An updated catalogue of salivary gland transcripts in the adult female mosquito, Anopheles gambiae
1 Department of Structural and Functional Biology, University
"Federico II", 80126 Naples, Italy
2 Parasitology Section, Department of Public Health, University "La
Sapienza", 00185 Rome, Italy
3 Medical Entomology Section, Laboratory of Malaria and Vector Research,
National Institute of Allergy and Infectious Diseases, National Institutes of
Health, 12735 Twinbrook Parkway, Rockville, MD 20852, USA
4 Department of Molecular Biology and Biochemistry and Department of
Biological Chemistry, University of California, Irvine, CA 92697,
USA
* Author for correspondence (e-mail: jribeiro{at}nih.gov)
Accepted 14 August 2005
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Summary |
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Key words: saliva, malaria, sialome, transcriptome
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Introduction |
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The `classical' process of learning the function of salivary gland products
in vector arthropods starts with the discovery of a biological activity in
crude homogenates, then isolation of the protein and, finally, description of
the DNA sequence coding for the protein primary structure. Recent advances in
transcriptome techniques led to the reversal of these steps in such a way that
the primary sequence of many putatively secreted salivary proteins are now
known; but for only a minority of these do we yet know the function and even
whether they are really secreted (Ribeiro
and Francischetti, 2003). In the case of the mosquito
Anopheles gambiae Giles, the main vector of malaria in Africa,
previous transcriptome analysis of nearly 500 expressed sequence tag (EST) and
signal sequence trap methods was used to identify genes expressed in the adult
female salivary glands (Arca et al.,
1999
; Francischetti et al.,
2002
; Lanfrancotti et al.,
2002
). Accordingly, a combined non-redundant (NR) set of 40
proteins has been proposed to be of a salivary secretory nature in An.
gambiae; we can assign a function based on experimental evidence for
fewer than 10 of these.
The recent elucidation of the genome of An. gambiae associated with high-throughput transcriptome analysis facilitates further gene discovery. In the current paper, we present the analysis of an additional set of 2396 salivary gland cDNA sequences (total of 3087 compared with previous set of 691 clones), resulting in the discovery of 33 new salivary gland proteins. An NR catalogue including 72 transcripts of which 71 code for proteins of a putative secretory nature is presented and discussed. It should be helpful in designing experiments to determine the function for the majority of these transcripts. To this end, we analyzed the tissue and sex specificity of 88 transcripts and found that 27 are either exclusively expressed or enriched in the salivary glands.
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Materials and methods |
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Bioinformatic tools used
ESTs were trimmed of primer and vector sequences, clusterized and compared
with other databases as described before
(Valenzuela et al., 2003). The
BLAST tool (Altschul and Gish,
1996
) and CAP3 assembler were used
(Huang and Madan, 1999
), as
well as the ClustalW (Thompson et al.,
1994
) and Treeview software
(Page, 1996
).
O-glycosylation sites on the proteins were predicted with the program
NetOGlyc
(http://www.cbs.dtu.dk/services/NetOGlyc/)
(Hansen et al., 1998
). We
submitted all translated sequences (starting with a Met) to the Signal P
server (Nielsen et al., 1997
)
to detect signal peptides indicative of secretion. For visualization of EST on
the An. gambiae genome, the EST and cluster sequences were mapped to
the An. gambiae genome using the Artemis tool
(Berriman and Rutherford, 2003
)
after downloading the GenBank-formatted files for the An. gambiae
chromosomes from Ensembl
(ftp://ftp.ensembl.org/pub/current_mosquito/data/flatfiles/genbank/).
Because the files for each chromosome or chromosome arm are partitioned into
several different files, a program written in Visual Basic was used to read
the GenBank-format files to obtain a single fasta-formatted file for each
chromosome or chromosome arm and a uniform rather than relative location for
each gene, producing a feature file that could be read by Artemis.
Accordingly, Artemis could read a single flat file and a single set of
features for each chromosome or chromosome arm instead of breaking up each
chromosome into several dozen pieces. The unique fasta files for each
chromosome were, in turn, broken into 30-kb fragments with 5 kb from previous
sequence to speed BLAST analysis. The EST and contigs were compared with this
fragmented-sequence genomic database by blastn
(Altschul et al., 1997
) and the
output transformed to a file compatible with Artemis using a program written
in Visual Basic. Sequence annotation was done with the help of AnoXcel
(Ribeiro et al., 2004
).
