Exploring the sialome of the tick Ixodes scapularis
1 Medical Entomology Section, Laboratory of Parasitic Diseases, National
Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda Maryland, 20892-0425, USA
2 Research Technologies Branch, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD 20892, USA
3 Center for Vector-Borne Disease, University of Rhode Island, Kingston
02881-0804, USA
* Author for correspondence (e-mail: jribeiro{at}nih.gov)
Accepted 17 June 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: salivary gland, proteome, electrophoresis, hematophagy, Lyme's disease, tick, Ixodes scapularis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tick saliva is also important in transmission of tick-borne pathogens for
several reasons; it may enhance pathogen transmission, hypersensitivity to
saliva may modify the site of inoculation of pathogens, and it may promote
non-viremic transmission of viruses by cofeeding (Jones et al.,
1987,
1990
;
Nuttall et al., 2000
;
Wikel et al., 1994
;
Wikel, 1996
). A protein of
unknown function (named SALP16) has been characterized by immunoscreening an
expression salivary gland cDNA library obtained from I. scapularis
nymphs (Das et al., 2000
), as
have 13 other immunodominant proteins from I. scapularis
(Das et al., 2001
).
The composition of I. scapularis saliva is interesting in the
study of the biology of parasitehost relationships, the discovery of
novel biologically active components, and the identification of novel vaccine
targets against I. scapularis-vectored diseases. Toward these goals,
we constructed a salivary gland cDNA library from blood-feeding I.
scapularis and randomly sequenced 735 clones that yielded 410 cDNA
clusters. Based on BLAST homology to other proteins in the non-redundant (NR)
database, the presence of conserved domains of the SMART
(Schultz et al., 2000) or Pfam
(Bateman et al., 2000
)
databases, and the presence of a signal peptide indicative of secretion in
these clones (Nielsen et al.,
1997
), we identified 100 clusters that are probably associated
with secretory products. From these, we obtained full-length information on 87
different clones, herein reported, 19 of whose expression was confirmed by
identification of their amino-terminal sequence in PVDF-transferred salivary
proteins separated by SDS-PAGE. While descriptive in nature, this paper raises
many hypotheses about the compositional diversity of blood-sucking arthropods
and identifies several novel sequences that could have biological activity and
possibly serve as vaccine targets.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ticks and tick saliva
Tick saliva was obtained by inducing partially engorged adult female I.
scapularis to salivate (3-4 days post-attachment to a rabbit) into
capillary tubes using the modified pilocarpine induction method
(Valenzuela et al., 2000).
Tick salivary gland extracts were prepared by collecting glands from partially
engorged female I. scapularis as described
(Valenzuela et al., 2000
).
Glands were stored frozen at -75°C until needed.
Salivary gland cDNA library construction
I. scapularis salivary gland mRNA was isolated from 25 salivary
gland pairs taken from adult females at days 3 and 4 after attachment to a
rabbit host. The Micro-Fast Track mRNA isolation kit (Invitrogen, San Diego,
CA, USA) was used to isolate mRNA, which was reverse transcribed to cDNA using
Superscript II RNase H-reverse transcriptase (Gibco-BRL, Gaithersburg, MD,
USA) and the CDS/3' primer (Clontech, Palo Alto, CA, USA). Second-strand
synthesis was performed using a polymerase chain reaction (PCR)-based protocol
with the SMART III primer (Clontech) as the sense primer and the CDS/3'
primer as antisense primer. These two primers create SfiI A and B
sites at the ends of the nascent cDNA. Double-stranded cDNA was immediately
treated with proteinase K (0.8µgµl-1) and washed three times
with water using Amicon filters with a 100 kDa cutoff (Millipore).
Double-strand cDNA was then digested with SfiI. cDNA was then
fractionated using columns provided by the manufacturer (Clontech). Fractions
containing cDNA of more than 400 base pairs (bp) were pooled, concentrated and
washed three times with water using an Amicon filter with a 100 kDa cutoff.
cDNA was concentrated and ligated into an 8-Triplex2 vector (Clontech). The
resulting ligation reaction was packed using the Gigapack Gold III from
Stratagene/Biocrest (Cedar Creek, TN, USA) following the manufacturer's
specifications. The library thus obtained was plated by infecting log-phase
XL1-blue cells (Clontech), and the amount of recombinants was determined by
PCR using vector primers flanking the inserted cDNA and visualized on agarose
gels with Ethidium Bromide. For more details, see Valenzuela et al.
(2002).
Sequence of Ixodes scapularis cDNA library
The salivary gland cDNA library was plated to approximately 200 plaques per
plate (150 mm diameter Petri dish). Randomly picked plaques were transferred
to a 96-well polypropylene plate containing 100µl of water per well. The
bacteriophage sample (5µl) was used as a template for a PCR reaction to
amplify random cDNA using 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. After
removal of primers, the PCR product 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. After cycle
sequencing the samples, a cleaning step was done using the multiscreen PCR
96-well plate cleaning system from Millipore. Dried samples were immediately
resuspended with 25µl 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 sequenced immediately on a CEQ 2000 DNA sequencer
(Beckman Coulter Inc.) or stored at -30°C.
Bioinformatics
Detailed description of the bioinformatic treatment of the data can be
found elsewhere (Valenzuela et al.,
2002). Briefly, primer and vector sequences were removed from raw
sequences, compared against the GenBank non-redundant (NR) protein database
using the standalone BlastX program found in the executable package at
ftp://ftp.ncbi.nlm.nih.gov/blast/executables/
(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 predicted translated proteins were
searched for a secretory signal through the SignalP server
(Nielsen et al., 1997
).
