Toward a catalog for the transcripts and proteins (sialome) from the salivary gland of the malaria vector Anopheles gambiae
1 Medical Entomology Section, Laboratory of Parasitic Diseases, National
Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, MD20892-0425, USA
2 Research Technology Branch, Twinbrook II, USA
* Author for correspondence (e-mail: jribeiro{at}nih.gov )
Accepted 1 May 2002
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Summary |
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Key words: Anopheles gambiae, mosquito, salivary gland, malaria, proteomics, transcriptome, genomics, blood-sucking insect
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Introduction |
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In an attempt to reveal the complexity of A. gambiae salivary
glands, a high-throughput approach designed to identify a large number of
cDNAs in the gland of this mosquito has been employed. Remarkably, only
approximately 15% of our cDNA isolates match A. gambiae sequences
previously reported (Arcà et al.,
1999); many of the remaining clusters have unknown functions.
Generation of a set of A. gambiae salivary cDNAs, in addition to the
Plasmodium genome currently available, may provide indispensable
tools for the systematic and comprehensive analysis of molecules that may play
an active role in the pathogenesis of malaria.
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Materials and methods |
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Mosquitoes
A. gambiae gambiae Giles mosquitoes were reared under the expert
supervision of Mr André Laughinghouse. Insectary rooms were kept at
26±0.5°C, with a relative humidity of 70% and a 16 h:8 h light:dark
photoperiod. Adult female mosquitoes used in the experiments were aged 0-7
days, took no blood meals, and were maintained on a diet of 10% Karo syrup
solution. Salivary glands from adult female mosquitoes were dissected and
transferred to 20 µl Hepes saline (HS; NaCl 0.15 mol l-1, 10
mmol l-1 Hepes, pH 7.0) in 1.5 ml polypropylene vials in groups of
20 pairs of glands in 20 µl of HS or as individual glands in 10 µl of
HS. Salivary glands were kept at -75°C until needed.
Salivary gland cDNA library construction
A. gambiae salivary gland mRNA was isolated from 80 salivary gland
pairs from adult females at days 1 and 2 after emergence using the
Micro-FastTrack mRNA isolation kit (Invitrogen, San Diego, CA, USA). The
polymerase chain reaction (PCR)-based cDNA library was made following the
instructions for the SMART cDNA library construction kit (Clontech, Palo Alto,
CA, USA). A. gambiae salivary gland mRNA (200 ng) was reverse
transcribed to cDNA using Superscript II Rnase H-reverse transcriptase
(Gibco-BRL, Gaithersburg, MD, USA) and the CDS III/3' PCR primer
(Clontech) for 1 h at 42°C. Second-strand synthesis was performed through
a PCR-based protocol using the SMART III primer (Clontech) as the sense primer
and the CDS III/3' primer as antisense primer. These two primers create
SfiIA and SfiIB sites at the ends of the nascent cDNA.
Double-strand cDNA synthesis was done on a Perkin Elmer 9700 Thermal cycler
(Perkin Elmer Corp., Foster City, CA, USA) using Advantage Klen-Taq
DNA polymerase (Clontech). PCR conditions were the following: 94°C for 2
min; 19 cycles of 94°C for 10 s and 68°C for 6 min. Double-strand cDNA
was immediately treated with proteinase K (0.8 µg µl-1) for
20 min at 45°C and washed three times with water using Amicon filters with
a 100 kDa cutoff (Millipore). Double-strand cDNA was then digested with
SfiI for 2 h at 50°C. The cDNA was then fractionated using
columns provided by the manufacturer (Clontech). Fractions containing cDNA of
more than 400 base pairs (bp) in size were pooled, concentrated, and washed
three times with water using an Amicon filter with a 100 kDa cutoff. The cDNA
was concentrated to a volume of 7 µl. The concentrated cDNA was then
ligated into a Lambda TriplEx2 vector (Clontech), and the resulting ligation
reaction was packed using Gigapack Gold III from Stratagene/Biocrest (Cedar
Creek, TN, USA) following the manufacturer's specifications. The obtained
library was plated by infecting log-phase XL1-Blue cells (Clontech) and the
amount of recombinants were determined by PCR using vector primers flanking
the inserted cDNA and visualized on a 1.1% agarose gel with ethidium bromide
(1.5 µg ml-1).
