whether the 800 nucleotides located immedi-
ately upstream of the putative apyrase starting codon were
able to drive the tissue-specific expression of a reporter gene in
D. melanogaster. Using PCR, we inserted the 800-bp AgApy
segment in front of the E. coli ␤-galactosidase gene in the
transformation vector pCaSpeR-AUG-␤gal (27). The resulting
construct pCaSpeR-Apy-␤gal, schematically represented in the
upper part of Fig. 6, was used for transformation. Flies from
different transgenic lines were histochemically stained for
␤-galactosidase activity, yielding essentially the same expres-
sion patterns. In all cases, weak staining was detectable in the
thoracic region after 5 to 6 h of incubation at 37 °C. Only after
longer incubations (⬎16 h), a more intense color appeared that
could be clearly ascribed to the staining of the glands. The

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staining was more intense in the Apy9 line that contains a
double insertion of the transgene. The glands were stained both
in adult males and females, with the blue color being especially
dark in the terminal, convoluted portion of Drosophila salivary
FIG. 5. Immunoblot of myc-tagged AgApy and AgApyL1 pro-
glands. No staining was detectable, even after extensive incu-
teins expressed in COS-7 cells. The supernatants were separated by bations, either in the larval glands or in the adult glands of the
SDS/polyacrylamide gel electrophoresis, transferred to nitrocellulose, recipient strain yw. As can be seen in Fig. 6, A and B, a staining
and stained with an anti-c-myc monoclonal antibody. The following was also visible in the midgut after long incubation periods.
samples were analyzed: 1, AgApy-myc; 2, AgApyL1-myc; 3, AgApyL1-
However, staining of the midgut of similar intensity was also
myc-Crat. Lane 4 contained the supernatant from untransfected cells.
Molecular weight standards are indicated. The DNAs analyzed are found both in the negative and in the positive control strains,
schematically shown above the blot. SP, signal peptide; myc, myc i.e. the recipient strain yw and the Lys␤-gal strain, which
epitope. contains a P element insertion carrying the ␤-galactosidase
under control of the salivary gland-specific lysozyme P pro-
abundance increases shortly after blood-feeding, when the sal- moter of D. melanogaster.2
ivary glands are depleted of the corresponding protein. This
pattern of expression would be consistent with the role of DISCUSSION
apyrase in blood-feeding. More complex is the expression pat- We have isolated from A. gambiae a cDNA that shows strong
tern of AgApyL1, which is clearly expressed in females and at similarity to the A. aegypti apyrase gene and to different mem-
different larval stages but also at lower levels in males and in bers of the 5⬘-nucleotidase family. The mRNA is specifically
early pupae. Finally, the expression of the AgApyL2 gene is expressed in the salivary glands of adult females, where it is
restricted to larval stages, suggesting that this member of the produced mainly in the gland cells constituting the distal-
apyrase/5⬘-nucleotidase family may play some specific function lateral lobes, a region of the mosquito glands known to express
during the larval stage, perhaps linked to nucleotide genes whose function is related to blood-feeding (2, 15, 33). The
metabolism. deduced protein contains at the amino terminus a putative
Expression of myc-tagged Recombinant cDNAs in COS-7 signal peptide and does not seem to carry any transmembrane
Cells—To ascertain whether the putative apyrase and the region; these data imply that it is secreted. This interpretation
apyrase-like 1 proteins are indeed secreted, we tagged them is supported by the finding that a myc-tagged recombinant
with an epitope that would enable their immunological detec- version of the protein is secreted in the cell culture medium
tion and followed the transient expression of the AgApy-myc when expressed in COS-7 cells. All these observations strongly
and the AgApyL1-myc constructs in COS-7 cells. In addition, suggest that the AgApy gene is likely to encode the A. gambiae
we also analyzed AgApyL1-myc-Crat, a construct derived from apyrase and is responsible for the previously described anti-
AgApyL1-myc, whose endogenous carboxyl-terminal domain platelet activity ascribed to the salivary apyrase (34).
was replaced by the carboxyl terminus of the rat 5⬘-nucleotid- We have also identified another protein belonging to the
ase (see boxed sequences at the carboxyl terminus in Fig. 2); apyrase/5⬘-nucleotidase family, and intriguingly, sequence
this domain is known to be responsible for the glycosylphos- comparison showed that it is significantly closer to the A.
phatidylinositol anchoring of this protein (31). After transfec- aegypti apyrase and to AgApy than to 5⬘-nucleotidases from
tion and overexpression in COS-7 cells, the supernatants were different organisms. In the absence of additional information
concentrated and analyzed by Western blot for the presence of regarding its biochemical activity, we named the corresponding
the recombinant proteins using an anti-c-myc monoclonal an- gene AgApyL1 (A. gambiae apyrase-like 1). As previously
tibody. As shown in Fig. 5 (lanes 1 and 2), both the AgApy-myc shown, the expression of AgApyL1 is enriched in female sali-
and the AgApyL1-myc proteins were found in the supernatant vary glands (15), yet the corresponding transcript is detectable
of COS-7 cells. However, no proteins could be detected in the also in other female tissues, at different larval stages, and at a
supernatant by the anti-c-myc antibody either in untransfected lower level in adult males. Such a pattern of expression would
5⬘-Nucleotidase Family Members from A. gambiae 23867

FIG. 6. Histochemical detection of E. coli LacZ expression in the salivary glands of transgenic D. melanogaster. The P element-based
vector used for Drosophila transformation is shown at the top. Flies were stained overnight with X-gal and dissected after the appearance of the
blue color. Shown are undissected (A) and partially dissected (B) fly of the Apy9 line carrying a double insertion of the transposon; the arrows point,
respectively, to the linear, intermediate portion, and to the terminal convoluted part of the salivary glands. C, gland dissected from an individual
of the Apy13 line, showing the stained terminal portion at higher magnification. Staining of the salivary glands was never observed in the control
line yw, whereas staining of the midgut, similar to the one visible in B, could be observed after overnight staining both in the yw and in the Lys␤-gal
lines.