Sequences were clustered using the BlastN program
(Altschul et al., 1990
) as
detailed before (Valenzuela et al.,
2002
), and the data presented in the format of
Table 1 in this paper. The
electronic version of the table has additional hyperlinks to ClustalX
(Jeanmougin et al., 1998
)
alignments as well as FASTA-formatted sequences for all clusters. The
electronic table is available upon request; e-mail:
jribeiro{at}nih.gov.
|
Full-length sequencing of selected cDNA clones
A sample (4 µl) of the -phage containing the cDNA of interest
was amplified using the PT2F1 and PT2R1 primers (same 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. Full-length sequences were again
compared with databases as indicated for the nucleotide sequences above, and
the data displayed as in Table
2, which has hyperlinks in its electronic version (available upon
request to
jribeiro{at}nih.gov).
|
SDS-polyacrylamide gel electrophoresis
NuPAGE 10% gels, 1 mm thick (Invitrogen), using reducing MES buffer, were
electrophoresed according to the manufacturer's recommendations to resolve
proteins in 60 µl of tick saliva. Salivary gland homogenates (SGH; 1.0
pairs per lane) were run in 12% gels under non-reducing conditions with
Bis-Tris buffer. To estimate the molecular mass of detected proteins,
SeeBlueTM markers from Invitrogen (myosin, bovine serum albumin, glutamic
dehydrogenase, alcohol dehydrogenase, carbonic anhydrase, myoglobin, lysozyme,
aprotinin and insulin, chain-B) were used. Samples were treated with NuPAGE
LDS sample buffer (Invitrogen). For amino-terminal sequencing of the salivary
proteins, the gels were transferred to PVDF membrane using 10 mmol
l-1 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
0.025% Coomassie Blue in the absence of acetic acid. Stained bands were cut
from the PVDF membrane and subjected to Edman degradation in a Procise
sequencer (Perkin-Elmer Corporation). To find the cDNA sequences corresponding
to the amino acid sequence obtained by Edman degradation, we wrote a search
program that checked these amino acid sequences against the three possible
protein translations of each cDNA sequence obtained in the mass sequencing
project. A more detailed account of this program is found elsewhere
(Valenzuela et al., 2002).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The complete Table 1 (available electronically; e-mail: jribeiro{at}nih.gov) containing 410 clusters was annotated to indicate whether each of the clusters is associated with a possibly secreted, probably housekeeping protein, or one of unknown function. These annotation and function assignments were based on both similarities to the NR or CDD databases and on whether the proteins indicate coding for a secretory signal peptide. We thus found 102 clusters possibly associated with secretory products. These 102 clusters account for a total of 310 sequences, or 42% of the cDNA database. Table 2 indicates the clusters possibly associated with secretory products, sorted alphabetically. The electronic version of the manuscript contains the tables for the clusters associated with probable housekeeping and unknown clusters, as well as links to all sequences, alignments and BLAST results.
Table 2 shows that, in
addition to the 13 proteins indicated above, there are several clusters
associated with anti-protease sequences or domains, such as
-2-macroglobulin and cystatin, and 28 clusters having the Kunitz domain
found in soybean trypsin inhibitor. Two serpins were also found, one of which
matches a previously reported I. ricinus sequence. One additional
cluster has the SMART TIL signature of trypsin inhibitors. Possible inhibitors
of platelet aggregation include disintegrins (four clusters) and
thrombospondin (five clusters). Three clusters code for proteins having
similarity to tick histamine-binding proteins, one of which has been already
described in I. scapularis.
A sequence matching the antimicrobial defensin was found, but this clone is truncated and does not have the distal 5' end of the starting methionine. Proteins or peptides with similarity to collagen or gap junction proteins are also represented, but their function is unknown. A serine carboxypeptidase, two serine proteases and metalloproteases appear to be secreted. More than 35 clusters are associated with proteins that are possibly secreted, but their function in tick feeding is not readily apparent. Also evident from Table 2 is the existence of several related proteins. Indeed, when the clustering of the database is done with a cutoff value of 1E-20 rather than 1E-60, several of these clusters collapse (for example, those labeled short proteins or those containing Kunitz domains), although the alignments indicate that these are composed of several different, but related, gene products (results not shown; see below).
Table 2A, available on request from the author (e-mail: jribeiro{at}nih.gov), contains information on clusters of sequences probably associated with housekeeping function. Three of these clusters, each containing only one sequence, all code for proteins of the 5'-nucleotidase family, a family previously associated with secreted salivary apyrase of mosquitoes. Of interest were also the finding of a sulfotransferase and an alkyl hydroperoxide reductase that could be linked to synthesis of sulfated products of secretion and salivary prostanoids, respectively.
Full-length sequence information on 87 clones
To obtain more information on this transcriptome collection, with emphasis
on the messages possibly associated with secreted proteins (the sialome set),
we obtained full-length sequence of 87 clones, the properties of which are
summarized in Table 3. 62 of
these sequences belong to seven distinct groups, obtained by comparing the
sequences against themselves using the BlastP program with a cutoff value of
1E-20 (see Materials and methods for more detail).
|
Peptide group 1
Peptides from group 1 consist of 22 sequences
(Table 3) representing the most
abundant family of messages in the salivary gland library (cluster 1;
Table 1). These sequences have
high similarity to the 14 kDa protein of I. scapularis (gi|
15428308), but have no other significant matches to the NR database. No motifs
were found when compared to CDD database. All sequences have a putative signal
peptide indicative of secretion, which end in the tripeptide Ala-Ala-Ala
(Fig. 1). Alignment of these 22
novel sequences with the 14 kDa protein (gi| 15428308)
(Fig. 1) indicates these
proteins belong to three closely related families, ETC, HNC and HDC, for the
amino-terminal sequence of the predicted mature peptides (see cladogram in
Fig. 1). These proteins have a
mature molecular mass predicted to vary from 9.1 to 11.5 kDa; most are basic
in nature due to a lysine-rich carboxy-terminal region. They all possess a
conserved sequence Asn-Gly-Thr-Arg-Pro, starting at position 5 of the putative
mature protein, which was detected twice by Edman degradation of protein bands
excised from gels subjected to SDS-PAGE from separated tick salivary proteins
(see below). Except for one sequence (ISL 1342), all have six conserved
cysteine residues. The function of these proteins remains elusive.
|
Peptide group 2
Peptides from group 2 (Table
3) are putative mature proteins varying in molecular mass from 6.5
to 8.4 kDa, of both basic and acidic pI. Four of the 13 proteins gave
significant matches to Kunitz domains, indicating they may be protease
inhibitors or otherwise interact with other protein domains. Most of the
peptides gave BlastP matches to the NR protein database, indicating similarity
to proteins annotated as protease inhibitors. Cysteine residues are conserved
in most peptides of this group, as well as a N-X-T preceding the third
conserved cysteine of the mature peptides
(Fig. 2). Remarkably, there is
significant conservation of the predicted signal peptide. In the first 24
amino acid positions, there are 12 positions that are identical or conserved
(excluding the initial methionine), whereas for the remaining 63 ungapped
positions there are 13 conserved positions. The 2-test
indicates these ratios to be significant at P=0.0223. This
conservation of the signal peptide was observed earlier in a family of
antimicrobial peptides of frog skin skin
(Charpentier et al., 1998
), and
in semenogelins, a family of mammalian semen proteins
(Lundwall and Lazure,
1995
).