Gel electrophoresis and Edman degradation studies
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of 20
pairs of homogenized An. gambiae adult female salivary glands was
performed using 1 mm-thick NU-PAGE 4% to 12% gels (Invitrogen). Gels were run
with MES buffer according to the manufacturer's instructions and the proteins
transferred to a PVDF membrane. The membrane was then stained with Coomassie
Blue in the absence of acetic acid; visualized bands (including a
negative-stained band) were cut and subjected to Edman degradation using a
Procise sequencer (Perkin-Elmer Corp., Foster City, CA, USA). More details can
be obtained in a previous publication
(Francischetti et al., 2002).
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 Visual Basic) that
checked these amino acid sequences against all possible reading frames of each
cDNA sequence obtained in the mass sequencing project. For details, see
Valenzuela et al. (2002b
).
Reverse transcription polymerase chain reaction (RTPCR) expression analysis
Salivary glands were dissected from 1- to 4-day-old adult females, frozen
in liquid nitrogen and stored at 80°C. Total RNA was extracted from
dissected glands, carcasses (i.e. adult females from which salivary glands had
been dissected) and adult males using the TRIZOL reagent (Invitrogen) and
treated with RNase-free DNase I. DNase-treated total RNA (50 ng) was amplified
using the SuperScript one-step RTPCR system (Invitrogen) according to
the manufacturer's instructions. Typically, reverse transcription (50°C,
30 min) and heat inactivation of the reverse transcriptase (94°C, 2 min)
were followed by 35 PCR cycles: 30 s at 94°C, 30 s at 65°C, 1 min at
72°C. For a subset of primer pairs, the annealing temperature was lowered
to 5560°C for optimal amplification. Twenty-five cycles were used
for the amplification of the product based on the ribosomal protein S7 mRNA
(rpS7) to keep the reaction below saturation levels and to allow a
more reliable normalization. The sequences of the oligonucleotide primers used
for rpS7 amplification were: Ag_rpS7-F-5' GGC GAT CAT CAT CTA
CGT GC 3' and Ag_rpS7-R-5' GTA GCT GCT GCA AAC TTC GG 3'.
The sequences of the other oligonucleotide primers are provided in the
supplemental material. Amplification reactions were analyzed on 1.2% agarose
gels stained with ethidium bromide.
Microarray analysis
Total RNA of five adult mosquitoes was extracted to prepare each sample. A
total of six samples three from non-blood-fed females and three from
sugar-fed male mosquitoes were analyzed. Isolated total RNA was
processed as recommended by Affymetrix, Inc. (Affymetrix GeneChip Expression
Analysis Technical Manual; Affymetrix, Inc., Santa Clara, CA, USA). Data
analysis was done with the Gene Chip Operational Software (GCOS) package.
Other procedures are exactly as described before
(Marinotti et al., 2005). The
microarray data are available at
http://www.angagepuci.bio.uci.edu/.
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Results and discussion |
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Because a significant number of contigs did not match any protein of the
An. gambiae proteome set, we considered the possibility that these U
class transcripts could be representing mostly untranslated (UTR) mRNA
regions. The PCR-based cDNA library used in this work supposedly provides for
full-length clones by use of a strategy using polydT primers and a modified
polymerase (Zhu et al., 2001)
when synthesizing the cDNA from RNA. The cDNA were directionally cloned into
the viral vector and sequenced only in the 5'
3' direction,
because extensions from the 3' end most often fail to cross the polyA
region. Accordingly, clones coding for 5' UTR could derive from
full-length clones with unusually long 5' UTR, because our average read
is larger than 400 nucleotides (nt). Alternatively, the sequenced cDNA could
correspond to the 3' UTR of the transcripts if the polymerase fell off
its template in the cDNA synthesis step or in the case of the transcript
having an Sfi restriction site, which is used during library construction.