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be compatible with a possible 5⬘-nucleotidase function. More- their salivary apyrase. Thus, AaApy and AgApyL1 are most
over, AgApyL1 is a secreted molecule, as suggested by the likely orthologous, and AaApy and AgApy are paralogous. Both
expression of the myc-tagged recombinant protein in cultured the exon-intron structure of the AaApy and AgApy genes and
mammalian cells. These observations raise the possibility that the difference in the length of their 3⬘-untranslated region
AgApyL1 may have 5⬘-nucleotidase activity and, when secreted would be in agreement with this hypothesis.
with the saliva and injected into the host skin, may play some We have also revealed the existence of a third member of the
role in blood-feeding. As was recently proposed for the salivary family, AgApyL2, which was identified by searching an A.
5⬘-nucleotidase of the sandfly L. longipalpis (35, 36), it may be gambiae genomic data base; it exhibits a larval-specific expres-
involved in the production of adenosine from the ADP and ATP sion profile. We do not know what function this gene may have
released from the injured host tissue. Adenosine is not only an during the larval stages and/or if there is any tissue- or organ-
antagonist of platelet recruitment, adhesion, and aggregation specific expression in the larvae; however, it could play some
but also a potent vasoactive agent (14); therefore, the hemo- role in connection to nucleotide metabolism. The existence of
static action of apyrase, which results in the production of multiple genes related in sequence to 5⬘-nucleotidase has been
AMP, would be amplified by the activity of a salivary 5⬘-nucle- demonstrated in other organisms (32), yet the significance of
otidase, capable of further converting the AMP to adenosine. this redundancy remains to be clarified.
We should stress here that we tried to assay for apyrase The putative apyrase gene represents the first salivary
and/or 5⬘-nucleotidase activity in concentrated cell superna- gland-specific gene isolated from the African malaria vector A.
tants containing, respectively, the AgApy-myc and the AgA- gambiae. The identification of control sequences capable of
pyL1-myc recombinant proteins, but we could not detect any conferring salivary gland-specific expression as well as a more
orthophosphate release according to the assay of Fiske and detailed understanding of the physiology of the glands are the
Subbarow (38). It is likely that the presence of the myc epitope necessary steps toward the development of malaria control
interferes with the correct folding or with the activity of the strategies based on the genetic modification of the mosquito
recombinant proteins. We have proposed, based both on the vector (33, 39 – 41). Salivary glands represent a crucial target
RT-PCR expression analysis and on the RNA in situ hybridiza- organ because malaria parasites can be transmitted to the
tion to salivary glands (15), that AgApy and AgApyL1 encode, vertebrate host with the saliva only after invading and travers-
respectively, an apyrase and a 5⬘-nucleotidase. However, at ing the salivary glands (40, 42). The availability of specific
this stage, we cannot rule out the possibility that they repre- promoters able to drive the expression of an “anti-parasite”
sent for example two apyrases or two secreted 5⬘-nucleotidases. gene in the glands may be of great help as part of a multi-step
The unambiguous assignment of the functions of these genes blocking strategy as soon as techniques for the introduction of
will require the purification of the corresponding proteins or foreign genes are available for A. gambiae. For these reasons
their expression in other in vitro systems (i.e. baculovirus ex- we tested the promoter activity of the region located immedi-
pression) that will preserve their enzymatic activity. ately upstream to the starting codon of the AgApy gene. We
Along with the A. aegypti apyrase, AgApy would represent used transgenic D. melanogaster rather than A. aegypti be-
the second apyrase gene isolated from a mosquito species. If cause transformation of the yellow fever mosquito is a rather
this is the case, then the observation that both enzymes are recent achievement and is a tedious technique. In comparison,
5⬘-nucleotidase family members suggests that the emergence of the transformation of the fruit fly is well established, and it can
the apyrase function adapted to blood-feeding may have origi- take advantage of a wide variety of transformation tools (43–
nated by a gene duplication event that took place before the 45). Moreover, promoter sequences both from chorion and silk
separation of Anophelinae and Culicinae from their common gland genes of the distantly related insect Bombyx mori (46, 47)
progenitor. This would imply a widespread occurrence of and also from midgut-specific genes of A. gambiae (48) have
apyrases of the 5⬘-nucleotidase type in different mosquito spe- been previously shown to be recognized and correctly expressed
cies. Moreover, from our data it appears that culicines and in D. melanogaster. Our observations show that some salivary
anophelines used different copies of the duplicated genes as gland-specific transcriptional regulatory elements are also con-
23868 5⬘-Nucleotidase Family Members from A. gambiae
served between A. gambiae and D. melanogaster. More specif- 11. Asai, T., Miura, S., Sibley, L. D., Okabayashi, H., and Takeuchi, T. (1995)
J. Biol. Chem. 270, 11391–11397
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13. Handa, M., and Guidotti, G. (1996) Biochem. Biophys. Res. Commun. 218,
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33116 –33122
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and S. D’Amelio for the help with the PAUP* 4.0b2 program and with 36. Ribeiro, J. M. C., Rowton, E. D., and Charlab, R. (2000) Insect Biochem. Mol.
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