A GPI-linked carbonic anhydrase expressed in the larval mosquito midgut
1 The Whitney Laboratory, University of Florida, Saint Augustine, FL 32080,
USA
2 Department of Fisheries and Aquatic Sciences, University of Florida, Saint
Augustine, FL 32080, USA
* Author for correspondence (e-mail: pjl{at}whitney.ufl.edu)
Accepted 20 September 2004
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Summary |
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Key words: GPI-linked carbonic anhydrase, mosquito, larva, Aedes, Anopheles, muscle, alkaline gut
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Introduction |
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The larval mosquito gut is known to perform both digestive and assimilation
functions (Clements, 1992). pH
values ranging from 7 to 11 (Dadd,
1975
) along the length of the mosquito gut are presumed to support
these different functions. Some regions of the mosquito gut are known to be
associated with various functions. The gastric caeca are responsible for ion
and water transport (Clements,
1992
). The anterior midgut is responsible for alkaline digestion,
while the posterior midgut absorbs nutrients
(Clements, 1992
). The
Malpighian tubules actively transport potassium and fluid
(Clements, 1992
). The midgut
contents of larval Aedes aegypti, the mosquito known to spread yellow
fever, have a pH as high as 11 in the anterior portion of the gut while the
two adjacent gut regions, the gastric ceaca and posterior midgut, have a pH
close to 8 (Zhuang et al.,
1999
). Carbonic anhydrases (CAs) catalyze the reversible hydration
of carbon dioxide (CO2) to bicarbonate
(HCO3) and therefore are predicted to function
within the anterior midgut region that surrounds the most alkaline pH.
However, it has been shown that a CA activity is present in the gastric caeca
and posterior midgut, but not the anterior midgut
(Corena et al., 2002
). Although
a CA enzymatic activity was not localized within the anterior midgut cells,
acidification (i.e. inhibition of alkalization) of the anterior midgut lumen
was observed upon incubation with a CA-specific sulfonamide inhibitor
(Corena et al., 2002
). This
result indicates that CA activity is indeed involved in the alkalization of
the mosquito gut lumen. Further studies of mosquito CA isoforms are being
pursued to better understand the alkaline gut system.
A more detailed characterization of a previously described CA of larval
Aedes aegypti (Corena et al.,
2002) is presented in this study, as well as preliminary analyses
of a homologous CA isoform cloned from Anopheles gambiae. These CA
enzymes share some characteristics of the mammalian CA IV isozyme, including a
glycosyl-phosphatidylinositol (GPI) link to the plasma membrane. Mammalian CA
IV enzymes have been found in dynamic organs such as kidney, lung, gut, brain,
eye and capillary endothelium (Chegwidden
and Carter, 2000
). The human CA IV isoform was found to be as
active as the CA II (the so-called `high activity' CA) isoform in carbon
dioxide hydration and even more active in bicarbonate dehydration
(Baird et al., 1997
). The
anterior mosquito midgut lacks a highly active cytosolic CA II-like isozyme
(Corena et al., 2002
).
Therefore, the presence of a highly active CA IV-like isozyme within the
mosquito gut may be able to provide the buffering capacity that is needed
within the highly alkaline anterior midgut. Our original hypothesis was that
the expression of CA controls the regionalized pH extremes found in the larval
mosquito midgut. Our results indicate that the distribution of CA alone cannot
fully explain the pH gradients found in the midgut.
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Materials and methods |
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Anopheles gambiae Giles eggs were obtained from the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia. Strict handling guidelines were followed with this particular species, which does not currently inhabit Florida, due to its inherent ability to acquire and transmit the causative agent of malaria, the parasitic protozoan Plasmodium. This Anopheles species was therefore reared in deionized water inside a locked incubator set at 30°C. A mesh screen served as a second barrier within the incubator while the sealed (but not airtight) containers harboring the Anopheles larvae served as the third barrier against escape. The Anopheles larvae were fed a Wardley tropical fish flake food (The Hartz Mountain Corp., Secaucus, NJ, USA). Early fourth instar larvae were chosen for all experiments. Ten to twelve days from the hatch day were required for this species to reach the early fourth instar. Late fourth instar larvae that went unused were sacrificed to prevent any chance of adult emergence.
Preparation and fixation of tissue
To dissect out the midgut, the heads of the cold-immobilized larvae were
pinned down using fine stainless-steel pins to a Sylgard layer at the bottom
of a Petri dish containing hemolymph substitute solution consisting of 42.5
mmol l1 NaCl, 3.0 mmol l1 KCl, 0.6 mmol
l1 MgSO4, 5.0 mmol l1
CaCl2, 5.0 mmol l1 NaHCO3, 5.0 mmol
l1 L-succinic acid, 5.0 mmol l1
L-malic acid, 5.0 mmol l1 L-proline,
9.1 mmol l1 L-glutamine, 8.7 mmol
l1 L-histidine, 3.3 mmol l1
L-arginine, 10.0 mmol l1 dextrose, 25 mmol
l1 Hepes, pH 7.0 adjusted with NaOH
(Clark et al., 1999). The anal
segment and the saddle papillae were removed using ultra-fine scissors and
forceps, and an incision was made longitudinally along the thorax. The cuticle
was gently pulled apart and the midgut and gastric caeca were removed. In some
cases, the gut contents enclosed in the peritrophic membrane slid out, leaving
behind the empty midgut. In other cases, it was necessary to remove the
peritrophic membrane and its contents manually. For enzyme histochemistry,
fixation was in 3% glutaraldehyde in 0.1 mol l1 phosphate
buffer, pH 7.3, overnight at 4°C
(Ridgway and Moffet, 1986
).