When each contig position was located in the An. gambiae genome and
the closest gene in the same orientation identified, we observed that 230
contigs were near the 3' end of predicted exons, while only 51 were near
the 5' region of predicted exons. A
2 test indicates
this difference to be highly significant (P<0.001). These 230
contigs containing 263 sequences, indicating that approximately 9% of the
database sequences were truncated. Additionally, 567 contigs overlapped with
predicted exon locations, including some that did not give significant blastx
matches to the An. gambiae proteome because they contained only a few
base pairs within the predicted exon. A few (13) contigs (see supplemental
spreadsheet at
http://www.ncbi.nlm.nih.gov/projects/omes/An_gambiae_sialome-2005/sup-tableI.xls,
worksheet `Nuclear', column AR) matched predicted intronic regions, usually on
large genes, possibly representing alternative splicing and/or the cloning of
unprocessed pre-mRNA. We also observed that many contigs coded for different
locations of the same gene. A non-redundant list of gene matches is provided
in the supplemental spreadsheets within the worksheet named `ENSANGP
list'.
Following visualization of these U-class contigs into the genome using the Artemis tool, we further excluded some potential hits to UTR either because the nearest match was too far from the gene (`too far' was considered a distance longer than the length between the start of the first exon and the end of the last exon) or because the contig was probably coding for a novel gene (25 occurrences; worksheet named `Nuclear', column AR search for `novel' on supplemental spreadsheet). We thus arrived at 177 contigs probably located in the 3' UTR region of predicted genes and 23 located at the 5' UTR. We have also observed that contigs matching 3' UTR tended to be on large genes. Indeed, the set of predicted An. gambiae genes identified by direct contig matches to an exon had an average gene length (measured from the beginning of the first exon to the end of the last exon) of 2733 nt and had 3.32 exons, while that of genes identified by their 3' UTR was more than twice as large (6112 nt) with an average of 5.07 exons. Both differences are highly significant (P<0.001) when compared by the MannWhitney rank sum test. It should also be considered that some of these putative UTR transcripts may code for not-yet-identified exons. Re-annotation of the database taking into consideration the 3' UTR matches increased significantly the number of probable H-class genes while decreasing those of the U class (Table 1).
Transcribed transposable elements (TE)
Two transcripts on our database (contigs 284 and 285; supplemental
spreadsheet at
http://www.ncbi.nlm.nih.gov/projects/omes/An_gambiae_sialome-2005/sup-tableI.xls#BM147)
possibly derive from transposable elements. Their translation products are
similar to those of Caenorhabditis elegans proteins annotated as
CCHC-type and RNA-directed DNA polymerase and integrase and to TY elements in
the GO database, and also possess rve Pfam domains indicative of reverse
transcriptases. These transcripts may indicate active ongoing transposition
activity in An. gambiae.
Transcribed bacteria-like gene products
A relatively large number of transcripts (34 sequences, organized into 11
contigs, representing 1.2% of the salivary EST originating from nuclear genes)
match three genes located contiguously in chromosome arm 3R that code for the
putative proteins ENSANGP00000027299, ENSANGP00000027791 and
ENSANGP00000029569) (Fig. 1).
Investigation of nearby genes identified another instance of a possible family
member (Fig. 2;
ENSANGP00000026834) without EST representation in our database. When the
program PSI-BLAST was used with protein sequence 29569 above for two
iterations, it gave a 0.0-e value with hypothetical protein WD0513 of
Wolbachia endosymbiont of Drosophila melanogaster
(gi:42520378) in addition to identifying all other proteins of the cluster.
Further iterations of the program retrieve many bacterial proteins annotated
as belonging to the Rhs family (Hill et
al., 1994). Although the automatic ENSEMBL translation predictions
indicate spliced products for the transcripts coding proteins 27791 and 26834,
the cDNA we sequenced did not confirm the predictions. The transcripts are not
spliced and show one single large open-reading frame
(Fig. 1). The likely
single-exon structure of these contiguous genes and their similarity to
bacterial proteins suggests that this protein family cluster arose by
horizontal transfer from a bacterial genome. Because horizontal gene transfer
could be mediated by transposable elements
(Syvanen, 1994
), we
investigated whether such sequences were present in the vicinity of these
genes. Indeed, two retro transposable element-like fragments, named TE5p and
TE3p, flank the region containing the bacterial genes, together with five
additional genes, as shown in Fig.