Sequencing of A. gambiae cDNA library
The A. gambiae salivary gland cDNA library was plated to
approximately 200 plaques per Petri dish (150 mm diameter). The plaques were
randomly selected and transferred to a 96-well polypropylene plate containing
100 µl of water per well. The plate was covered and placed on a gyratory
shaker for 1 h at room temperature. The phage sample (5 µl) was used as a
template for a PCR reaction to amplify random cDNA. The primers used for this
reaction were sequences from the TriplEx2 vector and were named PT2F1
(5'-AAG TAC TCT AGC AAT TGT GAG C-3'), which is positioned
upstream from the cDNA of interest (5' end), and PT2R1 (5'-CTC TTC
GCT ATT ACG CCA GCT G-3'), which is positioned downstream from the cDNA
of interest (3' end). Platinum Taq polymerase (Gibco-BRL) was
used for these reactions. Amplification conditions were: 1 hold at 75°C
for 3 min, 1 hold at 94°C for 2 min, and 33 cycles at 94°C for 1 min,
49°C for 1 min, and 72°C for 1 min and 20 s. Amplified products were
visualized on a 1.1% agarose gel with ethidium bromide. The concentration of
double-stranded cDNA was measured using Hoechst dye 33258 on a Flurolite 1000
plate fluorometer (Dynatech Laboratories, Chantilly, VA, USA). PCR reactions
(3-4 µl) containing between 100 and 200 ng of DNA were then treated with
exonuclease I (0.5 units µl-1) and shrimp alkaline phosphatase
(0.1 units µl-1) for 15 min at 37°C and 15 min at 80°C
on a 96-well PCR plate. This mixture was used as a template for a
cycle-sequencing reaction using the DTCS labeling kit from Beckman Coulter
Inc. (Fullerton, CA, USA). The primer used for sequencing (PT2F3) is upstream
from the inserted cDNA and downstream from primer PT2F1. The sequencing
reaction was performed on a Perkin Elmer 9700 thermocycler. Conditions were
75°C for 2 min, 94°C for 4 min, and 30 cycles of 96°C for 20 s,
50°C for 20 s and 60°C for 4 min. After cycle-sequencing the samples,
a cleaning step was done using the multiscreen 96-well plate cleaning system
(Millipore). The 96-well multiscreening plate was prepared by adding a fixed
amount (manufacturer's specification) of Sephadex-50 (Amersham Pharmacia
Biotech, Piscataway, NJ, USA) and 300 µl of deionized water. After 1 h of
incubation at room temperature, the water was removed from the multiscreen
plate by centrifugation at 750g for 5 min. After partially
drying the Sephadex in the multiscreen plate, the whole cycle-sequencing
reaction was added to the center of each well, centrifuged at
750g for 5 min, and the clean sample was collected on a
sequencing microtiter plate (Beckman Coulter Inc.). The plate was then dried
on a Speed-Vac SC 110 model with a microtiter plate holder (Savant Instruments
Inc, Holbrook, NY, USA). The dried samples were immediately resuspended with
25 µl of deionized ultrapure formamide (J. T. Baker, Phillipsburg, NJ,
USA), and one drop of mineral oil was added to the top of each sample. Samples
were either sequenced immediately on a CEQ 2000 DNA sequencing instrument
(Beckman Coulter Inc.) or stored at -30°C.
Sequence information cleaning
Raw sequences originating from the DNA sequencer were assigned one of five
letters in their result: ATCG for identified nucleotide bases, and N when the
sequencer program could not call a base. Usually the beginning and ends of the
sequences have a higher proportion of N calls. Sequences also contain primer
and vector sequences used in library construction. For this reason, raw
sequences were treated by a program written in VisualBasic 6.0 (VB) (Microsoft
Corp., Redmond, WA, USA) as follows. (i) Sequences were analyzed in their
first 80 bp for groups of four Ns, and, if found, the block of four Ns closer
to position 80 was used to trim the raw sequence from this 5' N-rich
region. (ii) For sequences longer than 110 bp, windows of 10 bp were screened
for the occurrence of four or more Ns above position 100. The positive window
with the smallest position value was used to trim the sequence from the
3' N-rich region. Sequences thus trimmed and having more than 10 % N
content were discarded. (iii) Good quality and trimmed sequences were then
searched for occurrence of the primers used in library construction (the SMART
III primer as well as the CDS/R primers). A moving window the size of the
primer was searched on the sequence for matches with the primer sequence. If
more than a 70 % match was obtained, or if a contiguous match longer than 50 %
of the length of the primer was observed, the sequence was trimmed at the
beginning or end of the window, depending on the expected position of the
primer. This simple algorithm avoided errors due to spurious insertions. (iv)
The trimmed sequence was `polished' by removing any trailing N residues. The
sequence final N content was assessed, as well as its AT content and length.
The final sequence was written to a FASTA-format file containing in its
definition line the actions taken by the program.