|
To further investigate the nature of the peptide group 2, we built a hidden
Markov model based on the alignment shown in
Fig. 2, using the -f switch to
allow for the presence of multiple domains in the resulting model. Search of
the NR database produced six matches with an E value of 5.4E-005 or lower,
three of which are the mouse, the rabbit and the human anticlotting protein,
tissue factor pathway inhibitor (TFPI). TFPI is a blood coagulation inhibitor
containing three tandem Kunitz domains; two of these domains have been
demonstrated to interact with Factor VIIa or Factor Xa
(Girard et al., 1989). Single
Kunitz molecules with specificity for Factor VIIa or elastase have also been
characterized in libraries from phage display
(Dennis and Lazarus, 1994
) and
from extracts of the parasitic worm Ancylostoma ceylanicum ceylanicum
(Milstone et al., 2000
),
respectively. The model also recognized another I. scapularis
salivary protein, SALP10, but with a higher (less significant) E value of
1.9E-4.
Peptide group 3
Group 3 cDNA sequences code for short peptides of mature molecular mass
ranging from 3.5-4.8 kDa of both basic and acidic nature
(Table 3). All sequences are
relatively glycine- and proline-rich. Some sequences give weak matches to
proteins in the NR database annotated as collagen; these possess two conserved
cysteine residues in the mature peptide and remarkable conservation of the
secretory signal peptide (Fig.
3). All amino acid sites of the predicted signal secretory peptide
are conserved, against 18 of 35 sites on the mature peptide. A
2-test is significant at P=0.0422. It is possible
some of these sequences are alleles of an extremely polymorphic locus or,
alternatively, that they represent different conserved loci. The possible
function of these peptides remains elusive.
|
Peptide group 4
Group 4 sequences code for putative mature peptides having four conserved
cysteine residues, molecular mass 7.9-8.7 kDa, of both basic and acidic
nature. All display strong similarity (BlastP against NR database) to a
protein from I. scapularis named SALP10 (gi| 15428348), and
weak similarities to mammalian tissue pathway inhibitor (TFPI) and
bungarotoxin. 19 of 21 first amino acids (excluding initial methionine) are
conserved (Fig. 4), as compared
to 33 of the 69 amino acids of the mature peptide. This difference is highly
significant (2-test, P<0.001), indicating higher
conservation of the signal peptide rather than the mature protein. An HMM
model made from the alignment shown in Fig.
4 retrieved only SALP10 from the NR database, with an E value of
1.9E-070 but no other significant matches.
|
Peptide group 5
Three sequences in group 5 (Table
3) code for proteins of mature molecular mass ranging from 33.7 to
35.5 kDa of a basic nature, and having 24 conserved cysteine residues
(Fig. 5). Comparisons with the
NR protein database using BlastP indicate similarities to proteins annotated
as protease inhibitors, including TFPI and the protein Ixolaris, an inhibitor
of Factor VIIa (Francischetti et al.,
2002). ISL228_Cluster344 has a Kunitz domain, as indicated by the
SMART database. These proteins probably code for anti-clotting compounds.
|
Peptide group 6
Group 6 represents sequences giving similarities to histamine-binding
proteins (Table 3,
Fig. 6). ISL1040_cluster233 has
no matches to the NR protein database but has a significant match by RPSBlast
to the Pfam histamine-binding domain, whereas ISL1276_cluster 363 has no such
match but instead has similarity to the tick Rhipicephalus
apendiculatus histamine-binding protein found in the NR protein database.
These two proteins are mildly acidic and have a mature molecular mass of 32.6
kDa and 34.6 kDa, respectively. It is probable that these proteins function by
binding histamine or other small ligands.
|
Peptide group 7
The two sequences in group 7 match a sequence deposited in the NR database
from I. scapularis and annotated as thrombospondin. The two predicted
mature sequences, with eight conserved cysteine residues, code for two
peptides of molecular mass 10.2 and 11.6 kDa, one basic and the other acidic
in nature. Their similarities to thrombospondin proteins are not apparent.
Both sequences have weak similarities to disintegrin metalloproteases, and
ISL373_cluster33 has the cysteine-rich domain of ADAM proteases as predicted
by the Pfam database. No RGD domains found in disintegrins are observed in
these sequences, nor in any of the other sequences reported in
Table 3.
Fig. 7 shows the alignment of
the two proteins with the Ixodes thrombospondin found in the NR
database. The role of the cysteine-rich domain of ADAM proteases is not known
but it is postulated to interact with integrins and/or other attachment motifs
of cells and matrix proteins (Hooper,
1994). Accordingly, these peptides could be involved in disruption
of platelet aggregation, cell-matrix interactions and/or inhibition of
angiogenesis (Roberts,
1996
).
|
The remaining 24 novel sequences presented in this paper can be grouped as: (i) similar to previously reported I. scapularis salivary proteins; (ii) a novel, shorter, protein with a Pfam histamine-binding motif, but not similar to other HBP found in the NR database (when compared by a BlastP search); (iii) five novel proteins coding for different inhibitors of proteolytic activity; (iv) six enzymes; and (v) ten proteins probably secreted and with unknown function.
Messages coding for proteins similar, but not identical, to
previously reported I. scapularis sequences
ISL1083_cluster9 is 95% identical to the previously reported 25 kDa
proteins of I. scapularis (not shown) and may represent an allele of
a highly polymorphic gene or another closely related gene. ISL1083_cluster9,
which does not display a histamine-binding motif, is highly similar to two
other proteins found in the NR database that are also from Ixodes
scapularis salivary gland cDNA libraries and annotated as
histamine-binding, 17 kDa proteins. The alignment of the four sequences shows
highly conserved areas, including the putative secretory signal peptide
(Fig. 8). The mature 17 kDa
protein is a truncated version of the other three proteins, containing two
conserved cysteine residues in the mature form, while the remaining proteins
have an additional four cysteine residues. These proteins may have a function
in blood feeding by binding small mediator molecules involved in hemostasis or
inflammation.
|
Novel putative protein containing the histamine-binding domain
ISL868_cluster49 has no similarities to proteins in the NR database but has
a histamine-binding motif, a predicted signal peptide, and the molecular mass
of the mature protein is 23.3 kDa. This molecular mass is similar to
ISL1083_cluster9 analyzed above, I. scapularis 25 kDa protein A, and
I. scapularis histamine-binding protein, to which ISL868 may be
distantly related.