2. TE5p, in particular, is located very close to the 5'-most
gene coding for ENSANGP00000026834 and could have originated the lateral
transfer. The BLAST alignments of TE5p and TE3p with described transposons are
shown in Fig. 3A,B. The
Wolbachia genus consists of Rickettsia-like organisms
infecting arthropods and conferring the phenomenon of cytoplasmic
incompatibility (Drancourt and Raoult,
1994
; Sinkins,
2004
). Of interest, Anopheles mosquitoes are resistant to
Wolbachia (Kittayapong et al.,
2000
; Ricci et al.,
2002
), and it is hypothesized here that these
Wolbachia-like transcripts may underlie such resistance.
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H-class gene products
Putative H-class genes were further classified according to their possible
function (Table 2). Results are
available online and can be searched on the columns labeled `Class' and
`Comments' (supplemental spreadsheet at
http://www.ncbi.nlm.nih.gov/projects/omes/An_gambiae_sialome-2005/sup-tableI.xls).
Not surprisingly, the most abundant gene class expressed constitutes members
of the protein synthesis machinery, which, together with transcription
machinery, protein modification and protein export, comprise 34% and 43% of
H-class contigs and sequences, respectively. Transporters and signal
transduction gene products are also highly represented in the library. EST
matching transporter proteins were found for several V-type ATPase subunits,
Na+/K+-ATPases, Ca2+-ATPases and several
families of solute carriers. V-type ATPases have been implicated in the
secretion of saliva in Diptera (Zimmermann
et al., 2003). Several transcripts coding putative receptors were
also found, including G-coupled proteins (ENSANGP00000023076), a kinase
associated with ß-adrenergic receptors (ENSANGP00000008658) and several
subunits of the NMDA/glutamate receptor family (ENSANGP00000018675,
ENSANGP00000025350 and ENSANGP00000021195). These may function in the
secretion signaling of the salivary glands.
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Among the transcripts coding for extracellular matrix components, we
highlight those coding for laminin (ENSANGP00000010745; a gene that needs to
be corrected in its intronexon borders), the heparin sulfate
proteoglycan perlecan (ENSANGP00000022422) and the enzyme chondroitin
N-acetylgalactosaminyltransferase, which is involved in the synthesis
of extracellular mucopolysaccharides (ENSANGP00000020105). These extracellular
constituents may be important for Plasmodium recognition of the
salivary glands, because sporozoites are known to recognize sulfated
polysaccharides (Pinzon-Ortiz et al.,
2001).
Several transcripts matched genes coding for transcription factors.
Table 3 lists some of interest
for the specialized function of the female salivary gland, including
transcription factors associated with expression of ER chaperones (XBP-1),
general transcription factors, and those associated with tissue
differentiation. In particular, two genes coding for Forkhead transcription
factors are indicated, as well as three involved in the Hairy pathway. The
Forkhead and Hairy transcription factors have been implicated in
Drosophila salivary gland differentiation and salivary protein
expression (Lee and Frasch,
2004; Mach et al.,
1996
; Myat and Andrew,
2000
,
2002
;
Myat et al., 2000
;
Poortinga et al., 1998
).
Expression of the gene coding for doublesex, which is associated with
sex-specific gene expression in Drosophila
(Baker et al., 1989
;
Baker and Wolfner, 1988
), is a
good candidate to explain the sexual dimorphism observed in adult mosquito
salivary glands.
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Updated catalogue of putative secreted salivary proteins
After identifying putative secreted proteins (supplemental spreadsheet at
http://www.ncbi.nlm.nih.gov/projects/omes/An_gambiae_sialome-2005/table4.xls),
we used this data set and the Artemis tool to identify novel proteins coded in
the An. gambiae genome. Indications of secreted polypeptides were
obtained with searches for the presence of signal peptides with the SignalP
program (Nielsen et al., 1997)
and of O-linked galactosylation sites (indicative of mucins) with the
NetOGlyc program (Hansen et al.,
1998
). A NR set of 72 putative proteins expressed in the salivary
glands of An. gambiae is presented in
Table 4; it includes 71
polypeptides predicted as secretory, 40 of which have been described
previously. Of these 40, seven are described now in full length. Thirty-three
proteins are indicated for the first time to be expressed in the salivary
glands of adult female mosquitoes. Of these 33 proteins, 29 were predicted by
the ENSEMBL annotation pipeline and four are novel. Of the 29 predicted by
ENSEMBL, 17 were re-annotated to fix the starting Met or stop codons.
