From the European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg 69117, Germany
Received for publication, August 9, 2002, and in revised form, November 25, 2002
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ABSTRACT |
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The genomic locus SRPN10 of the
malaria vector Anopheles gambiae codes for four
alternatively spliced serine protease inhibitors of the serpin
superfamily. The four 40- to 42-kDa isoforms differ only at their C
terminus, which bears the reactive site loop, and exhibit protein
sequence similarity with other insect serpins and mammalian serpins of
the ovalbumin family. Inhibition experiments with recombinant purified
SRPN10 serpins reveal distinct and specific inhibitory activity of
three isoforms toward different proteases. All isoforms are mainly
expressed in the midgut but also in pericardial cells and hemocytes of
the mosquito. The cellular localization of SRPN10 serpins is
nucleocytoplasmic in pericardial cells, in hemocytes and in a
hemocyte-like mosquito cell line, but in the gut the proteins are
mostly localized in the nucleus. Although the transcript levels of all
SRPN10 isoforms are marginally affected by bacterial challenge, the
transcripts of two isoforms (KRAL and RCM) are induced in female
mosquitoes in response to midgut invasion by Plasmodium
berghei ookinetes. The KRAL and RCM SRPN10 isoforms represent new
potential markers to study the ookinete midgut invasion process in
anopheline mosquitoes.
Successful transmission of malaria parasites to vertebrate
hosts requires completion of their sporogonic cycle within the mosquito vector. Therefore, in addition to factors such as feeding behavior or mosquito longevity, vector competence is determined by the
ability of the mosquito to support effectively the sporogonic development of a given Plasmodium species. The bird parasite
Plasmodium gallinaceum, for example, normally fails to cross
the midgut epithelium of the major human malaria vector Anopheles
gambiae, because its ookinete stages are lysed in the midgut
cells of the vector through an unknown mechanism (1). Even in naturally
occurring, well adapted mosquito/parasite combinations the parasites
experience severe losses. In fact, in the mosquito the parasite faces
not only the cellular and humoral immune responses of hemocytes and the
fat body but also the well documented local immune responses of the
midgut and salivary gland epithelial barriers (2). In the genetically
selected A. gambiae strain L3-5 the antiparasitic mechanisms reach an extreme form, in which complete refractoriness is
achieved through the melanotic encapsulation of early oocysts (3).
Because the mosquito's immune system was shown to react to the
presence of the parasite, major efforts have been devoted to dissection
of the molecular mechanisms underlying these responses (reviewed in
Refs. 4 and 5). Continuing efforts are guided by fundamental studies on
invertebrate innate immunity performed either in model experimental
systems such as Drosophila (6), or in systems that permit
consistent biochemical studies such as the Lepidoptera Manduca
sexta and Bombyx mori (7) or the horseshoe crab
Limulus (8).
Serine proteases play critical roles in the regulation of the
invertebrate innate immune responses. In Limulus, for
example, cascades of autocatalytically activated proteases triggered
either by lipopolysaccharide or glucans converge to activate the
hemolymph clotting system, which functions both in coagulation and in
defense against pathogens (9). Similarly in M. sexta, the
prophenoloxidase cascade (which catalyzes the formation of melanin
during the defense reaction and thus plays an important role in the
encapsulation of pathogens) is also initiated by proteolytic processing
(10).
Not only serine proteases but also their associated regulatory
serine protease inhibitors of the
serpin superfamily are important modulators of immune responses. The
proteases underlying the activation of the clotting cascade in
Limulus are known to be inhibited by regulatory serpins
(11), as are the Manduca and Hyphantria cunea prophenoloxidase-activating proteases (12, 13). The most dramatic evidence for the regulatory function of serpins comes from
Drosophila, where genetic dissection of complex pathways is
feasible. The Toll pathway has important roles not only in early
development but also in the antifungal response (14). Intracellular
signaling is initiated through the binding of the extracellular ligand
Spaetzle to the Toll receptor, after Spaetzle is proteolytically
cleaved by Persephone (15). This recently identified protease is
thought to be the end point of an antimicrobial recognition cascade
that controls activation of Spaetzle and, therefore, the Toll
pathway-mediated antifungal response. In necrotic mutants
(nec) the proteolytic cascade is deregulated, leading to the
uncontrolled cleavage of Spaetzle and to constitutive expression of the
antifungal peptide drosomycin (16). The necrotic
(nec) phenotype is associated with two mutations in a serpin
gene, the Spn43Ac locus (17). Several immune-responsive
serpins have been isolated from various insects, but their specific
functions remain unknown (18-21).
Serpins constitute a large group of functionally diverse serine
protease inhibitors that fold into a conserved structure and exhibit a
peculiar suicide substrate-like inhibitory mechanism through an exposed
reactive site loop (RSL)1
located toward the C terminus of the protein (22-24). Serpins have
been found in viruses and many groups of eukaryotes, including plants,
nematodes, arthropods, and vertebrates, but not prokaryotes or yeast
(23). Most members of this superfamily are inhibitory and
modulate serine proteases, subtilisins (25, 26), cysteine proteases
such as caspases (27), and papain-like cysteine proteinases and
cathepsins (28). In addition some serpins have lost their inhibitory
potential and perform other roles such as hormone transport, storage,
and blood pressure regulation (reviewed in Ref. 24).
We present the cloning and characterization of SRPN10, an
Anopheles mosquito serpin gene, which gives rise to four
alternative spliced serpin isoforms. These isoforms share homology to
other insect serpins but also to intracellular cytoprotective mammalian serpins of the ovalbumin family. We characterize biochemically the
inhibitory potential of three recombinant serpins and show that the
isoforms are expressed in tissues that participate in insect defense
reactions, such as hemocytes and the midgut epithelium. Finally, we
present evidence that at least two isoforms are transcriptionally up-regulated during parasite passage through the midgut, suggesting that they may be implicated in antiparasitic action or, alternatively, parasite tolerance.
Mosquito and Parasite Techniques--
The A. gambiae
strain G3 and the hemocyte-like cell line Sua 5.1* were reared and
cultured according to previous studies (Refs. 29 and 30, respectively).
Mosquito infections were performed as described previously (2), and the
mosquitoes were kept at 19 °C to allow parasite development until
RNA extraction.