Sequences coding for different protease inhibitors
Five predicted proteins appear to function as protease inhibitors.
ISL1095_cluster291, an -2-macroglobulin truncated clone with highest
similarity to the Limulus protein, also demonstrates very high
similarity to vertebrate proteins. These protein inhibitors are very large and
entrap the proteases that they inhibit; they may also bind to cytokines
(Armstrong and Quigley, 1999
;
Borth, 1992
). Because the clone
we describe in this paper is the truncated carboxyterminal region, we do not
know whether there is a signal peptide indicative of secretion coded in this
message.
ISL888_cluster62 codes for a secreted peptide with mature molecular mass of
11.9 kDa containing the cystatin domain of cysteine protease inhibitors; 15
kDa cystatin has been described previously in several nematodes
(Dainichi et al., 2001;
Hartmann et al., 1997
;
Manoury et al., 2001
). These
nematode cystatins inhibit the lymphocyte asparaginyl endopeptidase involved
in class II antigen processing in human B cells and inhibit T-cell
proliferation. A similar function may be served by ISL888_cluster62.
ISTA397_cluster68 is similar to the I. scapularis TFPI-like
molecule Ixolaris (alignment in Fig.
9), a molecule containing one complete and one incomplete Kunitz
domain (Francischetti et al.,
2002). ISTA397_cluster68 has the same number of cysteine residues
in the first and second Kunitz domains as does Ixolaris. ISTA397_cluster68 may
accordingly work also as a TFPI, or inhibit some other proteases such as
chymotrypsin or trypsin (Petersen et al.,
1996
).
|
ISL1156_cluster318 codes for a 10 kDa peptide with a Kunitz domain, having considerable similarity to other proteins from the NR database annotated as protease inhibitors of both vertebrate and invertebrate origins.
Finally, ISL1268_cluster360 codes for a mature protein of 20.8 kDa with a serpin motif, highly similar to Limulus coagulation inhibitor and to other serine protease inhibitors of both vertebrate and invertebrate origins. Interestingly, the mRNA has two open reading frames, both of which code for serpins, one with a typical secretory peptide, the other apparently leading to an intracellular protein. The specificity and activity of these putative protease inhibitors remain to be determined.
Sequences coding for different enzymes
Six clones are reported to code for enzymes. ISL1194_5nuc codes for a
protein with high similarity to invertebrate and vertebrate
5'-nucleotidases and apyrases. 5'-nucleotidases have a signal
peptide indicative of secretion, which causes the protein to be expressed
extracellularly, and a carboxy terminus in which a GPI anchor fixes the
protein to the extracellular side of the membrane
(Ogata et al., 1990). The GPI
anchor is attached to a conserved serine residue, followed by a stretch
containing 15 or 16 hydrophobic amino acid residues. Neither mosquito salivary
apyrase, a secreted enzyme, nor a 5'-nucleotidase of sand fly saliva,
has this conserved serine. These enzymes also lack the hydrophobic carboxy
terminus, allowing the enzyme to be secreted
(Champagne et al., 1995
;
Charlab et al., 1999
). Analysis
of the carboxy terminus of ISL1194_5nuc
(Fig. 10) shows that it does
not have the conserved serine found in mammalian and constitutive tick
5'-nucleotidases. Instead of 15-16 hydrophobic residues, it contains
only eight such residues. Furthermore, it contains four charged (K+E) and
three polar (T+S) residues, making the carboxy terminus unlikely to be
intramembranous. ISL1194_5nuc is thus possibly responsible for the previously
described salivary apyrase of I. scapularis
(Ribeiro et al., 1985
), or may
code for a secreted 5'-nucleotidase.
|
ISL1316_cluster379 codes for a serine carboxypeptidase containing a signal
peptide indicative of secretion. The specificity of this putative
carboxypeptidase is unknown. It probably does not code for the previously
described kininase activity of I. scapularis saliva, which has
kinetic characteristics of another family of peptidases, the angiotensin
converting enzymes (ACE) (Ribeiro and
Mather, 1998). ISL1316_cluster379 carboxypeptidase could, however,
be the salivary enzyme described previously to inactivate the serum
anaphylatoxins C3a and C5a (Ribeiro and
Spielman, 1986
).
ISL812_cluster188 codes for a protein with high similarity to proteins from
the NR database annotated as chymotrypsin, elastase, enterokinase and
enteropeptidase. The best protein match is from a protease from the tick
Haemaphysalis longicornis
(Mulenga et al., 1999).
ISL812_cluster188 putative protein has a strong signal anchor as determined by
the SignalP program. It probably is not secreted and serves a housekeeping
function.
ISL1033_cluster65 and ISL1324_cluster383 have very high similarity to a
hypothetical protein from the tick I. ricinus and to other proteins
in the NR database annotated as disintegrins and metalloproteases. Both have
the Pfam reprolysin motif indicative of a zinc metalloprotease family, most
commonly found in snake venoms (Hooper,
1994). Neither has a signal sequence indicative of secretion;
however, the amino-terminal sequences for both were found in protein bands of
one-dimensional electrophoresis of saliva samples (see below).
Finally, ISL939_cluster238 has very high similarity to Drosophila melanogaster NADH-ubiquinone oxidoreductase, a typical mitochondrial enzyme ranging in molecular mass from 69 to 75kDa, and to other proteins annotated as deoxyguanosine/deoxyadenosine kinases, consistent with the finding of a deoxynucleoside (DNK) motif from the Pfam database. DNK are 44 to 56 kDa enzymes described on both mitochondria and cytosol (http://brenda.bc.uni-koeln.de). ISL939_cluster238 codes for a putative protein containing a signal peptide indicative of secretion, with a mature molecular mass of 45.8 kDa. It is thus possible that ISL939_cluster238 codes for a secreted DNK in saliva with an unknown function in the tick feeding process.
Sequence coding for proteins of unknown function
Eleven additional clones were fully sequenced, either because they
represented abundant clones or because their partial sequence contained a
signal peptide indicative of secretion. Although all of these full-length
clones code for putative proteins displaying a signal peptide indicative of
secretion, no function was indicated when their sequences were compared to the
NR or CDD database. ISTB418_cluster 179 codes for a 4.3 kDa basic peptide with
similarity to human and murine proteins of unknown function. ISL942_cluster53
has similarity to a Borrelia burgdorferi protein (E value 1E-4) and
weak similarity to a tick histamine-binding protein (E value 0.006). This
putative protein, and that coded by ISL1270_cluster22, has a predicted mature
molecular mass of 22.5-22.6 kDa, similar to the protein described in
Table 3 as histamine-binding,
not group 5 (ISL868_cluster49). Alignments of these three putative proteins
reveal no obvious similarities (not shown).