Searches for known sequence similarities and known protein domains of
the cDNA sequences
To obtain information on the possible role of the cDNA sequences, the FASTA
file containing all the stripped sequences was blasted against the GenBank
nonredundant protein database (NR) from the National Center for Biotechnology
Information (NCBI) using the standalone BlastX program found in the executable
package at
ftp://ftp.ncbi.nlm.nih.gov/blast/executables/
(Altschul et al., 1997). The NR
database as well as the cumulative updates were regularly downloaded,
uncompressed with GUNZIP (found at
www.gzip.org/
), and formatted for Blast program use with the FORMATDB program
(executables also found at
ftp://ftp.ncbi.nlm.nih.gov/blast/executables/
) with the help of a program written in PERL code (software found at
www.activeperl.com
). NCBI sequences are indicated in this manuscript by their accession number
as gi|XXXX where XXXX is a unique identifier number. The resulting file
was parsed, and the best match was incorporated in the FASTA definition line
after the delimiter|. The sequences were next submitted to the
standalone program RPSBlast (Altschul et
al., 1997
) and searched against the Conserved Domains Database
(CDD) (found at
ftp://ftp.ncbi.nlm.nih.gov/pub/mmdb/cdd/
), which includes all Pfam (Bateman et al.,
2000
) and SMART (Schultz et
al., 2000
) protein domains. The RPSBlast result file was parsed as
above and the best match incorporated also into the FASTA definition line of
the sequence. When all sequences of a particular cluster were blasted against
the NR protein database (using the BlastX program), the best protein match was
searched for the species from which the NR database sequence originated. If
the species was not A. gambiae, or no matches to the NR were found,
the cluster was marked as representing a novel A. gambiae sequence
(indicated by Y under the column marked N (novel) in
Table 1). All cluster sequences
that gave a match to an A. gambiae protein sequence were further
individually inspected to verify whether the cDNA sequence represented nearly
the same information translated as the protein match or a closely related but
different protein. In this latter case, a Y would also be added to the results
in Table 1 in the N (novel)
column for the row of the cluster in question.
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Sequence clustering
The FASTA file containing all sequences was clustered by first blasting
(using the BlastN program) each sequence against the formatted database file
using a Blast cutoff score of 1E-60. The resulting file was used to
join in a single cluster all sequences that shared at least one common
sequence in the BlastN result. Thus, if sequence A had a 1E-60
match to B and B had a similar match to C, the three sequences would be joined
even if A had a less meaningful score in relation to C. The clustering program
also made individual FASTA-formatted files for each cluster, sorted in
descending order of sequence size. When these files contained two or more
sequences, they were used as input for the sequence alignment program CLUSTALW
(Higgins et al., 1996), which
was called automatically by the clustering program. CLUSTAL alignment files
were thus created for each cluster having two or more sequences. This
clustering program was also written in VB. Finally, a program was written in
VB that combines all the results to create
Table 1 of this paper, except
for the Function column. The output of this program is imported into a
Microsoft Excel spreadsheet. In the supplemental material,
Table 1 includes hyperlinks to
the best NR protein match in the NCBI site, all FASTA files for each
individual cluster, CLUSTAL alignment files for each cluster, when available,
and the FASTA file for the whole database. Each cluster was individually
analyzed for the probable function of its translation product and assigned a
`probably secreted', `probably housekeeping' or `indeterminate' function. This
decision was based on the best match to the NR protein database and related
sequences as searched online at the NCBI site
(www.ncbi.nlm.gov
) and on the SMART and/or Pfam matches, including searches of the nature of
the domains by online searches of the respective sites.
Full-length sequencing of selected cDNA clones
A portion (4 µl) of the lambda phage containing the cDNA of interest was
amplified using the PT2F1 and PT2R1 primers (conditions as described above).