For in situ hybridization and immunohistochemistry, dissected tissues
were fixed overnight in 4% paraformaldehyde in 0.1 mol l1
phosphate buffer, pH 7.2, or 4% paraformaldehyde in 0.1 mol
l1 cacodylate buffer pH 7.2, respectively. Some digital
images were acquired using a Leica DMR microscope equipped with a Hammamatsu
CCD camera (Shizouka Pref., Japan). Other images were gathered using a Leica
LSCM SP2 laser scanning confocal microscope (Exton, PA, USA). All images were
assembled using Corel Draw-11 software.
Bioinformatics
The National Center for Biotechnology Information (NCBI) website
(www.ncbi.nlm.nih.gov)
was used for the majority of the bioinformatical data presented in this study.
The first mosquito genome, Anopheles gambiae, was released in 2001
(Holt et al., 2002), and made
accessible to the public on the NCBI website. The basic local alignment search
tool (BLAST; Altschul et al.,
1990
) was employed for primer construction as well as analyzing
PCR products. The NCBI Blast Flies database
(www.ncbi.nlm.nih.gov/BLAST/Genome/FlyBlast.html),
together with the Ensembl database
(www.ensembl.org/Anopheles
gambiae/) were used to predict the number of CA genes in the
Drosophila melanogaster and Anopheles gambiae genomes by
inputting the Aedes aegypti CA as the search sequence.
Ensembl is a joint project between the European Bioinformatics Institute
and the Sanger Institute to bring together genome sequences with annotated
structural and functional information. The NCBI protein database (pdb) and the
BLAST were used in conjunction with the 3-dimensional structure viewer (Cn3D;
Hogue, 1997) for the
prediction of antibody accessible peptide regions in mosquito proteins. BLAST
analyses also confirmed that the chosen antigenic peptides were unique. The
conserved domain database (CDD;
Marchler-Bauer et al., 2002
)
and the conserved domain architecture retrieval tool (CDART;
Geer et al., 2002
) were used
to predict the function of our newly cloned mosquito proteins. Alignments were
produced using Clustal W (Thompson et al.,
1994
), as implemented in DNAman software (Lynnon Biosoft,
Vaudreuil, Quebec, Canada).
Cloning of CA from Aedes and Anopheles larval midgut
The strategies for cloning and sequencing the CA from Aedes
aegypti have been previously described
(Corena et al., 2002). To clone
the homologous CA from Anopheles gambiae, the Aedes CA
sequence was BLASTed against the Anopheles gambiae genome
(Holt et al., 2002
). The most
similar gene sequence was then used to derive exact primers
(5'-AACACTATCTTTTCAGAACCAG [forward primer];
5'-TAGTAGTACTATCGCTCCCA [reverse primer]) for PCR-based cloning using an
optimized protocol for invertebrate tissues (Matez et al., 1999). Amplified
cDNA pools from fourth instar Anopheles gambiae prepared as described
(ibid) were used as the basis for the PCR cloning.
In situ hybridization
Sense and antisense digoxygenin (DIG)-labeled cRNA probes were generated by
in vitro transcription using a DIG RNA labeling kit (Roche Molecular
Biochemicals, Indianapolis, IN, USA). The full-length Aedes CA was
subcloned using a PCR manufactured 5' SalI restriction site and
a 3' XhoI site. Full-length sense and antisense DIG cRNA probes
were produced according to the manufacturer's instructions.
These in situ experiments contained an additional pre-fixation
step. A glass electrode fitted to a micromanipulator was used to inject 4%
paraformaldehyde into the thoracic cavity, just behind the head. Successful
perfusion was easily identified by the cessation of the otherwise constant
muscle twitching along the length of the body. Then the exoskeleton was
removed by careful dissection. Subsequent steps for in situ
hybridization methods were adapted from Westerfield
(1994). The midguts were
washed with PBS at room temperature and then incubated in 100% methanol at
20°C for 30 min toensure permeabilization of the gut tissue. The
tissue was washed (5 min each wash) in 50% methanol in PBST [Dulbecco's
phosphate buffered saline (Sigma-Aldrich, St Louis, MO, USA) plus 0.1%
Tween-20], followed by 30% methanol in PBST and then PBST alone. The tissue
was fixed in 4% paraformaldehyde in 0.1 mol l1 phosphate
buffer for 20 min at room temperature and washed with PBST. The larval midguts
were digested with proteinase K (10 µg ml1 in PBST) at
room temperature for 10 min, washed briefly with PBST and fixed again, as
described previously.