Initial characterization of the proteome set of Ixodes
scapularis
To obtain further information on the salivary proteome set of I.
scapularis, electrophoresis of saliva and SGH were performed by
one-dimensional SDS-PAGE followed by transference of the proteins to PVDF
membranes, staining with Coomassie Blue, and submission of the cut bands to
Edman degradation. 15 and 19 bands yielded useful sequence information from
saliva and salivary gland gels, respectively (Figs
11,
12). With the exception of one
larger molecular mass band in the saliva gel (FEVGKDYYY...), and three
sequences on the SGH gel, we tentatively assigned all other sequences to a
gene product, as follows.
|
|
Host proteins
Sequences originating from proteins in saliva included two matching rabbit
albumin and one matching the -chain of rabbit hemoglobin. Similarly,
the SGH-derived sequences included both the
- and ß-chains of
rabbit hemoglobin as well as a sequence with high similarity to Ig-
light chain.
Amino-terminal sequences matching putative proteins coded by cDNA
sequences from cluster 1
Two sequences in each of the two gels fractionating saliva and SGH matched
putative proteins belonging to the most abundant cluster of cDNA sequences.
The observed amino-terminal sequences matched those predicted by the SignalP
program. Mature sequences from group 1 peptides start with either HX or ET,
followed by C-[QKRQ]-NGTRPAS (see above and
Fig. 1). Accordingly, the
sequences HNXQNG-TRPASEENREGXDY and HKXQNGTRPASEKNREGXDY were obtained from
protein bands of saliva separated by SDS-PAGE and corresponding to the
sequences of clones ISL1129 and TB222. Gels from SGH yielded the Edman
degradation products HNXQDGTRPASE and HNXKNGTRPASE, matching clones ISTA48 and
TA379 for which we do not have full-length sequences. Notably, although
proteins from group 1 (Table 3)
vary in molecular mass from 9.3 to 11.5 kDa, they all are located in the 20-24
kDa region in both gels. It is thus possible that the proteins of this cluster
make dimers through disulfide bridges even when the samples are run under
reducing conditions or, alternatively, they may be modified by
post-translation mechanisms such as glycosylation.
Amino-terminal sequences matching putative proteins coded by cDNA
sequences from cluster 14
Two proteins belonging to cluster 14 were also represented in both gels
and, in both cases, represented by the pair of sequences from clones ISTB346
and ISL914. The observed amino-terminal sequences are in agreement with the
mature peptide sequence predicted by the SignalP program. Although the mature
peptide predicted by ISL914_cluster14 is 7 kDa, it was found in the 10-12 kDa
regions of the reduced saliva gel and in the 30 kDa region of the non-reduced
SGH gel, indicating that these molecules may form multimers through disulfide
bridges. Alternatively, this peptide may have a compact structure in its
oxidized state that precludes sufficient binding of SDS, leading to less
charge and apparently higher molecular mass in the gel experiment
(Pitt-Rivers and Impiombato,
1968). No Asn glycosylation sites were found in ISL914_cluster14
or in ISTB346.
Amino-terminal sequence matching the tick anticomplement protein,
Isac
The sequence SEDGLE... obtained from saliva run in the SDS-PAGE gel and the
tripeptide SED on the SGH gel were found in a location with an apparent
molecular mass of 48 kDa (Figs
11,
12), matching the previously
reported inhibitor of the C3 convertase, Isac
(Valenzuela et al., 2000).
Isac has a molecular mass of 18.5 kDa but behaves in gel chromatography as
though it has a larger molecular mass than predicted
(Valenzuela et al., 2000
).
Amino-terminal sequences from salivary proteins matching putative
proteins within the metalloprotease reprolysin domain
Two amino-terminal sequences were obtained from the gel used to separate
tick saliva that match metalloproteases having the reprolysin domain. These
two clones (ISL1324 and ISL1033) were fully sequenced as described above.
ISL1033_cluster65 codes for a 44.1 kDa protein, while ISL1324_cluster383 codes
for a 46.1 kDa protein. The SignalP program does not predict these protein to
be secreted. The observed amino-terminal sequences represent unusually distant
sites from the starting methionine residue, at positions 49 and 72, predicting
mature proteins of 36.7 and 38.2 kDa and compatible with their migration on
gels (Fig. 11,
Table 3). These proteins may be
secreted by a different pathway from the other proteins, perhaps a product of
apocrine secretion (Aumuller et al.,
1999). They may also be the result of proteolytic processing of a
pro-enzyme. It is also possible that both clones are truncated at their
5'-end, where a conserved stretch of 169 residues is sandwiched between
the pre- and proproteinase in snake venom metalloproteases
(Jia et al., 1996
). Indeed,
ISL033_cluster65 is very similar to a hypothetical protein of I.
ricinus (gi|5911708), which contains a longer predicted
amino-terminal. These metalloproteases may be involved in digestion of skin
matrix constituents or fibrinogen, like the hemorrhagic metalloproteases of
snake venoms (Leonardi et al.,
1999
; Tortorella et al.,
1998
).
Presence in saliva of the peptide coded by clones TA242, ISL1014 and
ISL818
These clones were classified as being of unknown function because they did
not produce any significant matches when compared with protein NR or CDD
databases. Their aminoterminal sequences, as predicted by the SignalP program,
were found in protein bands of saliva separated by SDS-PAGE.
Calreticulin sequences of SGH proteins
The sequences DPTVYFK... and DPAIYFK..., found in protein bands from
SDS-PAGE-separated SGH, match the secreted calreticulin of the tick
Amblyomma americanum (gi|3924593) and rat calreticulin
(gi|11693172), respectively (Fig.
11). We have not found any sequence matching calreticulin in our
own library, which appears to be underrepresented for cDNA sequences coding
for proteins of molecular mass greater than 50 kDa. These two amino-terminal
sequences indicate that calreticulins, abundant intracellular proteins
(Nash et al., 1994), are
probably produced in I. scapularis salivary glands, although their
secretory nature is not obvious.