The PCR samples were cleaned using the multiscreen-PCR 96-well filtration
system (Millipore). Cleaned samples were sequenced first with PT2F3 primer and
subsequently with custom primers. Primer selection for complete sequence of
selected full-length cDNA was also assisted by a program (written in VB) that
identified unique primer sites within the sequences. To assemble the
sequences, the previously known sequence was blasted against the new sequence
using the standalone program b12seq found with the executable package provided
at the NCBI ftp site mentioned above. After identifying the regions of
overlap, the two sequences were joined. The program attempted to locate a
poly(A) region by using a 12-bp window in which 11 A residues would constitute
a poly(A) string. If no poly(A) was found, a new set of primers would be found
to continue extension of the cDNA. The program also generates CLUSTAL
alignments of all sequences and produces a consensus output and the three
possible translations of this unidirectionally cloned RNA. The final alignment
is adjusted by hand. If necessary, the original tracings of the DNA sequencer
are reviewed for critical base calls. The translated sequences are submitted
as a FASTA file to the SIGNALP server (at
http://www.cbs.dtu.dk/
services/SignalP/
) (Nielsen et al., 1997),
which responds by e-mail: indicating whether a signal peptide exists and its
location. A program written in VB interprets this SIGNALP result file and
removes the signal peptide, if it is predicted to exist, to create a mature
protein sequence. Molecular masses using average molecular masses for C, H, O,
N, P and S are calculated for all protein sequences, as are pI based on
reduced proteins, following the pKa for amino acids within proteins as
indicated before (Altland,
1990
; Bjellqvist et al.,
1994
). This program, combined with the program generating
Table 1 of this paper, produced
an output that can be read by the spreadsheet program Excel to produce
Table 2 in this paper. In the
supplemental material, available on request, hyperlinks are given to all
proteins.
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SDS-PAGE
A precast 16% polyacrylamide gel was used and run in Trisglycine-SDS
buffer. Alternatively, a NU-PAGE 12% Bis-Tris gel, 1 mm thick (Invitrogen),
was used and run in MOPS buffer according to the manufacturer's instructions.
To estimate the molecular mass of the samples, SeeBlueTM markers from
Invitrogen (myosin, bovine serum albumin, glutamic dehydrogenase, alcohol
dehydrogenase, carbonic anhydrase, myoglobin, lysozyme, aprotinin and insulin,
chain B) were used. Salivary gland homogenates were treated with SDS (2%) or
NU-PAGE LDS sample buffer (Invitrogen) without reducing conditions. 20 pairs
of homogenized salivary glands per lane (approximately 20 µg protein) were
applied when visualization of the protein bands stained with Coomassie Blue
was required. For amino-terminal sequencing of the salivary proteins, 20
homogenized pairs of glands were electrophoresed and transferred to
polyvinylidene difluoride (PVDF) membrane using 10 mM CAPS, pH 11.0, 10%
methanol as the transfer buffer on a Blot-Module for the Xcell II Mini-Cell
(Invitrogen). The membrane was stained with Coomassie Blue in the absence of
acetic acid. Stained bands were cut from the PVDF membrane and subjected to
Edman degradation using a Procise sequencer (Perkin-Elmer Corp.). To find the
cDNA sequences corresponding to the amino acid sequence, obtained by Edman
degradation of the proteins transferred to PVDF membranes from PAGE gels, we
wrote a search program (in VB) that checked these amino acid sequences against
the three possible protein translations of each cDNA sequence obtained in the
mass sequencing project. This program was written using the same approach
utilized in the BLOCKS (Henikoff and
Henikoff, 1994) or PROSITE
(Sibbald et al., 1991
)
databases. The program is very useful when mixed sequence information occurs,
for example, amino-terminal sequences deriving from a mix of equal peptides.
In this case, two different cDNA sequences may be unambiguously found as
matches.
Statistical tests
Statistical tests were performed with SigmaStat version 2.0 (Jandel
Software, San Rafael, CA, USA). KruskalWallis ANOVA on ranks was
performed, and multiple comparisons were done by the Dunn method. Dual
comparisons were made with the MannWhitney rank sum test.
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Results and Discussion |
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SDS-PAGE of A. gambiae salivary proteins
Fig. 1 shows the pattern of
separation of A. gambiae salivary protein by SDS-PAGE stained by
Coomassie Blue. The gel shows relatively low tissue complexity, with
approximately 15 clearly visible stained bands and many others lightly
stained. To identify these proteins, they were transferred to PVDF membranes
and the bands cut from the membrane and submitted to Edman degradation.
Amino-terminal information was successfully obtained for many of these bands,
and they were identified as SG6 (approx. 10 kDa apparent molecular mass),
A. gambiae D7-related proteins 1-3 (approx. 10-14 kDa apparent
molecular mass), similar to Glossinia morsitans antigen 5 (approx. 30
kDa), similar to Aedes aegypti D7 (approx. 33 kDa), similar to A.
aegypti 30 kDa allergen (approx. 36 kDa), HP 8 (CB1, 44 kDa), similar to
HP 9 (bB2, 46 kDa) and herein called HP 9-like, SG1-like 2 (approx. 48 kDa),
putative 5' nucleotidase (approx. 64 kDa) and SG1 (approx. 105 kDa).
Although the predicted translation products of some of these proteins have
been reported (Arcà et al.,
1999), amino acid sequencing has not been performed before. Edman
degradation for other bands was attempted unsuccessfully, either because the
protein's amino terminus was blocked, or because PTH-amino acids could not be
reliably identified.