Cloning, Characterization, and Cytogenetic
Mapping--
Degenerate primers corresponding to the third strand of
RNA Techniques--
Total RNA from mosquitoes was isolated using
TRIzol (Invitrogen). Northern blot analysis was carried out using
Hybond N+ (Amersham Biosciences) nylon membranes with 20 µg of total
RNA, following the manufacturer's instructions. Blots were hybridized
with radioactive DNA probes, labeled by random priming (Megaprime
labeling kit, Amersham Biosciences). For semiquantitative RT-PCR
experiments, first-strand cDNA synthesis was primed from total RNA
using oligo(dT) magnetic Dynabeads (Dynal) as described previously
(29), using 1-5 µl of resuspended beads as template. To amplify
specific products, serpin-specific primers (20 pmol) were used in
50-µl reactions with Amplitaq (0.25 unit, Roche Molecular
Biochemicals), 200 µM dNTP, 1.5 mM
MgCl2, through several thermal cycles (45 s at 94 °C,
45 s at 55/60 °C, and 30 s at 72 °C). During a pause at
72 °C, primers complementary to the ribosomal protein S7 gene were added, and the reaction was allowed to proceed for additional cycles,
leading to amplification of both the gene of interest and the internal
standard, and permitting normalization of the reaction. After
electrophoresis on agarose or polyacrylamide slabs, gels were stained
with the sensitive SYBR green dye (Molecular Probes) for 45 min and
analyzed with a fluorescence imager (Fuji). The primers used for
RT-PCRs were as follows. General SRPN10 primers: 5'-TTCTGGCTGAGCGAGACGGAATC and 5'-CTTTGTGGACGACTTTGGACACC;
KRAL-specific: 5'-GCTTGGATGATGGGGTCTTC, 5'-TCGTGGCGATTTGCTTGGGC;
RCM-specific: 5'-TACCGGTATGATCATGATGATGC, 5'-CGCGACCCACAAAGTAAACCATC;
FCM-specific: 5'-CCATGATCGCGGTGTCATTC, 5'-GGCATTTTACAGGTTTTTCC;
CAM-specific: 5'-CTGAAAGATTCGCAAGGAAACAT, 5'-CATACATACGGATGGATTAGTTA;
rpS7: 5'-GGCGATCATCATCTACGT and 5'-GTAGCTGCTGCAAACTTCGG.
Recombinant Serpin Expression and Affinity Purification--
The
ORF encoding the RCM-serpin isoform was amplified from an
Anopheles cell-line library with primers containing a
BamHI (5'-AAGGATCCATGGCCGACAATAGCAGCT)
and an SacI overhang
(5'-TTGGAGCTCGCTAAGCATCGATC) and cloned directionally
in a pQE-30 vector (Qiagen). This RCM-pQE30 plasmid was used as the
starting point to construct the expression vectors for the other serpin
variants. An internal PinAI/AgeI restriction
site, conveniently located immediately upstream of the splice site of
the CAM and RCM isoforms, was used in combination with a pQE-30
HindIII site to substitute the RCM RSL with the homologous
CAM sequence, isolated from a full-length library cDNA. For KRAL
and FCM isoforms, specific reverse primers with HindIII overhangs located at the 3'-end of the coding sequences
(5'-TGTGTAAAGCTTACAATTCCTCGTGGCGATTTGC and
5'-TTCGGAAGCTTGTTATGGCATTTTACAGGTTTTTC, respectively) were used in combination with a general forward primer,
5'-TTCTGGCTGAGCGAGACGGAATC, to amplify specific products from an
adult Anopheles library. The PCR products were then digested
with Eco47III and HindIII and directionally
cloned into the RCM-pQE30 acceptor plasmid, linearized with the same
restriction enzymes. All the expression constructs were sequenced
before transformation into Escherichia coli strain TG1. For
native protein purification, bacterial cultures were grown until
A600 0.7-0.8, induced with 0.4 mM
isopropyl-1-thio- Protease Inhibition Assays--
To measure the inhibitory
activity of Anopheles serpins, 10 and 100 pmol of purified
inhibitor were incubated with 10 pmol of a protease in 0.1 M Tris, pH 8.0, for 5 min in a 96-well microtiter plate
(Nunc) in duplicate series. As controls, 10 and 100 pmol of the
non-inhibitory serpin ovalbumin (Sigma), together with samples devoid
of inhibitor, were used. Then, 100 µl of a protease-specific chromogenic substrate solution was added to each well, and residual enzyme activity was immediately monitored in a Tecan Rainbow plate reader by following the kinetic plots of the absorbance at 405 nm.
Reagents were purchased from Sigma, and the enzymes and corresponding buffer substrates were: bovine pancreatic trypsin (Sigma) with 0.1 mM N-benzoyl-Phe-Val-Arg-pNA (B-7632)
in 100 mM Tris, pH 7.8, 50 mM NaCl, 5 mM CaCl2; human thrombin (T-6884) and
subtilisin Carlsberg (P-5380) with 0.1 mM
N-benzoyl-Phe-Val-Arg-pNA (B-7632) in 100 mM Tris, pH 7.8, 50 mM NaCl; porcine plasmin
(P-8644) with 0.1 mM
N-p-tosyl-Gly-Pro-Lys-pNA (T-6140,
dissolved in Me2SO) in 100 mM Tris, pH 7.8, 50 mM NaCl; porcine pancreatic elastase (E-0258) with 0.1 mM N-succinyl-Ala-Ala-Pro-Leu-pNA
(S-8511, dissolved in methanol) in 100 mM Tris, pH 7.8, 50 mM NaCl; bovine pancreatic chymotrypsin (C-7762) and
proteinase K with 0.1 mM
N-succinyl-Ala-Ala-Pro-Phe-pNA (S-7388, dissolved
in methanol) in 100 mM Tris, pH 7.8, 50 mM NaCl; porcine pancreatic kallikrein (K-3627) with 0.1 mM
N-benzoyl-Pro-Phe-Arg-pNA (B-2133) in 100 mM Tris, pH 7.8, 50 mM NaCl. The protease
inhibition assays at each concentration level (10 or 100 pmol) were
performed four times each: in two independent experiments using
different serpin preparations, and in two duplicates for each experiment.
