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First published online February 12, 2007
Journal of Experimental Biology 210, 733-740 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.001289
Identification and characterization of a salivary adenosine deaminase from the sand fly Phlebotomus duboscqi, the vector of Leishmania major in sub-Saharan Africa
1 Vector Molecular Biology Unit, Laboratory of Malaria and Vector Research,
National Institute of Allergy and Infectious Diseases, National Institutes of
Health, 12735 Twinbrook Parkway, Room 2E-22C, Rockville, MD 20852,
USA
2 Department of Veterinary Hygiene, Faculty of Agriculture, Yamaguchi
University, Japan
3 Preventive Medicine and Biometrics, Emerging Infectious Diseases,
Uniformed Services University of the Health Sciences, Bethesda,
MD20814
4 Laboratory of Parasitic Diseases, National Institute of Allergy and
Infectious Diseases, NIH, Rockville, MD 20852, USA
Author for correspondence (e-mail:
jvalenzuela{at}niaid.nih.gov)
Accepted 18 December 2006
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Summary |
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Salivary gland homogenates of P. duboscqi converted adenosine to inosine, suggesting the presence of ADA activity in the saliva of this species of sand fly; furthermore, this enzymatic activity was significantly reduced when using either salivary glands of recently blood-fed sand flies or punctured salivary glands, suggesting that this enzyme is secreted in the saliva of this insect. This enzymatic activity was absent from the saliva of P. papatasi. In contrast to other Phlebotomus sand flies, we did not find AMP or adenosine in P. duboscqi salivary glands as measured by HPLC-photodiode array. To confirm that the transcript coding for ADA was responsible for the activity observed in the saliva of this sand fly, we cloned this transcript into a prokaryotic expression vector and produced a soluble and active recombinant protein of approximately 60 kDa that was able to convert adenosine to inosine. Extracts of bacteria transformed with control plasmids did not show this activity. These results suggest that P. duboscqi transcripts coding for ADA are responsible for the activity detected in the salivary glands of this sand fly and that P. duboscqi acquired this activity independently from other Phlebotomus sand flies. This is another example of a gene recruitment event in salivary genes of blood-feeding arthropods that may be relevant for blood feeding and, because of the role of ADA in immunity, it may also play a role in parasite transmission.
Key words: sand fly, saliva, ADA, adenosine deaminase, insect saliva, Phlebotomus
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). Vasodilators,
anticoagulants and inhibitors of platelet aggregation are part of this
salivary mixture (Ribeiro and
Francischetti, 2003). Recently, with the technological advances in
DNA and protein sequencing, novel and unexpected molecules with potential
biological activities have been isolated from the saliva of blood-feeding
arthropods. Such molecules include hyalorunidase, nucleotidases, novel
apyrases, amine-binding proteins, tissue-factor pathway inhibitors and others
(Ribeiro and Francischetti,
2003). Another such protein is adenosine deaminase (ADA), which
was identified from transcripts of a salivary gland cDNA library of the New
World sand fly Lutzomyia longipalpis
(Charlab et al., 2000) and the
mosquitoes Culex quinquefasciatus and Aedes aegypti
(Ribeiro et al., 2001). This
protein or the transcript coding for this protein have also been identified in
other organisms including bacteria, fruit flies, mice and humans
(Charlab et al., 2001).
Adenosine deaminase (E.C. 3.5.4.4) catalyses the conversion of adenosine
and 2'-deoxyadenosine to inosine and 2'-deoxyinosine, respectively
(Cristalli et al., 2001). This
enzyme is evolutionarily conserved and has a beta alpha, 8 barrel structure
and zinc ion in the catalytic site (Wilson
et al., 1991). Adenosine deaminase deficiency in mice results in
the impairment of T and B cell function
(Resta et al., 1997) due to
the accumulation of adenosine resulting in severe combined immunodeficiency
(SCID).