Prehybridization of the tissue was accomplished by incubation in HYB solution [50% formamide, 5 x SSC (1 x SSC=0.15 mol l1 NaCl, 0.015 mol l1 sodium citrate buffer, pH 7.0), 0.1% Tween-20] for 24 h at 55°C. The larval midguts were transferred to HYB+ solution (HYB plus 5 mg ml1 tRNA, 50 µg ml1 heparin) containing 5 ng ml1 DIG-labeled probe and incubated overnight at 55°C. Excess probe was removed by washing at 55°C with 50% formamide in 2 x SSCT for 30 min (twice), 2 x SSCT for 15 min and 0.2 x SSCT for 30 min (twice). For detection, the tissue was incubated in PBST containing 1% blocking solution (Roche Molecular Biochemicals) for 1 h at room temperature. The tissue was incubated with anti-DIG-alkaline phosphatase (Roche Molecular Biochemicals) diluted 1:5000 in blocking solution for 4 h at room temperature. The tissue was washed with PBST and incubated in alkaline phosphatase substrate solution (Bio Rad Laboratories, Hercules, CA, USA) until the desired intensity of staining was achieved (23 h) with sense and antisense samples receiving identical incubations.
Real time polymerase chain reaction
Region-specific cDNA was produced from dissected mosquito tissue using the
Cells-to-cDNA standard protocol (Ambion INC, Austin, TX, USA). The gut regions
used to make the amplified cDNA pools were incubated in 50 µl of hot cell
lysis buffer for 10 min at 75°C. The lysed tissues were treated with 2 U
of DNase I for 30 min at 37°C. The DNase I was then inactivated by heating
to 75°C for 5 min. For the reverse transcription reaction, 10 µl of
cell lysate was combined with 4 µl dNTP mix (contains 2.5 mmol
l1 each dNTP) and 5 µmol l1 random
decamer first strand primer in 16 µl total volume. The mixture was
incubated at 70°C for 3 min and then chilled on ice for 1 min. This
mixture was then combined with 1 x reverse transcription (RT) buffer as
supplied with the enzyme, 1 U M-MLV reverse transcriptase, and 10 U RNAse
inhibitor, and incubated at 42°C for 1 h. The reverse transcriptase was
then inactivated by incubation at 95°C for 10 min. Primers were designed
using Primer Express software (Applied Biosystems; Foster City, CA, USA). The
SYBR Green PCR Master mix, which includes SYBR Green I dye, Amplitaq Gold DNA
Polymerase, dNTPs and buffer, was used for all real-time polymerase chain
reaction (PCR) investigations. Each cycle of PCR was detected by measuring the
increase in fluorescence caused by the binding of the SYBR Green dye to
double-stranded DNA using an ABI Prism 7000 Sequence Detection System (Applied
Biosystems). Initially, each primer set, including the control 18s ribosomal
RNA (GenBank accession no. M95126), was assessed to determine the optimal
concentration of primer to be used. All real-time experiments used the same
two-step cycling profile: 50°C for 2 min followed by 95°C for 10 min
and 40 cycles of 95°C for 15 s and 60°C for 1 min. Whole gut cDNA (100
µg l1) was used as template with 500 nmol
l1, 300 nmol l1, 100 nmol
l1, or 50 nmol l1 of each primer set and 1
x SYBR green I master mix in 25 liters total volume. Each reaction was
done in triplicate. The optimal concentration was then chosen based on the
amplification plots and the dissociation curves generated. Once a
concentration was chosen for each primer set, the efficiency of amplification
of that set was determined. Serial dilutions of whole gut cDNA were used as
template with the appropriate concentration of primers and 1 x SYBR
green I master mix in 25 µl total volume. The threshold cycle number (Ct)
was plotted versus the log of the template concentration and the
slope (m) and intercept (b) were determined. These pre-determinations were
then used in the standardized comparison of the amount of 18s transcript and
CA transcript in each of the cDNA samples tested. For each analysis a control
containing all of the necessary PCR components except the cDNA template was
run. To determine the relative expression level for each transcript analyzed,
the following equation was used: (Ctb)/m. The average log(ng) for each
transcript was then compared to the average log(ng) of 18s RNA transcript to
normalize the values. Then the expression levels were determined relative to
the transcript with the greatest normalized log(ng) value and expressed in a
bar graph using Microsoft Excel software.
Antibody production
An 18 amino acid peptide from the cloned Aedes CA sequence was
chosen for antibody production. In order to increase the probability that this
antibody would be specific for this particular CA sequence (in the event that
other CA isoforms were expressed in the larval mosquito gut), attempts were
made to synthesize an antigenic peptide that would be specific to this
isoform. The well-characterized mammalian CA isoforms served as a model when
trying to choose a unique CA peptide sequence. The comparison of the mosquito
CA with the mammalian isoforms yielded a peptide sequence from the amino (N)
terminus, where CA isoforms showed the most diversity, and least conservation.