Generation of Antiserum against the Common Serpin
Backbone--
Purified recombinant RCM-serpin (1.0 mg) was used to
immunize two rabbits in Ribi Adjuvant (RAS, Ribi Immunochem). Boosts were carried out every fourth week with 0.2 mg of antigen until final
bleeding. For antiserum purification, the RCM-serpin gene was fused
in-frame to a GST fusion vector pGEX-5x-1. The construct was sequenced
and transformed into an E. coli BL21(DE3) strain for protein
expression. The fused GST-serpin was purified through a
glutathione-Sepharose 4B matrix (Amersham Biosciences) according to the
instructions of the supplier, and eluted fractions were further
purified with a Mono Q ion-exchange column (Amersham Biosciences) using
a fast protein liquid chromatography device. The peak corresponding to
the GST-serpin fusion was collected, and 4 mg of protein was coupled to
CNBr-activated Sepharose 4B (Amersham Biosciences). The serpin
polyclonal antiserum was incubated with GST-serpin fusion Sepharose
overnight at 4 °C, packed in a column, and washed with PBS until
A280 was close to zero. Bound antibodies were
eluted with 200 mM glycine-HCl, 200 mM NaCl, pH
2.5, and fractions with A280 Immunoblotting and Immunocytochemistry--
Adult mosquito
protein extracts and dissected tissues were prepared essentially as
described (29). For immunoblotting, separated polypeptides were
transferred to Hybond-P membranes (Amersham Biosciences), blocked with
5% dry milk PBS/0.1% Tween 20, incubated with affinity-purified
serpin antiserum (1:1000), and detected by secondary horseradish
peroxidase-conjugated goat antibodies (anti-rabbit IgG 1:30000,
Promega), using a chemiluminescence kit (ECL detection system, Amersham
Biosciences). For whole mount gut and abdomen immunostainings,
dissected tissues were fixed for 45 min in ice-cold 4%
paraformaldehyde, washed twice in PBS, blocked for 1 h in PBT (1%
bovine serum albumin, 5% normal goat serum, 0.1% Triton X-100 in
PBS), and incubated overnight at 4 °C in blocking solution with
primary antibodies. Samples were washed three times in PBT for 20 min,
incubated with secondary antibodies, and mounted in 80% glycerol or
Pro-Long Antifade reagent (Molecular Probes) after three washes
in PBS. All samples were analyzed using a Zeiss LSM 510 confocal
microscope. Primary antibody dilutions: 1:1000 Sp22D; 1:333 serpin;
1:500 histones MAB052 (Chemicon). Secondary antibody dilutions: 1:1000
Cy3-, Cy2, Cy5-coniugated anti-rat, anti-mouse, anti-rabbit IgGs
(Jackson Laboratories); 1:1500 Alexa488-, Alexa546-conjugated
anti-mouse, anti-rabbit IgGs (Molecular Probes).
Cloning and Characterization of SRPN10--
To isolate mosquito
serpin genes, degenerate primers were designed based on conserved
LVNAVYF and IEVNEEGTEA sequences of both insect and vertebrate serpins
(23). A 450-bp fragment of expected size was amplified from an A. gambiae cell line cDNA library and subcloned into a Topo-TA
plasmid vector. After confirmation by sequencing, the cloned fragment
was used to screen the cell line library, and several positive clones
were picked and sequenced. All clones had an identical 5'-end
but formed two sequence groups at their 3'-end, indicative of
alternative splicing. Therefore, additional clones were analyzed and a
fourth instar cDNA larval library was also screened, leading to
identification of three distinct full-length serpin clones. They
correspond to alternatively spliced 3'-ends, which code for the exposed
reactive site loop (RSL) of the serpin.
Because multiple alternatively spliced isoforms had been reported for
another insect (32), a mosquito lambda DASH genomic library was
screened and used to derive the genomic sequence, thereby determining
the full range of possibilities for alternative splicing. Six
recombinant bacteriophage clones were isolated by high stringency
plaque hybridization with probes corresponding to the sequence of the
common serpin backbone and to the RSL of one isoform (CAM),
respectively, and analyzed using appropriate restriction enzymes. A
single bacteriophage clone spn
Several exons encompass the coding region present in the spn
In contrast to the alternatively spliced Manduca serpin-1
locus (33), each specific RSL exon codes for its own 3'-untranslated region. In-frame translational stop codons are found in each of the RSL
exons, followed at a distance ranging form 105 to 257 bp by a consensus
polyadenylation site AATAAA, with the exception of exon K in which an
obvious polyadenylation sequence is absent. The putative translation
initiation site is located in exon 1, with a conserved Kozak consensus
(A at
When analyzing the flanking regions of the locus, searching
for potential ORFs or additional exons encompassing previously undetected RSLs, sequences with homology to a retroviral elements were
noted at both ends (depicted with gray bars in Fig.
1A). Upstream of the serpin promoter a coding sequence with
significant homology to a retroelement polyprotein was located, whereas
downstream of exon C (encoding the last RSL) a sequence was found with
similarity to the reverse transcriptase-like protein of the Aedes
aegypti LINE transposon Juan-A (35). To check whether the
SRPN10 locus might encompass additional, distant RSL exons,
we checked the A. gambiae genome sequence that became
available on the web after this study was completed (scaffold
AAAB01008900.1). None were found in the 24.7-kb genomic sequence
spanning from exon C to the next predicted ORF (a putative Zinc-finger
DNA-binding protein). Thus, we are confident that the SRPN10
locus encodes only four alternatively spliced isoforms of the serpin superfamily.
Analysis of Sequence Similarities of SRPN10 Serpins--
Serpins
typically consist of 370-400 amino acid residues and fold into a
conserved structure, with an exposed reactive site loop (RSL) located
at the C terminus of the molecule. The RSL represents the accessible
"bait" region that mimics the cleavage consensus sequence of the
target protease. Upon cleavage of the RSL (at the so-called P1-P1'
scissile bond), serpins undergo a drastic conformational reorganization
in which part of the RSL and of the adjacent hinge region fold back
into the
BLAST analysis of the whole protein sequences revealed that SRPN10
serpins share high homology to other insect serpins, in particular the
Drosophila Sp-4 and Sp-6, with which it forms an orthologous
group according to bioinformatic analysis of complete mosquito and
fruit fly genomes (36). In addition, SRPN10 shows significant
similarity with the mammalian serpins belonging to the neuroserpin and
ov-serpin clades (data not shown).
The sequence and conformation of the RSL largely determines the
selectivity of inhibition. Thus, sequence alignments (Fig. 1D) and BLAST analysis of the serpin C termini comprising
the RSLs were particularly revealing. By these criteria, the four SRPN10 isoforms could be distinguished as follows. The RCM and CAM
isoforms resemble not only other insect serpins such as
Drosophila Sp-6, B. mori serpin-2 but also
neuroserpin and multiple intracellular cytoprotective ov-serpins
(e.g. human and bovine PI-6, mouse PTI-6, human PI-8 and
PI-9), which are involved in the inflammatory response and in the
modulation of pro-apoptotic proteases of epithelial and endothelial
tissues as well as of neutrophils and macrophages (37). The
Anopheles KRAL isoform and the Drosophila serpin
Sp-4 are characterized by multiple basic residues in the scissile bond and further down the C-terminal peptide sequence. Their C terminus is
distinguished by a short stretch of residues that closely resemble an
endoplasmic reticulum retention signal found also in
neuroserpins and in other serpin clades (38). Multiple basic residues
are also found in the C-terminal sequences of Hordeum and
Arabidopisis serpins, as well as in the chicken MENT
protein, which belongs to the intracellular ov-serpins. MENT is known
to induce higher order chromatin compaction and is an abundant
component in terminally differentiated hematopoietic cells (39). The
FCM isoform, characterized by a stretch of hydrophobic residues in the
reactive site, resembles most closely mammalian leukocyte elastase
inhibitors (intracellular ov-serpin) and two mouse stomach serpins. It
presents a Phe residue in the predicted scissile bond sequence, as is
the case for MENT and for a viral rabbitpox virus serpin SPI-1
(the latter is not shown in the figure). Searching for
Drosophila orthologs of SRPN10 serpins, we noticed that the
Sp-4 gene codes for 10 serpin splice combinations (accession numbers
AJ428880 to AJ428889), with the possibility of four alternative RSLs
(40). For convenience, we named the Sp-4 RSL variants according to the
amino acid residues located at the predicted scissile bond (ASM, TSL,
VMA, and KRAI, respectively; Fig. 1D). Protein sequence
alignments of the C-terminal regions of SRPN10 and Sp-4 show that Sp-4
KRAI may be considered an ortholog of Anopheles SRPN10 KRAL,
unlike the other Sp-4 isoforms. In fact, all the other RSLs diverge
significantly from the SRPN10 ones. Finally, additional mammalian and
insect serpin RSLs with low similarity to SRPN10 are presented in the
lowermost alignment of Fig. 1D. In conclusion,
SRPN10 Anopheles serpins exhibit remarkable sequence
homology not only to specific Drosophila serpins but also to
a set of vertebrate serpins of the neuroserpin and ov-serpin clades,
both at the whole protein level and in the exposed reactive site loop.