The role of ADAs in insects, particularly in the saliva of blood-feeding
insects, was proposed to be in the hydrolysis of adenosine, a molecule
involved in pain perception (Charlab et
al., 2001). The activity of this enzyme in blood-feeding insects
was demonstrated in the saliva of L. longipalpis
(Charlab et al., 2000), C.
quinquefasciatus and Ae. aegypti
(Ribeiro et al., 2001) and
from the activity of the recombinant salivary ADA from L. longipalpis
(Charlab et al., 2001).
Of interest, ADA enzymatic activity or the transcripts coding for this
enzyme were not present in the salivary gland of the sand flies P.
argentipes, P. papatasi, P. ariasi and P. perniciosus, which
belong to the genus Phlebotomus
(Anderson et al., 2006;
Charlab et al., 2000). Instead,
the saliva of P. papatasi and P. argentipes contains large
amounts of adenosine and adenosine monophosphate (AMP)
(Ribeiro and Modi, 2001).
Therefore, it appeared that ADA activity was only present in the saliva of
Lutzomyia sand flies and not in Phlebotomus sand flies.
Recently, transcriptome analysis of the salivary glands of the sand fly
Phlebotomus duboscqi, a sibling species of P. papatasi,
resulted in the identification of a transcript with homologies to ADA
(Kato et al., 2006). In the
present work, we tested whether there is ADA activity in P. duboscqi
and whether the identified transcript codes for this activity. Because P.
papatasi does not have ADA activity, but has large amounts of adenosine
and AMP in the saliva, we also tested for the presence of adenosine and AMP in
the saliva of P. duboscqi sand flies.
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Materials and methods |
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Salivary gland cDNA library
The P. duboscqi salivary gland cDNA library was made as previously
described (Kato et al., 2006).
Briefly, mRNA was isolated from 55 salivary gland pairs using the
Micro-FastTrack mRNA isolation kit (Invitrogen, San Diego, CA, USA). The
PCR-based cDNA library was made following the instructions for the SMART cDNA
library construction kit (BD-Clontech, Palo Alto, CA, USA) with some
modifications (Kato et al.,
2006). The P. duboscqi cDNA library was sequenced as
previously described using an Applied Biosystems 3730xl DNA Analyzer (Foster
City, CA, USA) and a CEQ 2000XL DNA sequencing instrument (Beckman Coulter,
Fullerton, CA, USA) (Kato et al.,
2006).
Phylogenetic analysis
Consensus protein sequences were compared to related sequences from sand
flies as well as non-sand fly species obtained from GenBank. Sequences were
aligned using ClustalX (Jeanmougin et al.,
1998) and manually refined using BioEdit sequence editing software
(http://www.mbio.ncsu.edu/BioEdit/page2.html).
Phylogenetic analysis was conducted on protein alignments using Tree Puzzle
version 5.2 (Schmidt et al.,
2002). Tree Puzzle constructs phylogenetic trees by maximum
likelihood, using quartet puzzling, automatically estimating internal branch
node support (1000 replications). Derived trees were visualized using MEGA
(Molecular Evolutionary Genetics Analysis) version 3.1
(http://www.megasoftware.net/)
(Kumar et al., 2004).
Enzymatic assays
Measurement of activity was performed in quartz microcuvettes using 60
µl samples (Starna Cells, Atascadero, CA, USA). 20 µmol
l–1 adenosine in PBS was added to the cuvette, followed by
addition of the enzyme source. After mixing the solution by pipetting, the
absorbance between 220 and 300 nm was monitored at 1.5 or 3.0 min intervals
using a Lambda 18 spectrophotometer from Perkin Elmer (Norwalk, CT, USA).