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D7 salivary proteins
The first member of the D7 protein family was described in the mosquito
Aedes aegypti (James et al.,
1991) and later found in virtually all mosquito
sialotranscriptomes. Short (
15 kDa) and long (
30 kDa) forms are
recognized. Long D7 forms also exist in sand flies
(Valenzuela et al., 2002a
).
The function of these proteins has not been verified, although one short D7
protein from An. stephensi, named hamadarin, was shown to prevent
kallikrein activation by Factor XIIa
(Isawa et al., 2002
).
Previously, one long D7 and five short D7 proteins were known in An.
gambiae (Arca et al.,
2002
; Francischetti et al.,
2002
). Table 4
shows these six proteins and two additional D7 proteins, all coded from
contiguous genes in chromosome arm 3R. The three long D7 genes follow
each other in the forward direction, the first two having four exons, but the
last having only three exons (Fig.
4). The five short-form genes follow the long D7 cassette
in reverse orientation, the first four having three exons, while the fifth has
only two exons. Notably, an apparently non-coding transcript (contig_709) maps
just 250 nt downstream of the short D7 cassette in the reverse
orientation of the gene. We speculate that this transcript may be associated
with regulation of D7 expression. Combined, these genes represented
574 sequences in our database, or nearly 20% of over 3000 EST. It is also
interesting to note that the last gene in each of the cassettes, i.e.
D7L3 and D7r5, was the least represented in terms of number
of EST, indicating that they are expressed at lower levels than their similar
neighboring genes. Moreover, in comparison to the other members of the
cluster, D7L3 and D7r5 differ in the number of exons, and their pattern of
expression is not restricted to female salivary glands
(Table 4). Evidence for the
synthesis of all but one of these proteins (named D7L3 in
Table 4) in the salivary glands
of female mosquitoes was found by Edman degradation of bands resolved by
SDS-PAGE.
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The SG1 family of Anopheline proteins
This family of salivary proteins, having mature molecular masses near 44
kDa, was previously described as SG1 or gSG1
(Arca et al., 1999;
Lanfrancotti et al., 2002
).
They do not yield significant similarities by blastp to other proteins in the
NCBI database except for other anopheline proteins, including the distantly
related TRIO protein. Six genes of this family are known in An.
gambiae, five of which reside in chromosome X, while the gene coding for
the TRIO protein is in the 2R chromosome arm (supplemental table 4 at
http://www.ncbi.nlm.nih.gov/projects/omes/An_gambiae_sialome-2005/table4.xls).
Two of these gene products are reported here in their full-length
configuration. Four of the five genes in the X chromosome are observed in a
tandem configuration, including one in reverse orientation
(Fig. 5). This family has a
relatively high EST representation in our database, with a total of 114 EST.
Except for gSG1a having two EST, all others had 10 or more EST
represented (Table 4). One
polyadenylated transcript (contig_78) was found mapping in anti-sense
orientation of SG1_like-3 (Fig.
5). Its possible significance is unknown. Alignment of the six
protein sequences is not very informative, except for a weak similarity region
in the middle of the protein (Fig.
6). A hidden Markov model made from the Clustal alignments of the
six proteins was used to search the NR protein database of NCBI. All retrieved
protein sequences were of anopheline origin (not shown). Evidence for
secretion of gSG1b, SG1 and SG1-like3_long was found by Edman degradation of
SDS-PAGE protein bands (supplemental table 4 at
http://www.ncbi.nlm.nih.gov/projects/omes/An_gambiae_sialome-2005/table4.xls).
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Mucins
Due to their putative high number of serines and threonines and their high
probability of having 10 or more O-linked
N-acetylgalactosamine, three proteins are identified as mucins, two
of which have been described previously and one of which is novel
(Table 4). All of these
proteins have homologues found in sialotranscriptomes of An.
stephensi, and one in Culex quinquefasciatus. These proteins
might function in the lubrication of the mosquito mouthparts. One of them
(SG3) has a weak indication of a chitin binding site domain, and it is
possible that it binds to the chitinous linings of the salivary ducts and
mouthparts. These predicted transcripts were found to be enriched in salivary
glands of females and were also found in male mosquitoes
(Table 4).
Other salivary-expressed genes coding for proteins or peptides of unknown function
Table 4 lists 33 peptides
and proteins with no hits or non-significant e values when compared with GO,
PFAM and SMART databases. Fifteen of them were not previously reported as
being expressed in the salivary glands of An. gambiae. Additionally,
three previously described messages are now reported in their full CDS form.