Specific Inhibitory Activity of Anopheles Serpin
Isoforms--
Recombinant serpin isoforms with a His tag were produced
in a bacterial expression system, and fractions containing a band of
the expected size, corresponding to the predicted authentic polypeptides, were purified using Ni-NTA matrices (Fig.
2A). An exception was the KRAL
isoform, where extensive processing resulted in the accumulation of a
lower mass product, possibly corresponding to the serpin after cleavage
of the RSL at the dibasic scissile bond.
The functionality of SRPN10 serpin isoforms was assayed in protease
inhibition tests, in which the enzymatic activity of commercially available proteases was monitored using chromogenic substrates. The
test proteases were mammalian digestive enzymes (trypsin, chymotrypsin,
and elastase), serine proteases from human blood (thrombin, kallikrein,
and plasmin), and subtilisin-like proteases (proteinase K, subtilisin
Carlsberg). Three of the isoforms proved to be inhibitors,
whereas KRAL was inactivated as expected. As can be seen in Fig.
2B, all four isoforms have small to medium hydrophobic
residues (Cys and Ala) at or near their scissile bond, suggesting that
they might be good inhibitors of elastase, which cleaves preferentially
at such target residues. In contrast, only RCM and KRAL have basic
residues at the predicted P1 site, making them potential inhibitors of
trypsin and thrombin, which prefer Arg or Lys at the N-terminal residue
of the target peptide bond. Consistent with these predictions, all the
testable isoforms, CAM, FCM, and RCM, are potent inhibitors of
elastase, whereas only RCM inhibits in addition trypsin as well as
thrombin (Fig. 2B). The pattern of chymotrypsin inhibition
is also interesting. This protease tends to cleave at hydrophobic
residues, with the catalytic efficiency improving as the side chain
increases in size. As predicted, it is efficiently inhibited by FCM,
which has a Phe residue in the scissile bond, and least so by the CAM isoform, which is characterized by the presence of small hydrophobic residues (Cys and Ala). Importantly, the CAM isoform proved to be an
effective inhibitor of bacterial subtilisin-like proteases, inhibiting
both subtilisin Carlsberg and proteinase K. This is intriguing, because
microbial subtilisin-like proteases are often associated with pathogenicity.
Expression and Localization of SRPN10 Isoforms--
The
distribution of the SRPN10-derived transcripts in adult
tissues was first monitored by RT-PCR (Fig.
3A), using as template RNAs
extracted from dissected adult thoraces, midguts and gut-free abdomens,
and a primer pair annealing to the sequences common to all isoforms, or
a combination of common and isoform-specific primers. Amplified
products were separated on agarose gels and visualized with a
fluorescence imager after SYBR green staining. Amplification of
ribosomal protein S7 transcripts served as an internal standard for
sample normalization. Serpin expression is highest in dissected midguts
(gt), with weaker expression levels in the thorax
(tx) and the gut-free abdomen (ab) (Fig.
3A). This is true both for all isoforms together and for
three individual isoforms. The exception is KRAL, which is enriched
both in the gut and in the gut-free abdomen. In the adult, RCM is the
most abundant of the four isoforms: its transcripts are readily
detected with 26 amplification cycles, whereas the other isoforms need four additional cycles to reach comparable amplification levels. Similarly, in the hemocyte-like mosquito cell-line Sua 5.1*, the RCM
transcripts are significantly more abundant than the other isoform
transcripts (data not shown).
Based on previous experience with the gut-free abdominal fraction, the
RT-PCR data suggested that SRPN10 serpin is produced in hemocytes as
well as the midgut. To determine the developmental profile as well as
the tissue distribution of total SRPN10 serpin, a polyclonal antiserum
was raised against a recombinant protein in which amino acid sequences
encompassing the common serpin backbone were fused to the His tag.
To determine the developmental profile of serpin levels, total protein
was extracted from different stages of the A. gambiae G3
strain, and the total protein content of each sample was equalized on
the basis of Bradford assays. The samples were then either treated with
SDS loading buffer and boiled for 5 min (Fig. 3B) or boiled
for 15 min in 8 M urea to promote the complete dissociation and denaturation of serpins and serpin-protease complexes (Fig. 3C) and were then immunoblotted with the serpin antiserum.
Serpins are known to bind to their target proteases, forming very
stable complexes resistant to SDS denaturation. The predicted molecular masses of the four intact isoforms range from 40 to 42 kDa, and the expected size of each isoform cleaved by its target protease was
~37 kDa. An immunoreactive band at this size range was detected in
protein extracts treated with 8 M urea (Fig.
3C). Additional higher molecular size bands were detected in
the same extracts in the absence of urea treatment and probably
represent serpin-protease complexes as well as uncleaved serpins (Fig.
3B). SRPN10 serpins are nearly undetectable in early embryos
(EE). Their levels increased in late embryos and the first
larval stage (LE and L1) and peaked at the fourth
larval stage (L4), thereafter declining in the early and
late pupal stages (EP and LP). In the adults,
1-day-old sugar-fed females (F) showed higher serpin content
than males of the same age (M), and a further increase in
serpin levels was recorded in females 24 h after a blood meal
(BF).