Molecular sieving-high-performance liquid chromatography
Molecular sieving–high-performance liquid chromatography (MS-HPLC)
was carried out using a Dionex Summit system and Chromeleon software (Dionex,
Sunnyvale, CA, USA). For analysis, 20 µl of sample was applied to a
Superdex Peptide PC 3.2/30 column (Amersham Biosciences, Piscataway, NJ, USA)
using 10 mmol l–1 NaPO4, 150 mmol
l–1 NaCl, pH 6.5 as the mobile phase at a flow rate of 150
µl min–1 for 30 min of separation. Detection was performed
using a photodiode array detector and a 3-D chromatogram generated using
Chromeleon software. Adenosine and AMP standards (500 pmol) were applied
separately. Single pairs of salivary glands from P. papatasi and
P. duboscqi were sonicated in PBS, clarified by centrifugation and
applied to HPLC.
Expression of P. duboscqi ADA
DNA fragments encoding mature P. duboscqi ADA protein were
amplified and inserted into the cloning site of the pCRT7/NT-TOPO vector
(Invitrogen). The primers used for PCR amplification of the mature ADA
encoding fragments were PDBL_P02_G04_VF
(5'-GTTTTGGACATTTCGAACATTA-3') and PDBL_P02_G09_VR
(5'-TGGCTCCAAATGATTCAGACA-3') for 2G4 (PduM73) and PDBL_P02_G09_VF
(5'-CTTTGAAAATTAAACCGAAACGA-3' and PDBL_P02_G09_VR for 2G9
(PduM74). Escherichia coli strain BL21(DE3)pLysS cells (Invitrogen)
were transformed with the recombinant plasmid and grown in LB broth containing
ampicillin (50 µg ml–1). Production of recombinant protein
was induced by addition of IPTG to a final concentration of 1 mmol
l–1 and at 27°C for 3 h. The recombinant protein was
purified from the supernatant of bacterial sonic lysate using a MagneHis
Protein Purification System (Promega, Madison, WI, USA) and dialysed with
Centricon Plus-20 (Millipore, Bedford, MA, USA) to remove imidazole from the
elution buffer before further enzymatic analysis.
Sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and western blotting
The samples were treated with NuPAGE LDS sample buffer (Invitrogen) and
analysed on NuPAGE 10% Bis-Tris gels (Invitrogen) with NuPAGE MES SDS running
buffer (Invitrogen). To estimate the molecular mass of the samples, SeeBlue
markers from Invitrogen (myosin, bovine serum albumin, glutamic dehydrogenase,
alcohol dehydrogenase, carbonic anhydrase, myoglobin, lysozyme, aprotinin, and
insulin, chain B) were used. After electrophoresis, the gels were stained with
SimplyBlueTM SafeStain Coomassie® (Invitrogen) or
SilverQuestTM Silver Staining (Invitrogen).
For the western blotting, the proteins in the gel were transferred to nitrocellulose membrane (Invitrogen) using NuPAGE transfer buffer (Invitrogen). After blocking with 5% milk in Tris-buffered saline containing 0.1% Tween-20, pH 8.0 (TBST), the membrane was incubated with alkaline phosphatase (AP)-conjugated anti-His6/G antibody (Invitrogen) for 1 h at room temperature. After three washes with TBST, the blots were developed by addition of 5-bromo-4-chloro-3-indolyl-1-phosphate and nitro blue tetrazolium for visualization.
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). The first transcript (PduM73; NCBI accession number
DQ835357) of 1846 bp codes for a secreted protein of 57.6 kDa with an
isoelectric point of 5.5 (Fig.
1); the second transcript (PduM74) of 1810 bp codes for a protein
of 57.2 kDa with an isoelectric point of 5.8 (data not shown). Multiple
sequence comparison of P. duboscqi ADA with homologues from dipterans
such as the sand fly L. longipalpis and the mosquitoes Ae.
aegypti, Ae. albopictus and Culex pipiens and from mammals such
as mice, rats and humans shows an overall low level of identity
(Fig. 2); however, the amino
acids forming part of the active site (His116, His118,
Ala121, Gly328, His355, Glu358,
Gly381, Asp440, Asp441) are highly conserved
(Fig. 2). The ADA from P.
duboscqi and from other insects is larger than the ADA from mice, rats or
humans; a large string of approximately 80 amino acids at the N-terminal
region is not present in the mammalian ADA. Additionally, the signal peptide
sequence is not present in the mammalian ADA
(Fig. 2). Phylogenetic analysis
of ADA from different organisms produced a tree with two distinct clades, one
containing ADA from dipteran blood-feeders and the other clade containing
other organisms including Leishmania, Plasmodium, Entamoeba, mice,
rats and humans (Fig. 3).