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cDNA library of the salivary gland of A. gambiae
To complement the data generated by SDS-PAGE and identify potentially novel
molecules in the salivary gland of A. gambiae, a cDNA library was
constructed and hundreds of independent clones randomly 5' sequenced.
When a cluster analysis of all 691 sequences from this library was performed
at e-60, 251 independent clusters were organized.
Subsequently, clusters were blasted against the nonredundant (NR) and protein
motifs databases. Signal peptides were predicted by submission of the cluster
sequences to the SignalP server, allowing the identification of putative
secretory (S) and housekeeping (H) cDNA. A comprehensive diagram depicting the
steps used for generation of the data is shown in
Fig. 2. The results are
presented as Tables
1,2,3,4.
The electronic versions of the tables, available on request, also contain: (i)
columns with hyperlinks to the best match of the NR database, (ii) links to NR
matches found for the cluster, (iii) matches to the conserved domain database
(CDD), (iv) FASTA-formatted files for each cluster, and (v) CLUSTAL alignments
of each cluster having two or more sequences.
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Fig. 3A shows that of the 691 sequences are concerned, 403 (58.3 %) code for putative S proteins, 165 (23.8 %) for housekeeping proteins and 123 (17.8 %) for proteins that could not be identified as housekeeping or secretory (unknown, U). Accordingly, cDNA for secretory proteins are highly represented in our library, suggesting that in vivo these molecules are preferentially expressed over H and U proteins. Fig. 3B shows that of the 251 clusters (including H, S and U), 127 (40.6 %) match sequences related to Drosophila melanogaster or other organisms; however, only 41 (16.3 %) have been assigned exclusively to the A. gambiae salivary gland. This indicates that 120 clusters (49.4 %) lack NCBI hits, although it is possible that related nucleotide sequences have been deposited as EST in other databases.
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cDNAs coding for putative housekeeping proteins
Table 1 describes A.
gambiae cDNA sequences with probable housekeeping function found in our
database. These include many different ribosomal proteins, t-RNA synthases,
cytochrome oxidase, elongation and translation factors, endoplasmic reticulum
proteins, NADH dehydrogenases, heat-shock protein, actin depolarization
factor, arrestin, aminotransferase, clatrin and porin gene product. Many
enzymes linked to respiratory metabolism or mitochondria proteins were
identified, including adenosine diphosphatase, ADP/ATP carrier protein,
unnamed protein product, V-ATPase and Na+K+ ATPase
subunits. Other enzymes or proteins were nucleoside diphosphate kinase,
ornithine decarboxylase, peptide chain release factor, prenylated rab
acceptor, RNAse L inhibitor proteasome protein, KE2 family, CG14525, Mago
Nashi protein, ß-glucosidase and condensation domain. Of interest,
clusters possessing matches to tetraspanin, hemopexin, heat-shock protein,
TRIO protein, multiprotein bridging factor (MBF) and antioxidant molecules
have also been found; their putative function is discussed below.
Tetraspanins, transmembrane proteins first discovered on the surface of
human leukocytes, have previously been identified in Drosophila
melanogaster, Caenorhabditis elegans, Apis mellifera and Manduca
sexta. This is the first description of tetraspanin in a mosquito. Their
function is not precisely known, but data from biochemical studies and
knockout mice suggest that they play a major role in membrane biology,
operating as molecular facilitators of diverse cellular functions from cell
adhesion to signal transduction (Todres et
al., 2000). Of interest, the tetraspanin CD9 associates with the
CD36, the Plasmodium falciparum receptor on platelets and endothelial
cells (Miao et al., 2001
).
Whether salivary gland tetraspanin has any role in cellparasite
interactions remains to be determined.
Also noteworthy is the identification of clones coding for proteins with
antioxidant function. This paper reports the first identification of hemopexin
(hpx) in the salivary gland of a blood-sucking insect. Hpx is a haem-binding
plasma glycoprotein that forms a line of defense against hemoglobin-mediated
oxidative damage during hemolysis
(Delanghe and Langlois, 2001).