The antiserum was then used for whole-mount stainings of dissected
adult tissues. High serpin levels were detected in selected hemocytes
attached to tracheae (Fig. 3D) or to the fat body (Fig. 3E) but not in the fat body itself (Fig. 3E,
fb). Consistent with the RT-PCR data, serpins were also
localized at high levels in the midgut cells, showing a predominant
nuclear localization (Fig. 3I). An additional class of cells
that stain strongly with the antibody are the scavenger (detoxifying)
pericardial cells (Fig. 3, panels G and H). In
all these three cell types serpin was detected in the nucleus. This is
better shown in Fig. 3, panels E and H, where an
antibody against the serine protease Sp22D (29), which is also
expressed in hemocytes and pericardial cells, was used in addition to
anti-serpin and antihistone antibodies. In the pericardial cells
serpins were detected in the nucleus (Fig. 3G and
arrowheads in Fig. 3H) and distributed throughout
the cytoplasm, whereas Sp22D is absent from the nucleus showing a
granular cytoplasmic localization (Fig. 3H). In hemocytes
Sp22D is restricted to a narrow subpopulation, whereas serpins are
present in a broader set of blood cells, only partially overlapping
with those which express Sp22D (data not shown). Similarly, in the
hemocyte-like cell line Sua 5.1* almost the entire cell population
(90%) showed nucleocytoplasmic serpin staining (Fig. 3F),
whereas Sp22D was present in secretory vesicles in only 5% of the
cells, as previously reported (29).
Ookinete Midgut Invasion Enhances Transcription from the SRPN10
Locus--
Because SRPN10 serpins are expressed in the midgut and
hemocytes of the mosquito, tissues that both have key roles in insect defense against pathogens, we wanted to investigate whether the expression levels of SRPN10 serpins are affected by challenge of
mosquitoes with bacteria and Plasmodium parasites. Serpins regulating the humoral response pathways in insects (for example the
Drosophila Spn43Ac) are expected to be secreted in the
hemolymph (16). However, aspects of insect defense are cell-mediated, and in M. sexta an intracellular hemocyte-specific serpin
was shown to be induced upon bacterial challenge (18).
After pricking with a mixture of heat-inactivated Gram+ and Gram
The same type of analysis was applied to female mosquitoes fed on
P. berghei-infected mice. Although no apparent differences of serpin transcript levels were detected in mosquitoes 18 and 20 h following an infected blood meal, a remarkable induction was visible
at 24 h and persisted at 48 h (Fig.
5A). This response coincides
with ookinete invasion and is midgut-specific, because no comparable
induction was evident in the gut-free carcasses of the same infected
mosquitoes. The results were confirmed by blot analysis of total RNA
extracted from naïve or infected midguts, dissected 23 h
after the blood meal (Fig. 5B).
To check whether the levels of all four isoforms are equally affected
during ookinete invasion, pairs of isoform specific primers were used
in RT-PCR analysis to amplify isoform specific transcripts (Fig.
5C). Although the levels of FCM and CAM transcripts in
mosquitoes fed on infected mice (black bars) did not diverge significantly from the levels present in the RNA of control blood fed
mosquitoes (gray bars), the transcript levels for RCM and especially KRAL were markedly enriched 24 h after the infective blood meal and remained higher than the control levels even 48 h later.
To exclude the possibility that this enrichment was due to
the parasite presence, by using KRAL- and RCM-specific primers we
attempted to amplify any putative contaminating band from genomic DNA
or from cDNA derived from in vitro cultured ookinetes
(data not shown). No signal was recorded, confirming that RT-PCR
results demonstrated an increase in the expression levels of RCM and
KRAL isoforms in the infected midguts.
The present work reports the cloning and characterization of an
A. gambiae serpin gene (SRPN10) that is
transcriptionally regulated during ookinete midgut invasion. Four
isoforms were derived from this gene by alternative splicing of exons
encoding distinct reactive site loops. This kind of genomic
architecture permits multiplication of the functionality of the gene by
increasing the number of target-specific bait regions and resembles the
organization found in some other insect serpin genes, such as the
M. sexta serpin-1 (12 splice variants (12)), the
B. mori serpin-1 (2 splice variants (21)), and the
Drosophila serpin Sp-4 (4 alternatively spliced RSLs
(40)).
Partial clones encoding serpin-like sequences were obtained recently in
gene discovery projects, aimed to identify immune-responsive molecules
in A. gambiae. Analysis of expressed sequence tags derived from a hemocyte-like cell line library revealed four clone clusters encoding putative serpins (41). One of these clusters, I10, corresponds
to SRPN10 serpins. Similarly, a differential display search for
immune-responsive genes in the adult females of A. gambiae
identified a fragment (AF203339) with a predicted sequence homology to
inhibitory serpins (42). Interestingly, in the salivary glands of the
mosquito vector A. aegypti, a secreted 48-kDa serpin was
identified, which possesses a hemostatic activity and is assumed to
inhibit the clotting Factor Xa during mosquito blood feeding (43).
We were able to show experimentally that at least three of the four
SRPN10 serpin variants are functional inhibitors of serine proteases.
This is consistent with features they share with inhibitory serpins,
which can generally be recognized by a consensus pattern of residues in
the hinge region: in inhibitory serpin P15 is usually glycine, P14 is
threonine or serine, and residues with short side chains, such as
alanine, glycine, or serine usually, occupy positions P12-P9. These
residues are essential for inhibitory activity, because they permit a
rapid insertion of the RSL into the A The similarity of SRPN10 serpins to mammalian ov-serpins, both in the
whole protein sequence and in some cases in the RSL amino acid
composition, leads to intriguing speculations as to their physiological
role. Ov-serpins reside in the cytosol and/or in the nucleus of
protease-secreting cells, including cytotoxic lymphocytes, monocytes,
and epithelial and endothelial cells (44). The physiological role of
ov-serpins is still emerging, but for many members of the family a
cytoprotective role is envisaged and thought to be exerted through the
modulation of pro-inflammatory and pro-apoptotic proteases (24).
Midgut epithelial cells invaded by ookinetes show features indicative
of apoptosis, such as loss of cell contacts, genomic DNA fragmentation,
and sometimes caspase activation (45, 46). In this context,
up-regulation of the inhibitory SRPN10 serpin gene may
reflect the activation of anti-apoptotic or cytoprotective mechanisms
during ookinete invasion.
Alternatively, the inhibitory activity of the CAM isoform against two
distinct bacterial subtilisin-like proteases may support another
working hypothesis, according to which SRPN10 serpin isoforms may
inhibit ookinete-derived proteases. It is known that ookinetes secrete
Sub2, a subtilisin-like protease, during the midgut invasion process
(45). Because a pivotal role in red blood cell invasion is predicted
for subtilisins secreted by the merozoite stages (47), a similar role
of Sub2 during ookinete midgut invasion is an intriguing hypothesis.
Provided that functional, purified enzyme becomes available, it would
be interesting to test the potential effect of all four SRPN10 serpins
toward Sub2. Production and testing of the KRAL isoform would be of
special interest, because it is strongly up-regulated during midgut invasion.
In agreement with the lack of an obvious signal peptide, we
demonstrated by immunofluorescence that Anopheles SRPN10
serpins have an intracellular nucleocytoplasmic localization,
principally in midgut cells, i.e. scavenger pericardial
cells and hemocytes, which are well known to mediate, respectively,
epithelial and cellular immune responses in the mosquito. Bacterial
challenge elicits only marginal up-regulation of serpin transcripts
(particularly of the KRAL isoform), in contrast to the
immune-responsive Spn43Ac Drosophila serpin (16, 17) and to
the hemocyte-specific Manduca serpin-2 (18).