The N terminus of our mosquito CA was predicted to have an extended loop
secondary structure. Unlike an alpha helix, an extended loop is more
accessible to antibody probing. Furthermore, three-dimensional analyses (Cn 3D
v4.1 NCBI) of predicted CA IV structures (human 1ZNC and mouse 2ZNC) predicted
that the N terminus is exposed and accessible. An 18 amino acid peptide was
therefore chosen from the N terminus of the Aedes CA sequence. This
peptide sequence (GVINEPERWGGQCETGRR) was sent to Sigma-Genosys (Woodlands,
TX, USA), where it was synthesized and conjugated to bovine serum albumin
(BSA). The synthetic peptideBSA construct and Freund's incomplete
adjuvant were injected into two rabbits to elicit an immune response. Prior to
injection, a blood sample from each rabbit was collected to serve as the
control pre-immune serum. Every 2 weeks a blood sample was collected from the
rabbits, the fraction of immunoglobulin G (IgG) pooled, and another dose of
the peptide-BSA construct administered. Three months after the initial
injections, the final bleeds were collected and used for all
immunohistochemical analyses.
Immunohistochemistry
The resultant antisera were used to determine the specificity of the
antibodies as well as to determine the localization of the larval mosquito
proteins. Dissected and fixed whole-mount mosquito guts were washed 6 x
in Tris-buffered saline (TBS), placed in pre-incubation medium (pre-inc; TBS
with 0.1% TritonX-100 and 2% bovine serum albumin) for a minimum of 1 h, and
then incubated in primary antibody (1:1000) overnight at 4°C. The guts
were then washed in pre-inc and incubated in FITC-conjugated goat anti-rabbit
(GAR) or Alexa-GAR secondary antibody (Jackson ImmunoResearch, West Grove, PA,
USA; 1:250 dilution) overnight at 4°C. The whole-mount preparations were
rinsed in pre-inc and mounted onto slides using p-phenylenediamine
(PPD, Sigma-Aldrich) in 60% glycerol. Draq 5 (Biostatus Limited, Shepshed, UK,
1:1000 dilution) was applied before mounting to visualize nuclear DNA. The
samples were examined and images captured using the Leica LSCM SP2 laser
scanning confocal microscope.
Live preparations were examined, following a similar procedure, to ensure
that antibodies were capable of localizing extracellular proteins only. In
this case, living larvae were pinned to Silgard dishes and the exoskeleton
opened and pinned back. The living gut preparation, which we have shown
remains functional for many hours (Boudko
et al., 2001), was exposed to antiserum or preimmune serum diluted
1:1000 in hemolymph substitute solution (HSS;
Clark et al., 1999
). The
samples were washed extensively in HSS and then fixed in paraformaldehyde as
described above, followed by labeling with fluorescent secondary antibodies.
The live gut assays were also performed to determine whether this specific CA
is tethered to the cell membrane via a GPI linkage. Ten live gut
preparations were incubated with phosphoinositol-specific phospholipase C
(PI-PLC, 5 units per ml in HSS; Sigma-Aldrich) for 3 h at 37°C. PI-PLC was
used as a tool in determining the presence of a GPI link. Controls in which
the guts were incubated in HSS alone were also performed. The guts were then
washed in HSS, fixed, and treated with primary and secondary antibodies as
described above.
CA protein expression
Recombinant Aedes CA was produced using the pET100 vector
(Invitrogen, Carlsbad, CA, USA). Specific primers were designed to amplify the
cDNA. The 3' primers included the sequence 5' to the hydrophobic
tail region. The 5' primers contain the sequence CACC preceeding the
native start codon for correct frame insertion. PCRs were performed using 1 U
of Platinum Pfx polymerase (Invitrogen), the gastric caeca cDNA
collections as template (200 ng), 1 x Pfx amplification buffer as
supplied with the enzyme, 1.2 mmol l1 dNTP mixture, 1 mmol
l1 MgSO4, and 0.3 µmol l1 of
each primer in a total volume of 50 µl. A three-step PCR protocol was used
consisting of 94°C for 2 min followed by 30 cycles of 94°C for 30 s,
55°C for 30 s, and 68°C for 1 min.
The resultant blunt-ended cDNA (4 µl from PCR mix) was ligated with the pET100 directional Topo vector (1 µl and 1 µl salt solution; Invitrogen) for 10 min at room temperature. Top 10 chemically competent E. coli (50 µl; Invitrogen) were transformed by incubating 3 µl of ligation mix with the cells for 30 min on ice, followed by a heat shock of 42°C for 30 s. SOC medium (250 µl; Invitrogen) was added to the cells and they were then incubated at 37°C for 30 min with shaking. The transformation mix (100 µl) was then plated on a LuriaBertoni-carbenicillin (LB-carb) plate (50 µg ml1) and incubated overnight at 37°C. Colonies were sequenced using Big Dye version 1.1 as described previously.