In fact, hpx complexes with heme noncovalently with high affinity
(Kd<1 pmol l-1) and shows much lower
peroxidase- and catalyse-like activity than the nonprotein heme. In addition,
hpx heme binds nitric oxide (NO) and carbon monoxide (CO) and may protect
against NO-mediated toxicity, especially in conditions of hemolysis. Hpx is
thus a molecule that safely carries heme. Perhaps it is present in the
salivary gland due to the synthesis of relatively large amounts of salivary
peroxidase, which function as a vasodilator. In addition, a clone was
identified coding for thioredoxin (Thrx), a molecule that plays a fundamental
role in maintaining a reducing cellular milieu together with Thrx reductase
(ThrxR) and NADPH (Holmgren and
Bjornstedt, 1995
). Interestingly, ThrxR has both Thrx and protein
disulphide isomerase (PDI) as substrate
(Nakamura et al., 1997
), and a
clone for PDI is in our library. We conclude that components of the Thrx
system are present in the salivary gland of A. gambiae and that they
may operate in concert with Hpx and other molecules to prevent haem-driven
free radical attack, considering that this organ is actively engaged in
hemeprotein synthesis. Finally, we have found clones coding for heat-shock
proteins, a family of proteins that functions as chaperones, or are involved
in cell defense against external stressors from various sources
(Lund, 2001
). In fact, a
general function of heat-shock proteins is to prevent protein misfolding and
aggregation in highly crowded cellular environments or under conditions of
denaturing stress (Young et al.,
2001
).
We have also found clones with sequence homology to signaling molecules.
TRIO is a multidomain protein that binds the lymphocyte activating receptor
transmembrane tyrosine phosphatase (PTPase) and contains a protein kinase
domain. It has been proposed that TRIO may orchestrate cell-matrix and
cytoskeletal rearrangements necessary for cell migration
(Lin and Greenberg, 2000).
Although we have found a signal peptide for A. gambiae TRIO, our
alignment with Drosophila TRIO (a protein of approx. 200 kDa with no
secretion sequence) makes it uncertain whether this mosquito form of TRIO is,
in fact, a secreted protein or a truncated protein with a false-positive
signal peptide. Finally, an open-reading frame with the complete coding region
for a protein with homology to Bombyx mori MBF without signal peptide
has been identified (Takemaru et al.,
1997
). MBF is similar to endothelial cell differentiation factor
(Dragoni et al., 1998
), an
intracellular protein that plays a role in regulation of human endothelial
cell functions including formation of blood vessels. The precise functions of
TRIO and MBP in the salivary gland of A. gambiae remain to be
determined.
cDNAs coding for putative secretory proteins
Table 2 shows clusters
probably associated with secreted products. Some match sequences have already
been reported for the A. gambiae salivary gland; however, several are
novel with database hits to genes unrelated to A. gambiae, or without
database hits.
A cDNA has been identified having an open reading frame with signal peptide
and sequence homology to calreticulin, an ubiquitous intracellular protein
present in the sarcoplasmic reticulum and involved in calcium homeostasis
(Johnson et al., 2001).
Calreticulin has also been identified extracellularly in the supernatant of
Epstein-Barr virus-immortalized cells; this secreted form has been shown to
inhibit angiogenesis, the biological process by which new blood vessels are
formed (Pike et al., 1998
).
This suggests that the saliva of A. gambiae may inhibit endothelial
cell proliferation, a proinflammatory event associated with host response to
injury, and other proinflammatory responses
(Griffioen and Molema, 2000
).
The CLUSTAL alignment of calreticulin from A. gambiae, Amblyomma
americanum and D. melanogaster is shown in
Fig. 4.
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Another full-length clone containing a typical secretion sequence and
homologous to selenoproteins has been identified. The selenoproteins
incorporate selenocysteine, a cysteine analog in which a selenium atom is
found in place of sulphur. Although this family of enzymes has been identified
in Bacteria, Archae and Eukarya, being common in mammals
(Behne and Kyriakopoulos,
2001), this is the first report of a clone coding for
selenoprotein being identified in insects. All the selenoproteins identified
thus far are enzymes, with the selenocysteine residue responsible for their
catalytic function. Both intracellular and plasma selenoproteins have been
identified, indicating that these enzymes are part of the cellular and plasma
antioxidant defense system (Behne and
Kyriakopoulos, 2001
). In fact, pro-oxidants have been involved
with processes related to inflammatory reactions, such as endothelial cell
injury (Varani and Ward, 1994
)
and platelet aggregation (Pignatelli et
al., 1998
); accordingly, we suggest that this putative secreted
form of selenoprotein may be involved in attenuation of these reactions. The
CLUSTAL alignment of selenoprotein from A. gambiae and D.
melanogaster is shown in Fig.
5.
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We have also found a partial-length clone with sequence homology to
salivary apyrase and 5' nucleotidase from A. gambiae
(Champagne et al., 1995;
Arcà et al., 1999
).