A remarkable property of SRPN10 is that two of its isoforms are
transcriptionally up-regulated upon ookinete midgut invasion. This
differentiates SRPN10 serpins from other described markers such as,
nitric-oxide synthase, defensin, and gram negative protein, which are transcriptionally regulated by both ookinete invasion and
bacterial challenge (2, 5, 48, 49).
Marker genes that are specifically regulated during ookinete invasion
are particularly valuable as tools to dissect and compare the
physiological responses triggered in the vector when infected with
different Plasmodium species or strains. A fragment
encoding a gene with sequence similarity to Only two of the serpin variants (the ones that utilize the most
upstream alternative exons) are enriched during ookinete midgut invasion. Combined with the results of primer extension experiments that indicate lack of alternative promoters, these observations point
to regulation at a step other than transcriptional initiation. The step
in question may affect transcriptional termination, splicing, or
relative stability of the mRNAs. The existence of distinct 3'-untranslated regions for each splice variant might be associated with differences in transcriptional termination or mRNA stability. Alternatively, ookinete invasion could result in preferential splicing
of KRAL- and RCM-serpin variants: cells penetrated by the parasite show
apoptotic phenotypes (45, 46), and it has been reported that cell death
can affect profoundly the splicing machinery, favoring maturation of
distinct gene products through specific responsive elements present in
their introns (51, 52).
Understanding the complex regulation of SRPN10 serpin expression
requires systematic studies, starting with the functional dissection of
the promoter region, which is characterized by the presence of multiple
regulatory elements, and proceeding with analysis of pre-mRNA
transcription and in vivo processing and stability. In
addition, the effects of purified SRPN10 serpin isoforms on in
vitro cultured ookinetes, or ookinete invasion in transgenic
midguts overexpressing or inhibiting specific isoforms, will be
necessary to clarify whether SRPN10 promotes or inhibits ookinete invasion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet A and to the P15-P9 hinge region (fSPI:
5'-AAYGCIGTSTAYTTYAARG; rSPI: 5'-GCYCCYTCYTCRTTIACYTC) were used at
20-pmol concentration in 50 µl of PCR reactions (1.5 mM MgCl2, 200 µM dNTP, Amersham Biosciences; 0.1 unit of Taq polymerase, Roche Molecular
Biochemicals) to amplify products of expected size from mosquito
cell-line and larval lambda ZAP-express cDNA libraries
(Stratagene). After subcloning and sequencing, positive fragments were
used to screen cDNA libraries for full-length clones. For
characterization of the SRPN10 genomic locus an A. gambiae lambda DASH (Stratagene) genomic phage library was
amplified, and the phage DNA was isolated using a Qiagen lambda prep
kit, following the manufacturer's instructions. The isolated phage DNA
was digested with EcoRI, XbaI, and
XhoI restriction endonucleases and subjected to Southern
blotting with radioactively labeled serpin probes. Identified fragments
containing the serpin locus were excised from gels, purified, and
cloned into a KS Bluescript plasmid. Several positive clones were
picked and sequenced. Cytogenetic mapping was performed with serpin RCM
isoform cDNA by in situ hybridization to polytene
chromosomes of the A. gambiae Suakoko strain (31).
-D-galactopyranoside, and grown for 4 additional hours at 30 °C. This procedure resulted in a high
solubility of all serpin isoforms. Bacteria were lysed in 300 mM NaCl, 50 mM NaH2PO4,
10 mM imidazole, pH 8.0, incubated 40 min on ice with 100 µg/ml lysozyme and 10 µg/ml DNase, passed through a French-press,
and centrifuged at 20,000 rpm for 40 min in a refrigerated Beckman
ultracentrifuge. The supernatants containing the soluble protein
fraction were recovered, incubated with 0.1% Triton X-100, and
gently mixed for 30 min at 4 °C. Soluble tagged serpins were
purified to over 80% purity with an Ni-NTA column (Qiagen) following
the manufacturer's instructions.
0.1 were pooled
and tested in immunoblots for activity.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G6.1 encompassed the whole gene locus
(12551 bp) in three adjacent EcoRI fragments (3917, 1772, and 6862 bp). The genomic structure of the locus is schematically
represented in Fig. 1A. It was
mapped to subdivision 21F on the left arm of the second chromosome
(2L) by in situ hybridization to polytene
chromosomes (Fig. 1B). The serpin gene was named
SRPN10, and its nucleotide sequence was deposited in the
GenBankTM data base with accession number AJ420785
(SPI21F).
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Fig. 1.
Four serpin isoforms arise from alternative
splicing of the SRPN10 gene. A, the
serpin SRPN10 genomic locus. Two probes indicated by
narrow black bars were used to isolate and characterize a
phage containing the whole locus. Restriction enzyme sites
(EcoRI, XbaI, and XhoI) are shown.
White boxes indicate the three exons forming the common
serpin backbone. Colored boxes represent alternatively
spliced exons (K, R, F, and C), which give rise to four splice variants
(KRAL, RCM, FCM, and CAM, respectively). Gray boxes indicate
the location of sequences coding for a retrotransposon-like element.
Potential upstream regulatory sites were identified with the Genomatix
Matinspector software available on the web and are presented if showing
over 90% sequence similarity to the core matrix of known eukaryotic
regulatory sequences. For abbreviations, see text. B,
in situ hybridization to polytene chromosomes, mapping the
serpin locus to the left arm of the second chromosome, subdivision 21F
(2L 21F). C, schematic representation of serpin variants.
The common backbone is depicted in white (cf.
exons 1, 2, and 3 in panel A) and the isoform-specific
reactive site loops are colored (cf. exons K, R,
F, and C in panel A). The number of amino acid residues in
each isoform is indicated. The serpin variants are named after the
first residue in the scissile bond region (P1 in panel
D). A matrix comparison of peptide sequence similarity and
identity of SRPN10 serpin isoforms is presented. Values indicate the
percent identity (pink) and similarity (light
gray) of the peptide sequences encompassing the RSL in different
isoforms (in parentheses are the corresponding values for
the entire protein sequences). D, sequence alignment of
SRPN10 serpins (in red lettering) and homologous serpins in
the C-terminal region encompassing the RSL. The hinge region is
indicated with a gray box, the putative scissile bond with
yellow, and a motif with high similarity to an ER
retention signal with pink. Abbreviations: Dm Sp,
Drosophila melanogaster serpin; Bm, B. mori; Em, Echinococcus multiocularis;
At, Arabidopsis thaliana; Hv ZX,
Hordeum vulgaris protein ZX; ACH,
anti-chymotrypsin; PAI, plasminogen activator inhibitor;
SCCA-1, human squamous cell carcinoma antigen 1;
LEI, leukocyte elastase inhibitor; PI-8, protease
inhibitor 8; PI-6, protease inhibitor 6; PTI-6,
mouse placental serine protease inhibitor 6 (also known as SPI-3);
SPB11, serpin B11; MENT, chicken
heterochromatin-associated protein; A1AT, A1 antitrypsin;
THBG, thyroxine-binding globulin; A1A4,
1-antitrypsin 1-4; KAIN, kallikrein inhibitor
(kallistatin).