Housekeeping and other protein sequences found in SGH proteins
The sequence AKDFIAGGVA matches those from cluster 64 with very high
similarity to the mitochondrial carrier enzyme ATP/ADP translocase. The
sequence MQIFV..., matching the cDNA clone ISL844 from cluster 201, has very
high similarity to ubiquitin. The amino-terminal sequence DPIMGYT... was not
found in the possible translations of our cDNA library but does match putative
oxidoreductases found in the NR protein database. Finally, the sequence
NEDLIL... does not match any possible translation product of our cDNA library
but does match the SALP17 protein from I. scapularis
(gi|15428298) at position 112. The protein sequence ARXDAYDNXSGIRARLH
matched clone TB210.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our library contains a remarkably large degree of redundancy, as shown by
the many related mRNAs, most of which are too different to be alleles from
polymorphic loci. In addition to those shown in Figs
1,2,3,4,5,6,7,8,9,10,
the previously reported salivary anticomplement protein (gi|8896135) is
82% identical to SALP20 (gi|5428300)
(Das et al., 2001). The long
evolutionary history of ticks may be responsible for this complex plethora of
related proteins. Indeed, when we sequenced similar salivary cDNA libraries
from sand flies (Charlab et al.,
1999
; Valenzuela et al.,
2001
), and mosquitoes
(Valenzuela et al., 2002
), we
found far less diversity of related molecules. This variability in the tick
salivary cDNA library is consistent with the reported high polymorphism of
salivary proteins among individual ticks analyzed by SDS-PAGE
(Wang et al., 1999
). The
adaptive role of this gene-duplication phenomenon may derive from divergence
of functions in duplicated genes. For example, a Kunitz-containing protease
inhibitor might evolve into another protease inhibitor of different
specificity, thus targeting another protease of the host blood-clotting
pathway. Another possible adaptive role for gene duplication is the generation
of different antigenicity epitopes within molecules of the same function,
allowing the tick to better evade host immune responses. It is interesting to
speculate whether each of these protein variants would have a differential
temporal expression. Because our cDNA library was made from 25 adult female
tick salivary glands removed from the tick 3-4 days after host attachment, and
because ticks vary up to 2 days in their total feeding time (5-7 days from
attachment to a rabbit), it is likely that our library represents an average
of messages translated within a broad range of physiologic ages. A microarray
experiment with messages obtained from ticks at different times
post-attachment could be used to detect individual messages produced at unique
times by individual ticks, thus testing the hypothesis of temporal switching
of similar salivary proteins in I. scapularis.
With regard to the related messages found in the salivary gland cDNA
library of I. scapularis, the higher conservation of signal peptides
found in peptide groups 2-4, compared with the remaining protein sequences, is
remarkable. This pattern was also found in secreted peptide families of
vertebrates (Charpentier et al.,
1998; Lundwall and Lazure,
1995
). Increased evolution of secreted rather than signal peptides
indicates possible conservation of a `secretion signal cassette' or strong
evolutionary pressure for variation of the secreted moiety, consistent with an
antigenic variation scenario.
This diversity of related salivary proteins, whether they vary from tick to
tick or temporally within individual ticks, will certainly pose an additional
burden in the attempts to develop a vaccine against tick salivary antigens
that may protect against tick-borne pathogens
(Valenzuela et al., 2001).
Defining invariant antigens, and/or using a cocktail vaccine approach will be
important for a successful vaccine development strategy.
With regard to the finding of host proteins in tick saliva and SGH, we
cannot rule out contamination by host blood trapped in the tick mouthparts by
tick regurgitation during saliva collection, or by tick-gut contents during
salivary gland dissection. Although our cDNA library did not contain a single
rabbit sequence match, and the tick mouthparts were thoroughly washed before
saliva collection, this does not eliminate the possibility of regurgitation.
Host Ig secretion in tick saliva has been reported before in other ticks with
Ig-binding proteins (IGBP) (Wang and Nuttall,
1995a,b
,
1999
), and is postulated to be
the carrier for this host protein through the tick midgut and salivary gland
epithelia. The biological reason for tick IGBP may be related to counteracting
the possible noxious effects of host Ig against midgut or hemocoel targets;
any other explanation for this seemingly wasteful secretion of host albumin
and hemoglobin is not immediately apparent. It is interesting to speculate
whether these host proteins are modified by the tick by glycosylation or by
other additions. Incorporation of such antigenic epitopes into self molecules
may be a strategy for tick suppression of host immunity against potentially
antigenic carbohydrate determinants. Further, hemoglobin degradation leads to
formation of hemorphins, opioid peptides active in the immune system and in
pain reception (Nyberg et al.,
1997
). Hemoglobin-derived peptides may also have antimicrobial
activities (Fogaca et al.,
1999
).
The functions of most tick sequences described in this paper are unknown.
Some, such as group 2, are relatively short peptides with single Kunitz
domains (Fig. 2,
Table 3). When compared with
snake dendrotoxins, which are also small peptides containing a single Kunitz
domain (Harvey, 2001),
similarities are apparent (Fig.
13) not only in the typical conservation of the Kunitz cysteine
residues but also in conserved glycine-rich and basic amino acid-rich regions.
These peptides may function as dendrotoxins that variously affect membrane
functions. These and other peptides are of a size amenable to either direct
synthesis or production by recombinant methods, and will eventually be tested
for their biological activities in various bioassays. Other biological
activities, such as the several antiproteases and metalloproteases, can be
identified with different enzyme assays. Our ongoing studies should increase
our understanding of how ticks successfully evade the hemostatic and immune
responses of their hosts.
|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215,403 -410.[Medline]
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J.,
Zhang, Z., Miller, W. and Lipman, D. J. (1997). Gapped BLAST
and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res. 25,3389
-3402.
Armstrong, P. B. and Quigley, J. P. (1999). Alpha2-macroglobulin: an evolutionarily conserved arm of the innate immune system. Dev. Comp. Immunol. 23,375 -390.[Medline]
Aumuller, G., Wilhelm, B. and Seitz, J. (1999). Apocrine secretion fact or artifact? Anat. Anz. 181,437 -446.[Medline]
Bateman, A., Birney, E., Durbin, R., Eddy, S. R., Howe, K. L.
and Sonnhammer, E. L. (2000). The Pfam protein families
database. Nucleic Acids Res.
28,263
-266.
Borth, W. (1992). Alpha 2-macroglobulin, a
multifunctional binding protein with targeting characteristics.
FASEB J. 6,3345
-3353.