Within the dipteran blood-feeders clade, sand flies form a distinct group,
separate from mosquitoes.
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). These data
suggest that the molecule responsible for this activity is secreted in the
saliva of this sand fly.
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Lack of adenosine and AMP in the saliva of P. duboscqi
It was previously shown that P. papatasi and P.
argentipes do not have transcripts coding for the enzyme ADA in their
salivary glands or the activity was not detected within their salivary glands
(Anderson et al., 2006;
Charlab et al., 2000); however,
it was shown that these sand flies have large amounts of adenosine and AMP
within their salivary glands (Ribeiro et
al., 1999). Although counterintuitive, due to the presence of ADA
activity, P. duboscqi salivary glands were tested for the presence of
adenosine and AMP by subjecting P. duboscqi SGH to MS-HPLC, and the
eluted products were detected by photodiode array detection. As expected, and
as previously shown, analysis of P. papatasi SGH resulted in the
presence of two major peaks with the same retention times as adenosine (18.5
min) and AMP (14.5 min), respectively (Fig.
6). By contrast, P. duboscqi SGH showed no peaks at the
retention times of adenosine and AMP (Fig.
6). Only a peak at 5 min was observed, which is the secreted
proteins from the salivary glands, as determined by the retention time and the
absorption spectra at 280 nm (Fig.
6). These data suggest that, in contrast to P. papatasi
and P. argentipes, P. duboscqi does not have AMP or adenosine in its
salivary glands and that it contains the active salivary ADA.
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Expression and activity of recombinant P. duboscqi salivary ADA
In order to determine if the ADA activity detected in P. duboscqi
SGH was related to the transcript coding for this enzyme, we cloned the two
transcripts coding for this protein into the PCRT7NT-TOPO bacterial expression
vector. The soluble expressed proteins were purified from the supernatant of
bacterial lysate by nickel magnetic beads, and an aliquot was subjected to
western blot analysis and detected using anti-histidine antibody. This
revealed a protein of approximately 60 kDa, which is the estimated molecular
mass of the predicted ADA including the 4-kDa N-terminal addition that
includes His6G and XpressTM peptide epitopes
(Fig. 7A, lanes 1 and 2). No
protein of this molecular mass was detected in the supernatant of bacteria
expressing the empty vector (Fig.
7A, lane 3). Furthermore, a soluble expressed protein with the
same migration pattern was detected by Coomassie blue staining
(Fig. 7B, lanes 1 and 2), and
this protein was not detected in samples from bacteria transformed with
control plasmid (Fig. 7B, lane
3). The purified soluble recombinant proteins were then tested for the
presence of ADA activity. Both sand fly recombinant proteins had a high level
of ADA activity as detected spectrophotometrically by the conversion of
adenosine to inosine (Fig.
8A,B). This activity was not detected in the supernatant of
bacteria transformed with control plasmid
(Fig. 8C). These data suggest
that the P. duboscqi transcript coding for an ADA is responsible for
the ADA activity detected in the saliva of this sand fly.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). This is
the first report of ADA in a sand fly from the genus Phlebotomus,
including data from transcriptome analysis from the salivary glands of P.
papatasi, P. ariasi, P. argentipes and P. perniciosus sand flies
(Anderson et al., 2006).