G6.1
genomic clone. Comparison with the three isolated cDNA clones showed that the first three exons (1-3) form the common backbone of
all splice variants. Exons R, F, and C code for the distinct isoform
specific C-terminal reactive site loops, known to be alternatively spliced from the cDNA studies. Careful search of the genomic
sequence revealed the presence of an additional in-frame exon (K),
encoding a fourth putative reactive site loop, suggesting an additional splice variant from this locus. Specific primers to exon K were designed and used in combination with a backbone primer to amplify from
a larval cDNA library a band, which upon sequencing was shown to
correspond to the KRAL transcript, thereby confirming the existence of
the fourth serpin isoform. Thus, seven exons of the SRPN10 gene were defined, separated by six introns of highly variable size
(85, 114, 125, 108, 2459, and 141 bp, respectively, for introns 1-6).
Exon/intron boundaries conform to the GT/AG splice donor/acceptor rule,
and all introns were characterized by the presence of a polypyrimidine
tract. The fifth intron is large (2459 bp), setting the last two RSL
exons F and C far apart from the rest of the gene. This intron sequence
was carefully searched for cryptic exons, looking for additional
putative RSL sequences, but none were found.
3, G at +4; Ref. 34). Consistent with the computational
prediction, a single major transcriptional initiation site was located
by primer-extension analysis, 25 nucleotides downstream of a well
conserved TATA box (data not shown). Using Genomatix Matinspector
software, a 1400-bp region upstream of the transcriptional initiation
site was explored for the presence of putative regulatory elements
(diagrammed in Fig. 1A). A CCAAT enhancer binding protein
(C/EBP
) sequence was located 10-bp upstream of the TATA box.
Other putative binding sites were found, for morphogenetic factors
implicated in embryogenesis (Dorsal (DL), Hunchback (HB), Deformed
(DFD), Krüppel (KR)), for the ecdysone-inducible DNA binding
proteins Broad Complex Z4 (BRZ4) and E74A, for GATA factors, for the
Activator Protein 1, and for an activator of the alcohol dehydrogenase
gene. Motifs with high similarity to a neuronal cis-element,
to nuclear factor AT, to a yeast stress response element, and to
binding sites for the ZESTE regulator and c-REL were also observed.
This complex organization of the putative SRPN10 promoter
region may reflect complex developmental and tissue-specific
transcriptional regulation and deserves further investigation. Two REL
family factors are also implicated in immune responses in
Drosophila (6).
-sheet of the backbone, conferring a more stable
conformation. When conformational change occurs before deacylation of
the Michaelis complex, formed between serpin and protease, the latter
is trapped in a very stable complex with the inhibitor (24). Thus,
serpins act as suicide substrates, and their specificity is largely
determined by the scissile bond located within the RSL. The SRPN10
serpins lack a signal peptide and share the first 335 N-terminal
residues, differing only in the last 44-60 C-terminal amino acid
residues, which encompass the RSL (Fig. 1, C and
D). The alternative splicing of the RSL coding exons
therefore potentially represents a functional multiplication of the
inhibitory range of a single serpin gene. The reactive site loops of
the four isoforms exhibit sequence conservation both at the nucleotide
and the amino acid level. In Fig. 1C we show a matrix
comparison of amino acid similarity and identity values between the
RSLs of the SRPN10 locus, which are lowest for the KRAL isoform.
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Fig. 2.
Protease inhibition assay. A,
SDS-PAGE of recombinant serpins. The proteins were produced in a
bacterial expression system and were purified through their N-terminal
His tags on Ni-NTA columns. After purification, bands of expected size
were detected on the gel, with the exception of the KRAL isoform that
is cleaved under native purification conditions. Lanes
marked i are extracts of bacterial cultures induced with 0.4 mM isopropyl-1-thio- -D-galactopyranoside,
whereas p are purified recombinant serpin isoforms.
B, SRPN10 protease inhibition assay. The indicated proteases
(10 pmol) were incubated for 5 min with an equimolar (10 pmol) or
10-fold higher concentration (100 pmol) of purified serpin. Proteases
were trypsin (TRYP), thrombin (THRMB),
chymotrypsin (CHY), porcine pancreatic elastase
(PPE), kallikrein (KAL), human plasmin
(PLAS), proteinase K (ProtK), and subtilisin
Carlsberg. Inhibition is reported as percent reduction of the rate
cleavage of a protease-specific chromogenic substrate (see
"Experimental Procedures"). Inhibition rates are relative to the
non-inhibitory serpin ovalbumin and reported values are averages of two
independent experiments (different serpin preparations have been used).
In each experiment and for each SRPN10 variant/protease combination,
duplicate assays were performed. Therefore, each mean and standard
deviation was calculated from four data sets. S.D. < 5%. Inhibition
rates of
15% were treated as insignificant (- -).
Asterisks denote that the KRAL isoform showed no inhibitory
activity, apparently due to proteolytic cleavage of the RSL during the
purification process.
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Fig. 3.
SRPN10 developmental profile and
localization. A, tissue expression profiles of SRPN10
transcripts. Total RNA was extracted from dissected midgut-free
abdomens (ab), midguts (gt), and thoraces
(tx) of adult female mosquitoes and subjected to RT-PCR. An
internal control corresponding to the ribosomal gene S7 was used for
normalization. The abundance of serpin transcripts was assayed both
with the general primers (upper panel) and with
isoform-specific primers (lower panels). The
numbers to the right of the panels report the
number of amplification cycles used in each experiment. B
and C, SRPN10 developmental protein profile. Samples derived
from different developmental stages were immunoblotted using the serpin
antiserum ( -SRPN10). Boiling in SDS loading buffer for 5 min is not
sufficient to dissociate the high molecular weight serpin-protease
complexes (panel B). Treatment with 8 M urea
succeeds in dissociating the inhibitor-protease complexes (panel
C). EE, early embryo 18 h; LE, late
embryo 42 h; L1 and L4, first and fourth
instar larval stages; EP, early pupae; LP, late
pupae; M, male adult mosquitoes; F, female adult
mosquitoes; BF, female adult mosquitoes 24 h after
blood feeding; 5.1*, Sua 5.1* mosquito cell line. Equal
amounts of total protein were loaded, as calculated using a Bradford
assay (Bio-Rad) prior to treatment with SDS or urea.