Champagne, D. E., Smartt, C. T., Ribeiro, J. M. and James, A. A. (1995). The salivary gland-specific apyrase of the mosquito Aedes aegypti is a member of the 5'-nucleotidase family. Proc. Natl. Acad. Sci. USA 92,694 -698.[Abstract]
Charlab, R., Valenzuela, J. G., Rowton, E. D. and Ribeiro, J.
M. (1999). Toward an understanding of the biochemical and
pharmacological complexity of the saliva of a hematophagous sand fly
Lutzomyia longipalpis. Proc. Natl. Acad. Sci. USA
96,15155
-15160.
Charpentier, S., Amiche, M., Mester, J., Vouille, V., Le Caer,
J. P., Nicolas, P. and Delfour, A. (1998). Structure,
synthesis, and molecular cloning of dermaseptins B, a family of skin peptide
antibiotics. J. Biol. Chem.
273,14690
-14697.
Dainichi, T., Maekawa, Y., Ishii, K., Zhang, T., Nashed, B. F.,
Sakai, T., Takashima, M. and Himeno, K. (2001).
Nippocystatin, a cysteine protease inhibitor from Nippostrongylus
brasiliensis, inhibits antigen processing and modulates antigen-specific
immune response. Infect. Immun.
69,7380
-7386.
Das, S., Banerjee, G., DePonte, K., Marcantonio, N., Kantor, F. S. and Fikrig, E. (2001). Salp25D, an Ixodes scapularis antioxidant, is 1 of 14 immunodominant antigens in engorged tick salivary glands. J. Infect. Dis. 184,1056 -1064.[Medline]
Das, S., Marcantonio, N., Deponte, K., Telford, S. R., 3rd,
Anderson, J. F., Kantor, F. S. and Fikrig, E. (2000). SALP16,
a gene induced in Ixodes scapularis salivary glands during tick
feeding. Am. J. Trop. Med. Hyg.
62, 99-105.
Dennis, M. S. and Lazarus, R. A. (1994). Kunitz
domain inhibitors of tissue factor-factor VIIa. I. Potent inhibitors selected
from libraries by phage display. J. Biol. Chem.
269,22129
-22136.
Dickinson, R. G., O'Hagan, J. E., Shotz, M., Binnington, K. C. and Hegarty, M. P. (1976). Prostaglandin in the saliva of the cattle tick Boophilus microplus. Aust. J. Exp. Biol. Med. Sci. 54,475 -486.[Medline]
Fogaca, A. C., da Silva, P. I., Jr, Miranda, M. T., Bianchi, A.
G., Miranda, A., Ribolla, P. E. and Daffre, S. (1999).
Antimicrobial activity of a bovine hemoglobin fragment in the tick
Boophilus microplus. J. Biol. Chem.
274,25330
-25334.
Francischetti, I. M., Valenzuela, J. G., Andersen, J. F.,
Mather, T. N. and Ribeiro, J. M. C. (2002). Ixolaris, a novel
recombinant tissue factor pathway inhibitor (TFPI) from the salivary glands of
the tick, Ixodes scapularis: identification of factor X and factor Xa
as scaffolds for the inhibition of factor VIIa/tissue factor complex.
Blood 99,3602
-3612.
Gillespie, R. D., Dolan, M. C., Piesman, J. and Titus, R. G.
(2001). Identification of an IL-2 binding protein in the saliva
of the Lyme disease vector tick, Ixodes scapularis. J.
Immunol. 166,4319
-4326.
Girard, T. J., Warren, L. A., Novotny, W. F., Likert, K. M., Brown, S. G., Miletich, J. P. and Broze, G. J., Jr (1989). Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor. Nature 338,518 -520.[Medline]
Hartmann, S., Kyewski, B., Sonnenburg, B. and Lucius, R. (1997). A filarial cysteine protease inhibitor down-regulates T cell proliferation and enhances interleukin-10 production. Eur. J. Immunol. 27,2253 -2260.[Medline]
Harvey, A. L. (2001). Twenty years of dendrotoxins. Toxicon 39, 15-26.[Medline]
Higgs, G. A., Vane, J. R., Hart, R. J., Porter, C. and Wilson, R. G. (1976). Prostaglandins in the saliva of the cattle tick, Boophilus microplus (Canestrini) (Acarina, Ixodidae). Bull. Ent. Res. 66,665 -670.
Hooper, N. M. (1994). Families of zinc metalloproteases. FEBS Lett. 354, 1-6.[Medline]
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.[Medline]
Jia, L. G., Shimokawa, K., Bjarnason, J. B. and Fox, J. W. (1996). Snake venom metalloproteinases: structure, function and relationship to the ADAMs family of proteins. Toxicon 34,1269 -1276.[Medline]
Jones, L. D., Davies, C. R., Steele, G. M. and Nuttall, P. A. (1987). A novel mode of arbovirus transmission involving a nonviremic host. Science 237,775 -777.[Medline]
Jones, L. D., Davies, C. R., Williams, T., Cory, J. and Nuttall, P. A. (1990). Non-viraemic transmission of Thogoto virus: vector efficiency of Rhipicephalus appendiculatus and Amblyomma variegatum. Trans. R. Soc. Trop. Med. Hyg. 84,846 -948.[Medline]
Leonardi, A., Aragon-Ortiz, F., Gubensek, F. and Krizaj, I. (1999). Partial primary structure of a fibrinogenase from the venom of the snake Lachesis stenophrys. J. Chromatogr. A 852,237 -243.[Medline]
Lundwall, A. and Lazure, C. (1995). A novel gene family encoding proteins with highly differing structure because of a rapidly evolving exon. FEBS Lett. 374, 53-56.[Medline]
Manoury, B., Gregory, W. F., Maizels, R. M. and Watts, C. (2001). Bm-CPI-2, a cystatin homolog secreted by the filarial parasite Brugia malayi, inhibits class II MHC-restricted antigen processing. Curr. Biol. 11,447 -451.[Medline]
Milstone, A. M., Harrison, L. M., Bungiro, R. D., Kuzmic, P. and
Cappello, M. (2000). A broad spectrum Kunitz type serine
protease inhibitor secreted by the hookworm Ancylostoma ceylanicum.J. Biol. Chem. 275,29391
-29399.