In the present work, we have demonstrated the presence of ADA activity in
the saliva of P. duboscqi and we also demonstrated that the soluble
recombinant protein produced from the transcript coding for this enzyme
exhibited ADA activity. Phylogenetic analysis placed P. duboscqi ADA
in the same clade with ADA from other blood-feeding arthropods. This group
belongs to the ADGF/CECR1 family of proteins identified previously in
Sarcophaga peregrina, Lutzomyia longipalpis, Drosophila, Aplysia and
humans (Dolezelova et al.,
2005). This sub-family of ADAs has an extended N-terminus region
and is targeted for secretion. These data suggest that P. duboscqi
acquired this activity independent of other Phlebotomus sand flies.
The question remains as to what the role of this protein is in blood feeding.
It was previously speculated that the activity may be related to the
hydrolysis of adenosine, an important component in pain perception and in
immunity (Charlab et al.,
2001). What is puzzling is that other Phlebotomus sand
flies do not have ADA in their salivary glands but they have large amounts of
adenosine and AMP, very active vasodilators and platelet inhibitors. Neither
adenosine nor AMP was present in the saliva of P. duboscqi, as
demonstrated in here. Lutzomyia longipalpis also lacks adenosine and
AMP in its saliva; however, it has maxadilan, a very potent vasodilator
(Ribeiro et al., 1989).
Maxadilan was not identified in the P. duboscqi cDNA library
(Kato et al., 2006). Therefore,
it appears that P. duboscqi may contain a novel vasodilator that will
replace the lack of vasodilatory activities exerted by AMP and adenosine in
other Phlebotomus sand flies. The fact that ADA is present only in
P. duboscqi and not in other Phlebotomus sand flies examined
to date emphasizes the ability of blood-feeding arthropods to acquired
independent strategies to overcome or modulate the host hemostatic,
inflammatory and immune system.
ADA has an important role in immunity as a result of the effects of
adenosine, 2-deoxyadenosine and the hydrolytic product of these compounds
(Cristalli et al., 2001).
Further work will be necessary to determine the effect of this enzyme in
parasite transmission. It may be possible that this enzyme changes the
environment in the skin where the Leishmania parasite is deposited by
the sand fly. Inosine is the primary metabolite of adenosine by ADA. Inosine
has been shown to inhibit the production of proinflammatory cytokines
including TNF-
, IL-1, IL-12, MIP1- and INF
in stimulated
macrophages and spleen cells (Hasko et al.,
2000). Additionally, adenosine and inosine can alter cutaneous
vasopermeability by activating A3 receptors on mast cells
(Tilley et al., 2000). Then it
may be possible that inosine may favor a Th2 environment that will benefit
parasite establishment in the skin of the mammalian host.
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Acknowledgments |
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Footnotes |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Anderson, J. M., Oliveira, F., Kamhawi, S., Mans, B. J., Reynoso, D., Seitz, A. E., Lawyer, P., Garfield, M., Pham, M. and Valenzuela, J. G. (2006). Comparative salivary gland transcriptomics of sandfly vectors of visceral leishmaniasis. BMC Genomics 7,52 .[CrossRef][Medline]
Charlab, R., Rowton, E. D. and Ribeiro, J. M. (2000). The salivary adenosine deaminase from the sand fly Lutzomyia longipalpis. Exp. Parasitol. 95, 45-53.[CrossRef][Medline]
Charlab, R., Valenzuela, J. G., Andersen, J. and Ribeiro, J. M. (2001). The invertebrate growth factor/CECR1 subfamily of adenosine deaminase proteins. Gene 267, 13-22.[CrossRef][Medline]
Cristalli, G., Costanzi, S., Lambertucci, C., Lupidi, G., Vittori, S., Volpini, R. and Camaioni, E. (2001). Adenosine deaminase: functional implications and different classes of inhibitors. Med. Res. Rev. 21,105 -128.[CrossRef][Medline]
Dolezelova, E., Zurovec, M., Dolezal, T., Simek, P. and Bryant, P. J. (2005). The emerging role of adenosine deaminases in insects. Insect Biochem. Mol. Biol. 35,381 -389.[CrossRef][Medline]
Hasko, G., Kuhel, D. G., Nemeth, Z. H., Mabley, J. G.,
Stachlewitz, R. F., Virag, L., Lohinai, Z., Southan, G. J., Salzman, A. L. and
Szabo, C. (2000). Inosine inhibits inflammatory cytokine
production by a posttranscriptional mechanism and protects against
endotoxin-induced shock. J. Immunol.