The purified plasmids (10 ng each) were transformed into BL21 Star (DE3) cells (Invitrogen) for CA expression. However, after SOC addition and incubation, the culture was transferred to fresh LB-carb (10 ml) and grown overnight at 37°C with shaking. The next day, 1 ml of culture was transferred to 100 ml of fresh LB-carb and was grown at 37°C with shaking. Optimization experiments were performed in order to facilitate the production of the greatest quantity of CA protein. For production of CA protein, isopropylthio-ß-galactoside (IPTG, 1 mmol l1 final concentration; Stratagene, La Jolla, CA, USA) was added when the culture had attained an optical density of 0.5 at a 600 nm wavelength. Achieving this density took about 1.5 h of growth at 37°C and 200 revs min1. Zinc, in the form of zinc sulfate (0.5 mmol l1 final concentration), was added along with the IPTG to facilitate the proper conformation of an active zinc-binding CA protein. In order to optimize the duration of the induced growth phase, samples were collected every hour for 6 h. These samples were analyzed on an SDS-PAGE 412% Bis-Tris gel to compare CA protein content. 4 h of growth was determined to be ideal for the production of the truncated Aedes CA.
Total protein was collected using the Probond Purification System according to the manufacturer's instructions for soluble proteins (Invitrogen). The cells were harvested by centrifugation, sonicated in native buffer (250 mmol l1 NaPO4, 2.5 mol l1 NaCl; Invitrogen) with lysozyme (1 mg ml1; Sigma-Aldrich), and centrifuged again to collect a crude protein extract. The supernatant was applied to a Probond nickel column (Invitrogen) and washed free of non-specific binding contaminants. The nickel column binds the CA protein due to the added histidine tag, a repeat of six histidine residues within the pET100 expression vector that is inserted after the carboxy (C) terminus of the CA protein. CA was eluted by adding imidazole (250 mmol l1; Invitrogen) to the column, which competes and displaces the histidine tag. Eluted fractions were separated on an SDS-PAGE 412% Bis-Tris gel (Invitrogen). Separated proteins were electroblotted to nitrocellulose membranes and then analyzed for total protein and then by immunostaining by standard methods.
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Results |
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Sequence comparisons of CA IV-like isoforms
Using exact primers deduced from the Anopheles gambiae genome, we
also cloned a CA IV-like cDNA from the malaria mosquito. This CA isoform
(Ensembl gene ID: ENSANGG00000018824, chromosome 2L) is partially predicted by
the Ensembl CA protein family (ENSF00000000228) as one of 14 gene family
members found in the Anopheles gambiae genome. These cloned mosquito
cDNAs from Aedes aegypti and Anopheles gambiae are 61%
identical in amino acid residues and show the greatest likeness to the
mammalian CA IV isozyme. In contrast to the mammalian CA IV, which is encoded
by 7 exons (Sly and Hu, 1995),
only three exons make up the Anopheles CA isoform. Alignment of the
mosquito CA IV-like isoforms from Aedes and Anopheles with
various mammalian CA IV isozymes shows amino acid similarities between these
CA isoforms (Fig. 1). The
multiple leucine residues within the N terminus of the mammalian CA IV
propeptides that comprise the signal sequence are also found in the
Aedes and Anopheles CA IV-like isoforms. One important
feature of the mosquito CA IV-like sequences is the conserved alignment of
G-69 (human CA IV numbering) with the human, bovine and rabbit CA IV
sequences. This particular amino acid residue has been changed to glutamine
(Q) in rat and mouse CA IV, which results in reduced enzyme activity (Tamai et
al.,
1996a
,b
).
Additionally, all of the CA IV sequences, including the mosquito isoforms,
display a hydrophobic tail region. In addition to the conserved CA IV-like
features of GPI-linked proteins, there are also conserved cysteine residues
(C28 and C211, human CA IV numbering) between all of these CAs
(Fig. 1). It has been
determined via cysteine labeling, proteolytic cleavage and sequencing
that these two cysteine residues, in the human CA IV, form a disulfide bond
(Waheed et al., 1996
). A
second disulfide bond is present in the mammalian CA IVs between residues C6
and C18 (human CA IV numbering; Waheed et
al., 1996
). This second pair of cysteine residues, and hence the
resultant disulfide bond, is not present in either of the mosquito
isoforms.
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In situ hybridization for CA localization
In situ hybridization analyses indicate that the Aedes
aegypti CA message is expressed most heavily within the epithelial cells
of the gastric caeca and posterior midgut
(Fig. 2). An antisense cRNA
probe corresponding to the entire cDNA sequence generated strong cytoplasmic
staining of the proximal gastric caeca, while the distal cap cells (*) were
void of label consistent with previously published histochemical staining
(Corena et al., 2002)
(Fig. 2B). Anterior to the
gastric caeca, a strong localization was evident in a small subset of cardia
cells that encircle the tissue, forming a collar
(Fig. 2B). These `collar cells'
are clearly different from the surrounding cells in this same area. This
technique also highlighted a set of specific epithelial cells that are found
only in a subset of the posterior midgut. These CA-positive cells form a ring
of about five cells in width that circumscribe the lower-posterior gut region
(Fig. 2A,C). CA message was
also localized to longitudinal and circular muscle fibers of the anterior and
posterior midgut (Fig. 3).