These enzymes play a determinant role in controlling nucleotide concentrations
in the blood and preventing platelet aggregation by destroying ADP, a
pro-aggregatory molecule necessary for completion of platelet aggregation
triggered by most physiological agonists
(Francischetti et al., 2000
;
Gachet, 2000
). We have also
sequenced a clone with homology to anophelin from A. albimanus, a
tight-binding inhibitor of thrombin
(Francischetti et al., 1999
).
Calreticulin, selenoprotein, apyrases, 5' nucleotidase, anophelin and
peroxidase (Ribeiro and Valenzuela,
1999
) are some of the candidate molecules that provide the
redundant anti-hemostatic `barrier' to prevent host defenses triggered by
blood feeding. Finally, we have identified for the first time in the salivary
gland of A. gambiae a clone coding for a protein similar to A.
aegypti amylase (sugar digestion)
(Grossman and James, 1993
) and
confirmed the presence of transcripts for lyzozyme in this same tissue
(gi:894206) and most likely involved in bacterial cell-wall digestion
(Rossignol and Lueders, 1986
;
Gao and Fallon, 2000
).
A clone with a Pfam match for the mucin-like domain has been encountered.
This protein has 41 amino acid residues in the signal peptide, which is
atypical for the proteins coded by our library; whether this protein is a true
full-length clone or a truncated form remains to be determined. Nevertheless,
studies on host-pathogen interactions have led to the discovery of various
cell surface-associated and secretory mucins. Mucins and mucin-like molecules
have recently been described in several protozoan parasites at different
life-cycle stages. It is now becoming evident that mucins in parasites are
involved in cell-cell interaction and cell surface protection, thus helping
the parasite to establish infection (Hicks
et al., 1999). Whether A. gambiae salivary mucin-like
protein could somehow modulate parasite infectivity or help to lubricate
insect mouthparts remains to be determined.
Several clusters abundantly expressed in the salivary gland from insects
other than A. gambiae have been identified in our library. Among
these, three cDNA clusters related to proteins of the antigen 5 family
(Schreiber et al., 1997) were
found. These proteins have been designated here `antigen 5-related protein 1'
(A5R1) (homologous to antigen 5 from Glossinia morsitans)
(Li et al., 2001
), `antigen
5-related protein 2' (A5R2) (homologous to antigen 5 from D.
melanogaster) (Megraw et al.,
1998
) and `antigen 5-related protein 3' (A5R3) (homologous to
antigen 5 from Lutzomyia longipalpis)
(Charlab et al., 1999
).
Antigen 5 belongs to the larger CAP family of proteins that has such members
as mammal cysteine-rich secretory proteins (crisp), nematode Ag5-Ag3, vespid
antigen 5 and plant pathogenesis-related proteins. These secreted proteins
share a core sequence of about 200 amino acids whose precise function remains
largely unknown. The CLUSTAL alignment of antigen 5-related protein from
A. gambiae, G. morsitans, D. melanogaster and L. longipalpis
is shown in Fig. 6.
|
We have also found for the first time a cluster with sequence homology to
30 kDa allergen from A. aegypti
(Brummer-Korvenkotio et al.,
1996). Although the precise function of 30 kDa allergen is
currently unknown, it is clear that allergic reactions to mosquito bite are of
increasing clinical concern. In fact, cutaneous reactions usually involving
both IgE- and lymphocyte-mediated hypersensitivity are common with insect
bites, and systemic reactions including angioedema, generalized urticaria,
asthma and anaphylactic shock have been reported
(Almeida and Billingsley, 1999
;
Peng et al., 2001
).
Accordingly, identification of these potential allergens could lead to their
use as markers of bite exposure or, eventually, as antigens for use in
immunotherapy (Bousquet et al.,
1998
). The CLUSTAL alignment of 30 kDa allergen from A.
gambiae and A. aegypti is shown in
Fig. 7.
|
We have also confirmed the presence of previously described D7-related 1-4
transcripts (Arcà et al.,
1999) in our library. In addition, a novel full-length D7-related
protein containing a typical signal peptide was found, herein designated
A. gambiae D7-related 5 protein. Furthermore, a novel D7 sequence
that codes for a translated mature protein of approx. 33 kDa with high
similarity to A. aegypti D7 protein
(James et al., 1991
) has also
been encountered. A report on the D7 family of salivary proteins in several
blood sucking diptera has been recently published
(Valenzuela et al., 2002
).
Accordingly, the putative function of the D7 family is unknown, but the high
sequence similarity to odorant-binding proteins suggests that these proteins
are carriers for small ligands presumably involved in vector/host interactions
(Steinbrecht, 1998
). The
CLUSTAL alignment for the long form of D7 from A. gambiae and A.
aegypti D7 is depicted in Fig.