Mulenga, A., Sugimoto, C. and Onuma, M. (1999). Characterization of proteolytic enzymes expressed in the midgut of Haemaphysalis longicornis. Jpn. J. Vet. Res. 46,179 -184.[Medline]
Nash, P. D., Opas, M. and Michalak, M. (1994). Calreticulin: not just another calcium-binding protein. Mol. Cell. Biochem. 135,71 -78.[Medline]
Nielsen, H., Engelbrecht, J., Brunak, S. and von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10,1 -6.[Abstract]
Nuttall, P. A., Paesen, G. C., Lawrie, C. H. and Wang, H. (2000). Vector-host interactions in disease transmission. J. Mol. Microbiol. Biotechnol. 2, 381-386.[Medline]
Nyberg, F., Sanderson, K. and Glamsta, E. L. (1997). The hemorphins: a new class of opioid peptides derived from the blood protein hemoglobin. Biopolymers 43,147 -156.[Medline]
Ogata, S., Hayashi, Y., Misumi, Y. and Ikehara, Y. (1990). Membrane-anchoring domain of rat liver 5'-nucleotidase: identification of the COOH-terminal serine-523 covalently attached with a glycolipid. Biochemistry 29,7923 -7927.[Medline]
Pitt-Rivers, R. and Impiombato, F. S. A. (1968). The binding of sodium dodecyl sulphate to various proteins. Biochem. J. 109,825 -830.[Medline]
Petersen, L. C., Bjorn, S. E., Olsen, O. H., Nordfang, O., Norris, F. and Norris, K. (1996). Inhibitory properties of separate recombinant Kunitz-type-protease-inhibitor domains from tissue-factor-pathway inhibitor. Eur. J. Biochem. 235,310 -316.[Abstract]
Regoli, D. A. and Barabe, J. (1980). Pharmacology of bradykinin and related kinins. Pharmacol. Rev. 32,1 -46.[Medline]
Ribeiro, J. M. and Mather, T. N. (1998). Ixodes scapularis: salivary kininase activity is a metallo dipeptidyl carboxypeptidase. Exp. Parasitol. 89,213 -221.[Medline]
Ribeiro, J. M. C. (1987). Ixodes dammini: Salivary anticomplement activity. Exp. Parasitol. 64,347 -353.[Medline]
Ribeiro, J. M. C. (1989). Role of saliva in tick/host associations. Exp. Appl. Acarol. 7, 15-20.[Medline]
Ribeiro, J. M. C. (1995). Blood-feeding arthropods: Live syringes or invertebrate pharmacologists? Infect. Agents Dis. 4,143 -152.[Medline]
Ribeiro, J. M. C., Evans, P. M., MacSwain, J. L. and Sauer, J. (1992). Amblyomma americanum: Characterization of salivary prostaglandins E2 and F2alpha by RP-HPLC/bioassay and gas chromatography-mass spectrometry. Exp. Parasitol. 74,112 -116.[Medline]
Ribeiro, J. M. C., Makoul, G., Levine, J., Robinson, D. and Spielman, A. (1985). Antihemostatic, antiinflammatory and immunosuppressive properties of the saliva of a tick, Ixodes dammini.J. Exp. Med. 161,332 -344.[Abstract]
Ribeiro, J. M. C., Makoul, G. and Robinson, D. (1988). Ixodes dammini: Evidence for salivary prostacyclin secretion. J. Parasitol. 74,1068 -1069.[Medline]
Ribeiro, J. M. C. and Spielman, A. (1986). Ixodes dammini: Salivary anaphylatoxin-inactivating activity. Exp. Parasitol. 62,292 -297.[Medline]
Ribeiro, J. M. C., Weis, J. J. and Telford, S. R., III. (1990). Saliva of the tick Ixodes dammini inhibits neutrophil function. Exp. Parasitol. 70,382 -388.[Medline]
Roberts, D. D. (1996). Regulation of tumor
growth and metastasis by thrombospondin-1. FASEB J.
10,1183
-1191.
Schultz, J., Copley, R. R., Doerks, T., Ponting, C. P. and Bork,
P. (2000). SMART: a web-based tool for the study of
genetically mobile domains. Nucleic Acids Res.
28,231
-234.
Tortorella, M. D., Pratta, M. A., Fox, J. W. and Arner, E.
C. (1998). The interglobular domain of cartilage aggrecan is
cleaved by hemorrhagic metalloproteinase HT-d (atrolysin C) at the matrix
metalloproteinase and aggrecanase sites. J. Biol.
Chem. 273,5846
-5850.
Valenzuela, J. G., Belkaid, Y., Garfield, M. K., Mendez, S.,
Kamhawi, S., Rowton, E. D., Sacks, D. L. and Ribeiro, J. M.
(2001). Toward a defined anti-Leishmania vaccine
targeting vector antigens: characterization of a protective salivary protein.
J. Exp. Med. 194,331
-342.
Valenzuela, J. G., Charlab, R., Mather, T. N. and Ribeiro, J.
M. (2000). Purification, cloning, and expression of a novel
salivary anticomplement protein from the tick, Ixodes scapularis.J. Biol. Chem. 275,18717
-18723.
Valenzuela, J. G., Pham, V. M., Garfield, M. K., Francischetti, I. M. and Ribeiro, J. M. C. (2002). Toward a description of the sialome of the adult female mosquito Aedes aegypti. Insect Biochem. Mol. Biol., in press.
Wang, H. and Nuttall, P. A. (1995a). Immunoglobulin G binding proteins in male Rhipicephalus appendiculatus ticks. Parasite Immunol. 17,517 -524.[Medline]
Wang, H. and Nuttall, P. A. (1995b). Immunoglobulin-G binding proteins in the ixodid ticks, Rhipicephalus appendiculatus, Amblyomma variegatum and Ixodes hexagonus.Parasitology 111,161 -165.[Medline]
Wang, H. and Nuttall, P. A. (1999). Immunoglobulin-binding proteins in ticks: new target for vaccine development against a blood-feeding parasite. Cell. Mol. Life Sci. 56,286 -295.[Medline]
Wang, H., Kaufman, W. R. and Nuttall, P. A. (1999). Molecular individuality: polumorphism of salivary gland proteins in three species of ixodid tick. Exp. Appl. Acarol. 23,969 -975.[Medline]
Wikel, S., Ramachandra, R. N. and Bergman, D. K. (1994). Tick-induced modulation of the host immune response. Int. J. Parasitol. 24,59 -66.[Medline]
Wikel, S. K. (1996). Host immunity to ticks. Ann. Rev. Entomol. 41,1 -22.[Medline]