Following the longitudinal muscle fibers, in close association, are distinct
nerve fibers that also display strong CA labeling
(Fig. 3). Epithelial cells of
the anterior midgut were clearly void of signal beneath the labeled muscle and
nerve cells. Specific staining was also evident however within the abdominal
ganglia of the nervous system (CNS) and peripheral nerve tissue
(Fig. 4). No labeling was seen
in the Malpighian tubules. Sense probes showed no labeling (not shown).
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Real-time PCR analysis of Aedes aegypti CA IV-like transcripts
Real-time PCR was used to compare the levels of Aedes aegypti CA
mRNA within specific tissue regions of the larvae. 20 fourth instar Aedes
aegypti larvae were dissected and the head, gastric caeca (GC), anterior
midgut (AMG), posterior midgut (PMG), and Malpighian tubules (MT) were pooled.
RNA was isolated from each tissue sample for subsequent real time PCR
analysis. Aedes aegypti ribosomal RNA (GenBank accession number
M95126) was used to normalize the quantity of transcript from each sample. The
results are presented in graph format in
Fig. 5. This technique found
the gastric caeca to contain the greatest quantity of CA message within the
gut sections (Fig. 5). The head
section contained roughly half as much message as the gastric caeca
(Fig. 5). The localization of
CA IV-like message within the larval head supports the in situ
hybridization finding of CA message within CNS tissue. The anterior midgut,
posterior midgut and Malpighian tubule collections showed much lower levels of
CA message (Fig. 5).
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Immunolocalization of CA IV-like protein in the mosquito gut
The N-terminal peptide sequence (GVINEPERWGGQCETGRR, see
Fig. 1) was chosen from the
Aedes aegypti CA sequence as an antigen for antibody production. The
resultant antiserum was used to analyze recombinant protein expressed in
bacteria and to immunolocalize the CA IV-like isoform within the mosquito gut.
The pre-immune serum was used as a control for all experiments.
Fig. 6 shows a western
immunoblot analysis of the cloned CA expressed in E. coli. XPress
epitope antibody (Invitrogen Inc.) identifies the expressed recombinant
protein band (Fig. 6B) and the
same band then labels intensely with the rabbit anti-CA peptide serum
(Fig. 6B).
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Whole-mount preparations of larval mosquito guts were immunostained with the anti-CA serum diluted 1:1000. These preparations were counterlabeled with TRITC-conjugated Phalloidin and DRAQ-5 to label muscle (actin) and nuclei (DNA), respectively. Fig. 7 shows laser scanning confocal images of whole gut preparations at two magnifications. The most prominent staining was of a specific subset of the muscles, which encircle the gut epithelial tube. It has long been known that larval mosquito gut is contained within a tightly associated tubular meshwork of muscles (e.g. see chapter 5 in Clemens, 1992). Immunostaining with the antibodies to the CA IV-like CA from Aedes aegypti defines a subdivision in the muscle basket: CA positive and CA negative muscle fiber bundles (Fig. 7). The labeling of the muscle fibers is on the plasma membrane surface of the muscles, and in direct contact with the hemolymph and the basal side of the anterior midgut epithelium. The conservation of the peptide epitope (used in generating this antibody) between different mosquito species (see Fig. 1; 14 of 18 amino acids conserved between Aedes aegypti and Anopheles gambiae), led us to test the immunostaining capacity in a number of larval mosquito species. In each of five species that we tested (Aedes aegypti, Aedes albopictus, Anopheles gambiae, Anopheles quadramaculatus, Ochlerotatus taeniorhynchus), similar discrimination of a subset of gut muscles was seen (not all shown). The overall pattern of CA-positive muscles in Aedes aegypti was such that in the anterior half of the gut, the lateral quadrants of the gut tube were bounded by a meshwork of CA-positive muscles (both circular and longitudinal) that covered approximately one quarter of the circumference of the gut each. This regularly arranged mesh of muscle ran approximately two thirds of the length of the midgut from caecum to the pylorus. In the midst of the posterior midgut region, this grouping of muscles dissipates. On the dorsal and ventral sides of the gut tube, only a few longitudinal muscles labeled for the CA and this labeling proceeds throughout the full length of the midgut. In all cases, the CA-positive muscles were accompanied by CA-negative muscles. Once the presence of the lateral arrangement of CA-positive muscles in the anterior gut is recognized, it then becomes a simple matter to see these distinct but overlapping muscles relative to the remainder of the muscle basket even in the absence of CA staining. That is to say, that the CA-positive muscles create a higher density of basket muscles on the lateral aspects of the anterior gut tube that can be recognized with actin staining alone in Aedes aegypti (Fig. 7B). In other larval mosquitoes, a similar distinction between CA-positive and CA-negative muscles exits. In Anopheles gambiae, the lateral CA-positive muscles have a rounded posterior extreme and hence appear somewhat wing-shaped (Fig. 7DF). In the posterior midgut of Anopheles gambiae, very little labeling of any muscle fibers occurs with the CA antibody. All species examined showed lateral groupings of CA-positive muscles intermixed with CA-negative muscles in the anterior midgut. The posterior midgut musculature varied in CA-labeling from very few positive muscles to mostly positive muscles between species. It is impossible to state the functional distinction between the two classes of gut muscles at this point. However, both peristaltic and antistaltic contractions are known to occur in larval mosquito gut and perhaps the two muscle types contribute differentially to these gut movements. Immunolabeling of the gastric caeca and posterior midgut was also seen, although frequently obscured by the labeling of the basket muscles surrounding the gut epithelial tube. Immunoreactivity was also found within the neural ganglia and immunoreactive nerve fibers that traverse the ventral gut in punctate clusters (Fig. 8). There was no immunoreactivity in the Malpighian tubules.
