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