164,1013
-1019.
Jeanmougin, F., Thompson, J. D., Gouy, M., Higgins, D. G. and Gibson, T. J. (1998). Multiple sequence alignment with Clustal X. Trends Biochem. Sci. 23,403 -405.[CrossRef][Medline]
Kato, H., Anderson, J. M., Kamhawi, S., Oliveira, F., Lawyer, P. G., Pham, V. M., Sangare, C. S., Samake, S., Sissoko, I., Garfield, M. et al. (2006). High degree of conservancy among secreted salivary gland proteins from two geographically distant Phlebotomus duboscqi sandflies populations (Mali and Kenya). BMC Genomics 7,226 .[CrossRef][Medline]
Kumar, S., Tamura, K. and Nei, M. (2004).
MEGA3: integrated software for molecular evolutionary genetics analysis and
sequence alignment. Brief. Bioinform.
5, 150-163.
Resta, R., Hooker, S. W., Laurent, A. B., Jamshedur Rahman, S. M., Franklin, M., Knudsen, T. B., Nadon, N. L. and Thompson, L. F. (1997). Insights into thymic purine metabolism and adenosine deaminase deficiency revealed by transgenic mice overexpressing ecto-5'-nucleotidase (CD73). J. Clin. Invest. 99,676 -683.[Medline]
Ribeiro, J. M. (1987). Role of saliva in blood-feeding by arthropods. Annu. Rev. Entomol. 32,463 -478.[CrossRef][Medline]
Ribeiro, J. M. and Francischetti, I. M. (2003). Role of arthropod saliva in blood feeding: sialome and post-sialome perspectives. Annu. Rev. Entomol. 48, 73-88.[CrossRef][Medline]
Ribeiro, J. M. and Modi, G. (2001). The salivary adenosine/AMP content of Phlebotomus argentipes Annandale and Brunetti, the main vector of human kala-azar. J. Parasitol. 87,915 -917.[CrossRef][Medline]
Ribeiro, J. M., Vachereau, A., Modi, G. B. and Tesh, R. B.
(1989). A novel vasodilatory peptide from the salivary glands of
the sand fly Lutzomyia longipalpis. Science
243,212
-214.
Ribeiro, J. M., Katz, O., Pannell, L. K., Waitumbi, J. and Warburg, A. (1999). Salivary glands of the sand fly Phlebotomus papatasi contain pharmacologically active amounts of adenosine and 5'-AMP. J. Exp. Biol. 202,1551 -1559.[Abstract]
Ribeiro, J. M., Charlab, R. and Valenzuela, J. G.
(2001). The salivary adenosine deaminase activity of the
mosquitoes Culex quinquefasciatus and Aedes aegypti. J.
Exp. Biol. 204,2001
-2010.
Schmidt, H. A., Strimmer, K., Vingron, M. and von Haeseler,
A. (2002). TREE-PUZZLE: maximum likelihood phylogenetic
analysis using quartets and parallel computing.
Bioinformatics 18,502
-504.
Tilley, S. L., Wagoner, V. A., Salvatore, C. A., Jacobson, M. A. and Koller, B. H. (2000). Adenosine and inosine increase cutaneous vasopermeability by activating A(3) receptors on mast cells. J. Clin. Invest. 105,361 -367.[Medline]
Wilson, D. K., Rudolph, F. B. and Quiocho, F. A.
(1991). Atomic structure of adenosine deaminase complexed with a
transition-state analog: understanding catalysis and immunodeficiency
mutations. Science 252,1278
-1284.
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