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Phospholipase-C treatment
In order to validate that the CA IV-like isoform cloned from Aedes
aegypti is indeed GPI linked to the membrane, live fourth instar
Aedes aegypti and Anopheles gambiae larvae were subjected to
phosphoinositol-specific phospholipase C (PI-PLC) treatment and subsequent
immunohistochemistry. This enzyme specifically cleaves the GPI-anchor and
therefore severs GPI-linked proteins from the plasma membrane. Larvae
subjected to PI-PLC treatment showed a dramatic decrease in CA antibody
immunoreactivity along the midgut muscle and nerve fibers, as compared to the
non PI-PLC treated controls (not shown). This evidence supports the
bioinformatical finding that the mosquito CA IV-like isoform is in fact
GPI-linked to the outer plasma membrane.
To further substantiate the cell-surface localization of the muscle CA compartment, living larvae were dissected in HBSS and laid open. The living gut tissue was then exposed to antibodies followed by washing, fixation and subsequent localization of antibody binding with secondary antibodies. As with pre-fixed tissue, the antibodies specifically labeled the specific gut muscles described previously (not shown). High magnification confocal microscopy also shows the muscle-surface labeling clearly when viewed as a single z-plane in cross section (Fig. 9).
|
Fig. 10 shows a Clustal alignment of the two CA IV-like mosquito CA sequences plus the putative homologue from Drosophila aligned with all known human CA isoforms. Conservation of critical amino acids such as histidines known to be involved in coordination of zinc in the active site are all present in the insect CAs but vary in the human genes, which produce inactive CA-related proteins (Fig. 10). Also, it is very interesting to note that the insect CAs have a shortened active site sequence relative to human forms (Fig. 10, broken red line). Thus it is possible that the active site in the Dipteran CAs may be sufficiently different to provide an avenue for the development of very specific CA inhibitors that might be used in mosquito control strategies.
|
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Discussion |
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In this study we have shown that the basket of muscles surrounding the
larval mosquito midgut is complex and contains at least two distinguishable
populations of muscle fibers: CA-positive and CA-negative. Prominent
expression of CA on the surface of the muscles may have a role in midgut
alkalization. As noted before, the alkaline region of the larval mosquito
midgut is restricted to the anterior half of the gut tube. In this region the
gut pH can be as high as 11 (Zhuang et
al., 1999). It is widely thought that the alkaline buffer is most
likely to be carbonate (perhaps potassium carbonate;
Boudko et al., 2001
), so it
stands to reason that a CA activity should be involved in alkalization.
Furthermore, we have previously shown that inhibition of CA activity blocks
anterior midgut alkalization (Corena et
al., 2002
). Nevertheless, enzyme histochemistry (ibid),
real-time PCR, in situ hybridization and immunolocalization studies
all show there to be little or no CA in the anterior midgut epithelial cells.
This very strongly suggests that the bicarbonate source of the anterior midgut
carbonate buffer, originates by the action of CA in cells other than the
anterior midgut cells themselves. This leaves at least two possibilities: the
bicarbonate may be produced and secreted into the gut luminal fluid by the
gastric caeca cells and is then stripped of its extra proton once it reaches
the anterior midgut, or it may be transported from the hemolymph into the
lumen by the anterior midgut cells. The CA IV-like enzyme that we have
localized to a specific subset of muscles specifically associated with the
anterior region of the midgut could possibly contribute to anterior gut
alkalization by maintaining the highest possible concentrations of bicarbonate
in the hemolymph in the immediate vicinity of the anterior midgut (thus
supporting the local epithelial transport hypothesis). Previous findings from
this laboratory have shown a strong efflux of chloride from the AMG epithelium
(Boudko et al., 2001
). Since
chloride transport is frequently part of an exchange with bicarbonate, a net
influx of bicarbonate may indeed be characteristic of the AMG epithelium. In
the absence of a CA specifically expressed in the AMG cells, transported
bicarbonate may simply be shuttled to the gut lumen where it could then be
deprotonated to the double anion carbonate. Carbonate has a pKa in excess of
10 and is likely to be a major contributor to the alkaline luminal pH
(Boudko et al., 2001
).