Thirty of these 33 proteins have a signal peptide indicative of secretion,
although it should be noted that their final destination could be the ER or
Golgi complex. Except for the transcript coding for the protein described
before as cE5 (Arca et al.,
1999), which is the homologue of the An. albimanus
antithrombin peptide named anophelin
(Valenzuela et al., 1999
), we
have no information that could indicate the function of these gene products.
Some of these proteins apparently result from gene duplication events, such as
those listed in Table 4 as: (1)
hyp15 and hyp17, coding for basic peptides (pI>11.0) of
4.7 kDa and residing contiguously on chromosome X; (2) hyp10 and
hyp12, coding for slightly acidic peptides of
7.5 kDa residing
contiguously on chromosome arm 3R; (3) hyp8.2 and hyp6.2,
apparently unrelated in sequence similarity but coding for mature peptides of
7.6 and 6.2 kDa and residing contiguously on chromosome arm 2L; (4)
SG2 and SG2A, coding for mature peptides of 9.5 and 15.5
kDa, apparently unrelated in sequence similarity, residing close to each other
on 2L and (5) hyp4.2 and hyp13, coding for mature peptides
of 4.2 and 3.6 kDa on chromosome arm 2R. These pairs of genes show identical
or very similar patterns of expression (see
Table 4) and possibly reflect
examples of gene duplication and divergence of function
(Sankoff, 2001
), as do the D7,
SG1 and AG5 families described above.
Of these 33 salivary gland-expressed genes, 22 appear to code for proteins
found only in anopheline mosquitoes, five are common to Culicidae, one is
known to occur also in Drosophila and four are more generally
conserved. Together with the six members of the SG1 family, there are 28 gene
products that appear to be unique to anophelines and could be used as
antigenic markers of anopheline exposure for epidemiologic studies, as done
previously for ticks and sand flies (Barral
et al., 2000; Schwartz et al.,
1990
).
Among the gene products unique to mosquitoes (including anophelines and
culicines), we report here the full-length information for the 30_kD protein.
The 30_kD transcript sequence produced nearly 200 EST matches from our
database, being the second most abundantly expressed gene in the salivary
glands of An. gambiae. Splice variants of this protein are apparent
from the different assemblies of these EST. Transcripts coding for members of
this acidic protein family, first identified as the 30-kDa Aedes
allergen (Simons and Peng,
2001) and also named GE-rich protein
(Valenzuela et al., 2003
),
were found in all previously described transcriptomes of both culicine and
anopheline mosquitoes. Another unique mosquito protein family is represented
by hyp55.3 (Table 4).
Additionally, two An. gambiae genes code for a protein similar to a
salivary Culex protein annotated as a putative 14.5-kDa salivary
peptide. Although these two Anopheles proteins appear to be related,
their corresponding genes are located in different chromosomes. The protein
indicated as SG2a also has homology to a Culex putative salivary
protein.
One single EST identified an An. gambiae gene coding for a protein
with 49% identity to Drosophila retinin, a protein of unknown
function expressed in the insect eye. Genes of a more general conserved nature
expressed in An. gambiae salivary glands include the previously
described selenoprotein, the hypothetical proteins named in
Table 4 as hyp14.6 and hyp1.2,
and calreticulin. Although calreticulin functions as a chaperone in the ER,
and the An. gambiae salivary calreticulin has a carboxy-terminal
sequence HDEL suggestive of retention in the ER, proteins of this family have
anti-thrombotic functions in the extracellular compartment
(Nash et al., 1994;
Nauseef et al., 1995
;
Pike et al., 1998
;
Sontheimer et al., 1995
) and
have been described in the saliva of ticks
(Jaworski et al., 1995
). Its
possible function in the saliva of ticks and Anopheles mosquitoes
remains to be investigated.
For this broad class of proteins, we found evidence of synthesis of gSG7, gSG7-2 and the 30_kDa peptides by Edman degradation of salivary gland peptides resolved by SDS-PAGE.
Enzymes
Several transcripts coding for enzymes are identifiable. These enzymes are
probably associated with four groups of functional activities.