8.
|
A number of other A. gambiae sequences have also been reported and
they code for the so-called salivary gland (SG) proteins (SG1-8)
(Arcà et al., 1999); our
library has clones identical to signal peptide-containing SG 1-like 2, SG 2,
SG 3, SG 5, SG 6 and SG 7, in addition to SG 1-like proteins herein called SG
1-like 3 and SG 1-like 4, and an SG 7-like molecule herein called SG 7-like 1.
We could not identify transcripts for SG 1, SG 4 and SG 8. These proteins have
unknown functions. Of interest, nine unique sequences coding for the so-called
A. gambiae hypothetical proteins (HP) have been reported and
designated cE5, c8, c4, c10, c6, A36B, Df2, CB1 and bB2
(Arcà et al., 1999
). In
our library, we have identified sequences similar or identical to c10, bB2 and
cE5. Although cE5 has been designated as a hypothetical protein before, this
molecule has been more recently characterized as a potent inhibitor of
thrombin (Francischetti et al.,
1999
). In this regard, we have identified by Edman degradation the
N-terminal sequence compatible with CB1; in addition, a bB2-like protein
containing the sequence X6SDSEEA (X6SDSDEA in bB2) was
found (Fig. 1).
Finally, the A. gambiae cDNA library has a number of hypothetical proteins characterized by an open reading frame and a putative signal peptide with no database hits.
cDNAs coding for protein that could not be characterized as
housekeeping or secretory
Table 3 shows that for a
significant number of clones, no significant match to the NR database was
found, nor was indication of a signal peptide obtained. Accordingly, these
sequences could represent partial housekeeping or secretory cDNA or,
alternatively, truncated cDNA.
A catalog for the cDNA from the salivary gland of A.
gambiae
To gather the maximum amount of information about the putative secreted
proteins from the A. gambiae salivary gland, the sequences presented
in Table 2 that were classified
as `novel', with or without database hits, were resequenced to obtain, when
applicable, their full-length cDNA. The full-coding sequences with database
hits were then blasted to the NR protein database and SignalP server to,
respectively, confirm sequence novelty and the presence of a signal peptide
(Nielsen et al., 1997). In the
event a signal peptide was predicted to exist, the molecular mass and the pI
of the mature protein were also calculated and, when possible, the function
annotated. The same approach was performed for other A. gambiae
salivary gland cDNAs whose sequences have been reported or deposited in
GenBank. The clones without database hits with an open reading frame and a
putative signal peptide were subjected to the same bio-informatic analysis and
were designated hypothetical proteins (HP), as suggested before (Arcà
et al., 1998). In an attempt to provide a uniform and comprehensive
classification of these hypothetical proteins, we suggest designating each
such HP by a given number, beginning with 1. The nine previously described HP
have been designated herein HP1HP9 and the eight novel proteins
described in this paper have been named HP10HP17 (see
Table 4). Formal
characterization of such proteins and their biological function remains to be
determined.
Taking into account the 21 novel A. gambiae sequences described herein in addition to 25 previously described, there are 46 different salivary gland cDNAs coding for putative secreted proteins, most of them (42 sequences) being full-length clones with a clear signal peptide. The four remaining partial clones have been classified as secretory, based on their high sequence similarity to other unambiguously characterized extracellular proteins. Accordingly, SG 4 was not included in Table 4 since no signal peptide could be detected for this protein. Interestingly, we have found in our library the cDNAs corresponding to most proteins whose amino terminus had previously been identified by Edman degradation (Fig. 1, Table 4). In contrast, and as expected, the amino terminus of many putative proteins coded by secretory cDNA shown in Table 4 could not be identified, either because the protein is expressed in low-copy number or because of technical limitations inherent to Edman degradation. In some cases, the apparent molecular mass of some proteins detected by SDS-PAGE (Fig. 1) is different from that predicted by the cDNA (Table 4). This is most likely due to protein glycosylation or formation of dimers that have not been appropriately separated by SDS.
To our knowledge, Table 4 is the first attempt to create a comprehensive catalog of the cDNAs from the A. gambiae salivary gland coding for putative secretory proteins. It is clear from this set of cDNAs that many proteins could not have their putative function annotated. Eventually, however, such a catalog will contain a nonredundant set of full-coding cDNA sequences covering every A. gambiae salivary gland cDNA and possibly each salivary protein function. Thus, this transcript and protein catalog could form part of a large-scale and comprehensive functional analysis of mosquito genes and, together with information derived from Plasmodium spp. genome, could be an essential tool for understanding the molecular basis of malaria.
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