Our results also show that the CA IV-like CA in mosquito larvae is
expressed in the tracheal system. Human CA IV was first purified to
homogeneity from lung tissue (Zhu and Sly,
1990), where this cell-surface form of CA contributes to the
elimination of gaseous CO2 from the bicarbonate form transported by
red blood cells. It is quite feasible that the GPI-linked CA of the mosquito
larva expressed in the trachea performs a similar function. That is, tracheal
GPI-linked CA may act to convert ionic and aqueous forms of the ubiquitous
aerobic waste product into a gas for elimination by diffusion through the
tubule system (e.g. Clements,
1992
).
Although the mosquito CA isoforms display similar features to mammalian CA
IV enzymes, such as a 5' signal sequence, a hydrophobic 3' tail
and extracellular GPI expression, there is one striking difference in the
amino acid composition of mosquito CA isoform active sites. The active site
within all of the 14 characterized mammalian CA isoforms is tightly conserved.
Three histidine residues (His-94, His-96 and His-119) are essential for CA
activity through their coordinated binding of a required zinc molecule. The
absence of one or more of these histidine residues results in inactive
proteins called CA-related proteins (CA-RPs), as found in mammalian CA
isoforms VIII, X and XI (Tashian et al.,
2000). The mosquito CA IV-like isoforms contain all three of the
required histidine residues, along with all of the other 13 highly conserved
residues found in most other CAs (refer to figs
1 and
10,
Tashian, 1992
; Sly et al.,
1995; Tamai et al.,
1996a
,b
).
However, as the alignment shows in Fig.
1, there is a conserved gap within the mosquito isoform active
sites that is not present in any of the mammalian active sites. Because this
shortened active site was found in mosquito but was not found in any mammalian
CA isoform, we expanded our bioinformatics analyses. The Drosophila
melanogaster genome was found to contain 14 putative CA genes
(ENSF00000000228), the same number found in Anopheles gambiae. Only
one out of the 14 CA isoforms was discovered to contain the identical number
of deleted amino acids within the same active site region (refer to
Fig. 10). This
Drosophila CA sequence (accession number CG3940-PA) may also be a
GPI-linked isoform, due to the presence of a leucine-rich 5' signal
sequence and hydrophobic tail region.
We have previously shown that the application of CA-specific inhibitors
dramatically decreases the alkalinity of the gut (i.e. pH), and in fact is
lethal to the larval mosquitoes (Corena et
al., 2002). We now present evidence that a CA found in the
mosquito gut is most similar to the mammalian CA IV isozyme but contains a
novel active site motif unlike any of the mammalian CA isoforms
(Fig. 10). The finding of a
novel CA active site within the mosquito may facilitate the construction of a
mosquito-specific CA inhibitor for use in larval mosquito control. We are
hopeful that our ongoing mosquito CA crystallization project will yield
further significant structural differences from the mammalian CA IV structure.
These differences could then be utilized in the formulation of a
mosquito-specific CA inhibitor.
Out of the 14 mammalian CA isoforms identified thus far as cytosolic, membrane-bound, secreted and mitochondrial, only CA IV has a GPI link to the cell membrane. The localization of this highly active mammalian isozyme to dynamic tissues such as the gut, brain, kidney and lung supports the important catalyst role of CA for the reversible hydration of CO2. It should not be surprising that the gut of a mosquito, a highly alkaline and fluctuating system, has been found to contain a presumably active CA IV-like isoform as well. The single amino acid substitution of glycine-69 to glutamine is unique to rodent (rat and mouse) CA IV, and was found to be responsible for their reduced activity rate of only 1020% of the human CA IV enzyme (Tamai et al., 1996). Mutating glutamine-69 to glycine within the rodent sequence resulted in almost three times greater CA activity (Tamai et al., 1996). Unlike the rodent sequences, both of the mosquito CA IV-like sequences display the high-activity glycine residue (Human CA IV numbering, refer to Fig. 1).
The task ahead is to decipher if a GPI-linked CA is better equipped to
function in a highly dynamic system than other CA isoforms. Perhaps the GPI
link affords the mosquito CA enzyme a characteristic advantage in buffering
such an alkaline pH through its exclusively extracellular expression. Residing
at the plasma membrane intrinsically affords this isozyme the best location
for monitoring CO2 and HCO3
concentration and flux in the hemolymph in the insect open circulatory system.
Indeed, mammalian CA IV isoforms are expressed on membrane surfaces where
large fluxes of CO2 and/or HCO3 are
expected (Sly, 2000). The most
compelling ability of GPI-linked proteins is that they are known to elicit
second messengers for signal transduction
(Brown and Waneck, 1992
). The
alkaline pH of the larval mosquito gut was found to drop within 23 min
after being narcotized or just simply handled
(Dadd, 1975
). This `handling
effect' lends itself to the prediction that larval mosquitoes exert nervous
control over the generation of the gut lumen's pH. Since a GPI-linked CA was
localized within the mosquito gut and CNS tissue it seems possible that a
GPI-linked CA may regulate the pH of mosquito guts through nervous control and
a connection to a signal cascade.
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Acknowledgments |
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