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Tissue and sex transcriptions specificity
While the function of most putative salivary proteins and peptides is
presently unknown, determination of their tissue and sex specificity may help
to direct further research to characterize these gene products. To this end,
we used RTPCR on total RNA extracted from female salivary glands,
female carcasses (i.e. adult females from which salivary glands had been
removed) and adult males. Eighty-eight mRNA, mostly encoding secreted
polypeptides, were selected on the basis of either their sequence similarity
or absence of any similarity to known proteins. Nine additional mRNA
previously analysed by RTPCR (AgApy, AgApyL1, D7r1, gSG1, gSG3, gSG6,
gSG7, gSG10, cE5/anophelin) were also included as controls for the
amplification reactions (Arca et al.,
1999; Francischetti et al.,
2002
; Lanfrancotti et al.,
2002
). We have also analysed the sex-dependent expression of the
genes shown in Table 4 using
the Affymetrix microarray chip for comparison with the RTPCR results.
Of the 72 gene products shown in Table
4, three are not represented in the Affymetrix gene set. The
combination of these two independent analyses allows the delineation of three
categories. The first is represented by genes either expressed at
approximately the same level in the three tissues examined or less abundantly
transcribed in female glands: proteins encoded by these ubiquitous genes are
presumably involved in housekeeping functions. Approximately one-third of the
genes analysed (25/72) belong to this group; a few representatives are shown
in Fig. 7A. In the microarray
experiment, these genes should show equal expression in males and females,
and, indeed, the average of the log of the hybridization signal ratio between
sugar-fed females and males for this set of genes was 0.11±0.07 (mean
± S.E.M.; N=24), a result not
significantly different from 0 (a value indicating equal hybridization signal,
because 100=1). These ubiquitously expressed genes may play
housekeeping roles related to glandular function, such as calreticulin, as
well as general immune mechanisms, such as the case of the lyzozymes and
prophenoloxidase activating serine proteases.
The second category of tissue-specific expression consists of 34 genes represented by 17 transcripts that are either female salivary gland specific (marked as `SG' in Table 4) (Fig. 7B) or whose expression is enriched in the female salivary glands (the 17 additional genes are marked as `Enrich' in Table 4) (Fig. 7C). Microarray experiments indicated a highly significant differential ratio of expression between sexes in both subgroups. The SG set had a mean log of ratios of 1.45±0.22 (N=17), and the Enrich set had an average ratio of 1.24±0.19 (N=15; two genes are missing on the microarray chip), indicating a geometric average increase of transcript expression of 28- and 17-fold, respectively, when female transcript expression is compared with that of males. Additionally, the hybridization signals for each probe set were analyzed with an algorithm (GCOS) that designated the presence or absence of the corresponding transcript. Again, in most cases, the transcripts identified as female specific by RTPCR were confirmed by the microarray data.
The female-enriched or female-specific salivary gland genes are likely to play a role in blood feeding as anti-hemostatics or immunomodulators. Nineteen of the 34 genes are either newly described here or had not been previously analyzed for their expression specificity. Jointly, these genes include all eight members of the D7 family, the AG5 protein gVAG, all six members of the SG1 family, the protein/peptides named 30_kDa, gSG7-2, hyp17, gSG7, hyp8.2, gSG6, hyp14.5, gSG5, hyp15, gSG8, hyp37.7, hyp6.2, the enzyme salivary peroxidase, 5'-nucleotidase, apyrase, the serine proteases sal_serpro1, sal_tryp_XII, Sal_serpro2, the salivary epoxy hydrolase and the salivary galectin.
The third subgroup of genes includes those expressed in female salivary glands as well as in adult males, with absent or irrelevant expression in female carcasses (Fig. 7D). Microarray experiments indicated that the average log of the ratio of the hybridization intensities between females and males was not significantly different from 0 (0.13±0.08; N=12). We assume that these genes are salivary gland specific and expressed both in male and female glands. The corresponding gene products are probably involved in sugar feeding, antimicrobial activity or in more general physiological gland functions. Overall, 13 genes appear to be part of this group, including those encoding three mucins, and the proteins/peptides hyp55.3, hyp10, hyp12, sg2, sg2a, gSG9, hyp6.3, and the sugar-digesting enzymes amylase and maltase (Table 4). With the exception of enzymes that may help sugar digestion in the mosquito crop and midgut, and the mucins that might help maintenance of the food canal, the function of the remaining gene products of this group is presently unknown.