D-I, immunolocalization by confocal microscopy.
SRPN10 is stained in red in all the panels. Nuclei are
green in panels D-G, whereas the serine protease
Sp22D is blue in panel E and green in
panel H. D, a group of hemocytes (hc)
is attached to a trachea, with one expressing SRPN10. E, two
hemocytes attached to the fat body (fb, note the
characteristic lipid inclusions of this tissue) one with
nucleocytoplasmic SRPN10 (red) staining, the other with
cytoplasmic Sp22D (blue) staining. F, most of the
hemocyte-like cells of line Sua5.1* show nucleocytoplasmic SRPN10
staining. G, low magnification view of pericardial cells
with strong SRPN10 staining, which is absent from fat body cells
(fb). H, magnified view of a binucleated
pericardial cell showing nucleocytoplasmic serpin staining
(arrowheads indicate the nuclei) and patchy Sp22D staining
(absent in the nuclei). I, in the A. gambiae
midgut, SRPN10 serpins are mostly located in the nucleus of the
epithelial cells. In panels D and E the
transmitted light channel (differential interference contrast filter)
was combined with the fluorescent channels of the confocal microscope.
Scale bars = 10 µm.
bacteria, mosquito females were dissected and the levels of serpin
transcripts were analyzed by RT-PCR using as template total RNA
isolated at successive time points after bacterial challenge (Fig.
4A). Although transcriptional
up-regulation of the antimicrobial gene defensin was evident 12 h
after pricking, the bacterial challenge had no substantial effect on
the total level of all SRPN10 serpin transcripts combined and for each
serpin variant, as monitored separately by using pairs of common and
isoform specific primers (quantified in Fig. 4B). Only the
KRAL splice variant showed a modest and transient up-regulation after
bacterial challenge (Fig. 4B).
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Fig. 4.
RT-PCR analysis of SRPN10
regulation after bacterial challenge. A, SRPN10
transcripts of female mosquitoes were analyzed by RT-PCR 2, 5, 12, and
24 h after bacterial challenge. Results were compared with
unchallenged controls (C) and normalized against S7.
Induction of the immune marker defensin served as a positive challenge
control (upper panel). The regulation of specific SRPN10
splice variants (lower panel) was assayed with
isoform-specific primers. The number of amplification cycles used for
each primer combination is indicated. B, average induction
rates of each of the four isoforms, according to the mean fluorescent
values of specific bands in four separate experiments. Error
bars indicate S.E.
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Fig. 5.
SRPN10 up-regulation during
ookinete invasion. A and B,
midgut-specific SRPN10 induction after ookinete
invasion. A, RT-PCR analysis of common backbone SRPN10
transcripts assayed in midgut and carcass 18, 20, 24, and 48 h
after an infective (i) or non-infective (c) blood
meal. B, RNA blot analysis of SRPN10 RNAs in dissected
midguts of infected (i) or control (c)
female mosquitoes 23 h after the blood meal. The ribosomal protein
gene S7 was used as loading control in both experiments. C,
differential mRNA abundance of SRPN10 splice variants after
ookinete midgut invasion. RT-PCR of midgut transcripts assayed 18, 20, 24, and 48 h after an infective (i, black
bars) or non-infective blood meal (c, gray
bars) with isoform-specific primers. Amplification products were
resolved on agarose or polyacrylamide gels and stained with SYBR green
dye. After normalization to S7, the intensity of each band was measured
with a fluorescence imager. The bars indicate the
average -fold induction of serpin transcripts relative to a
non-infective blood meal 18 h after feeding (triplicate
experiments). Error bars indicate S.E. In the lower
panels photographs of the RT-PCR products of a representative
experiment are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet, facilitating the
conformational change that is necessary for the inhibitory activity of
the serpin to be manifested (23). In SRPN10 serpins these essential
residues are conserved (shaded gray in Fig. 1D),
suggesting the potential for inhibitory activity, which in fact was
demonstrated for all three variants that could be tested in
vitro. We were unable to test the KRAL serpin isoform, because it
is proteolytically cleaved, presumably by endogenous bacterial
proteases, during the production and purification procedure. Consistent
with the distinct composition of the RSLs, we also showed that the
different isoforms exhibit specific protease inhibition spectra
in vitro. These biochemical assays did not aim to identify the physiological target(s) of SRPN10 serpins but rather to establish their inhibitory potential. However, we detected high molecular mass
complexes in immunoblots using a specific anti-serpin antibody, which
are dissociated under harsh denaturing conditions, suggesting that also
in vivo SRPN10 serpins are associated with target proteases.
2-macroglobulin was
shown to respond strongly to malaria parasite infection and not to
bacteria (42). Several genes that are differentially regulated and may be involved in the defense reaction of the A. gambiae midgut
toward P. falciparum have been recently isolated by
differential display (50). Curiously, SRPN10 serpins were not among
them. This might be due to the different combination of experimental
organisms (A. gambiae and P. falciparum) or to
the low midgut infection rates in that study (not exceeding 15 oocysts
per infected midgut). In our experimental combination (A. gambiae and P. berghei), high infection rates were
achieved. Additional studies are necessary to distinguish whether
SRPN10 serpin up-regulation takes place only in cells that are invaded
by the parasite or is a general response of the midgut after heavy
infection. Our highly specific
-SRPN10 antibody is a very valuable
tool for such studies.
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ACKNOWLEDGEMENTS |
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We thank Claudia Blass for cytogenetic mapping of SRPN10 cDNA and Belen Minana for assistance with DNA sequencing of the SRPN10 locus. We are grateful to Prof. V. Scarlato for support (to A. D.) during the writing of the article and to Prof. H. Jiang, Dr. E. Levashina, Dr. G. Christophides, and Dr. G. Lycett for fruitful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Program Project Grant PO1-AI44220-2.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ420785 (SPI21F).
Current address: Dipartimento di Biologia Evoluzionistica
Sperimentale, Università di Bologna, Via Selmi 3, Bologna 40126, Italy.
§ To whom correspondence may be addressed. Tel.: 49-6221-387-200; Fax: 49-6221-387-211; E-mail: dg-office@embl-heidelberg.de.
¶ To whom correspondence may be addressed (current address): Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology Hellas, Vassilika Vouton, P. O. Box 1527, GR 71110, Heraklion, Greece. Tel.: 0030-2810-391149; Fax: 0030-2810-391104; E-mail: loukeris@imbb.forth.gr.
Published, JBC Papers in Press, November 26, 2002, DOI 10.1074/jbc.M208187200
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ABBREVIATIONS |
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The abbreviations used are: RSL, reactive site loop; ORF, open reading frame; GST, glutathione S-transferase; PBS, phosphate-buffered saline; RT, reverse transcriptase; pNA, p-nitroanilide; Ni-NTA, nickel-nitrilotriacetic acid.
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