Although the results obtained from the microarray experiment generally
agree well with those from the RTPCR experiments, we found some
noticeable discrepancies in the results for the genes sal_galectin,
hyp14.5, Sal_serpro2, sal_tryp_XII, Ag_epoxy_hydrolase and
AG5-related-4. One of the reasons for the observed incongruity may be
related to alternative splicing of the gene products. Indeed, RTPCR
expression analysis followed by cloning and sequencing of the amplified
fragments suggested that the salivary galectin (not shown) and sal_tryp
XII genes produce different polypeptides by alternative splicing. In the
case of female-gland-specific sal_tryp XII, a band 126 bp in length
is obtained, as expected, when female salivary gland RNA is used as template,
whereas larger products are amplified from RNA extracted from carcasses and
males (Fig. 8). Sequence
analysis indicated the longer product to be a transcript that retains a 98-bp
intron carrying an in-frame stop codon. This would give rise to a hypothetical
truncated product of 112 amino acids in place of the putative trypsin-like
protease produced in female salivary glands (431 amino acids). It is possible
that this tissue- and sex-specific splicing may have a regulatory role,
producing a functional protease only in the saliva of An. gambiae
females. As suggested above, if secreted, it may influence the clotting and/or
the complement cascades of vertebrate hosts. Microarray hybridization
experiments using a set of 10 or 11 short probes, as used in the Affymetrix
chip, cannot distinguish between these splice variants, making such
comparisons invalid (Carter et al.,
2005). On the other hand, the incongruence between RTPCR
experiments and microarray data may point to differentially spliced genes that
may have importance in tissue translation selectivity
(Black, 2003
).
|
Two of the nine genes included in this analysis as controls showed an
expression pattern slightly different from what has been reported before, most
probably because new primer pairs and different amplification conditions were
employed. The expression of gSG7 appeared enriched in female glands,
rather than equally expressed in female salivary glands and in males, as
previously reported (Lanfrancotti et al.,
2002), whereas cE5 showed very little expression in
carcasses in comparison to what was observed previously
(Arca et al., 1999
). In
Table 4, they have been
classified according to the more recent RTPCR results, although it
should be kept in mind that their expression pattern may be in-between these
categories. Very good overlap with previous analyses was obtained with the
other seven genes used as controls (AgApy, AgApyL1, D7r1, gSG1, gSG3,
gSG6 and gSG10). It is also interesting that cE5 is a homologue
of anophelin, a potent anti-thrombin peptide found in the salivary glands of
the New World mosquito An. albimanus
(Francischetti et al., 1999
;
Valenzuela et al., 1999
);
however, in An. gambiae, cE5 is not found selectively in female
glands, as shown here and previously (Arca
et al., 1999
), raising the possibility that it may exert a
different function in Old World mosquitoes.
Concluding remarks
Using high-throughput transcriptome analysis, we significantly expanded the
An. gambiae salivary gland transcript repertoire. Thirty-three novel
putative salivary proteins were identified, and the full-length sequences of
seven previously identified partial cDNA were reported. Moreover,
tissue-specific expression studies on selected clones allowed us to identify
27 additional genes that are either enriched or specifically expressed in the
salivary glands. The information obtained in the course of this analysis,
combined with the results from previous studies, allowed us to compile an
updated catalogue that includes a total of 72 transcripts, mainly encoding
putative secreted products. Forty-seven of these transcripts encode proteins
that may play essential physiological roles, as indicated by their exclusive
or preferential expression in female and/or male salivary glands. This
catalogue makes the mosquito An. gambiae the arthropod disease vector
for which the most complete salivary transcriptome is available. On the other
hand, the fraction of genes included in this list for which we know or can
postulate a function is surprisingly small, emphasizing how much we still have
to learn about bioactive molecules from the saliva of blood-feeding
arthropods. We believe that this updated catalogue should help our continuing
effort of understanding the evolution of blood sucking in vector arthropods
and the discovery of novel pharmacologically active compounds.
![]() |
Acknowledgments |
---|
This work was supported in part by grants from the European Union to M.C. and B.A. (BioMalPar N 503578), MIUR/COFIN funds to Vincenzo Petrarca and B.A., the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health to J.M.C.R., and by the UND/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), ID A20314 to O.M.
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