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Previous Article | Next Article 
Infection and Immunity, December 2003, p. 6995-7001, Vol. 71, No. 12
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.12.6995-7001.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Monoclonal Antibody MG96 Completely Blocks Plasmodium yoelii Development in Anopheles stephensi
Rhoel R. Dinglasan,1* Iesha Fields,2 Mohammed Shahabuddin,2 Abdu F. Azad,1 and John B. Sacci Jr.1
Department
of Microbiology and Immunology, School of Medicine, University of
Maryland, Baltimore, Maryland
21201,1
Laboratory of Malaria and
Vector Research, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, Maryland
208922
Received 13 June 2003/
Returned for modification 11 August 2003/
Accepted 3 September 2003
 |
ABSTRACT
|
|---|
In
spite of research efforts to develop vaccines against the causative
agent of human malaria, Plasmodium falciparum, effective
control remains elusive. The predominant vaccine strategy focuses on
targeting parasite blood stages in the vertebrate host. An alternative
approach has been the development of transmission-blocking vaccines
(TBVs). TBVs target antigens on parasite sexual stages that persist
within the insect vector, anopheline mosquitoes, or target mosquito
midgut proteins that are presumed to mediate parasite development. By
blocking parasite development within the insect vector, TBVs
effectively disrupt transmission and the resultant cascade of secondary
infections. Using a mosquito midgut-specific mouse monoclonal antibody
(MG96), we have partially characterized membrane-bound midgut
glycoproteins in Anopheles gambiae and Anopheles
stephensi. These proteins are present on the microvilli of midgut
epithelial cells in both blood-fed and unfed mosquitoes, suggesting
that the expression of the protein is not induced as a result of blood
feeding. MG96 exhibits a dose-dependent blocking effect against
Plasmodium yoelii development in An. stephensi. We
achieved 100% blocking of parasite development in the mosquito
midgut. Preliminary deglycosylation assays indicate that the epitope
recognized by MG96 is a complex oligosaccharide. Future investigation
of the carbohydrate epitope as well as gene identification should
provide valuable insight into the possible mechanisms of ookinete
attachment and invasion of mosquito midgut epithelial
cells.
|
INTRODUCTION
|
|---|
The life cycle of the malaria parasite itself is complex and involves
asexual development within the vertebrate host as well as both sexual
and asexual development within its primary insect vector, the
Anopheles (Diptera: Culicidae) mosquito. Plasmodium
gamete fertilization and the formation of ookinetes occur within the
midgut of the Anopheles mosquito. This is followed by the
crucial step of the escape through the peritrophic membrane that
encloses the blood meal and invasion of midgut epithelial cells, which
are key determinants for successful development and amplification of
the parasite in the mosquito. For this reason, the midgut plays a
special role as the first insect tissue barrier, and therefore the
interaction between parasite and vector host at this site may provide
promising molecular targets for vaccine
intervention.
Most of the malaria vaccines currently
under development target forms that are found in humans, such as
preerythrocytic and erythrocytic stages of the parasite
(2,
4). More recently,
vaccines are being developed not only against the sexual stages
(7,
13,
19,
26) but against the
mosquito vector as well
(1,
2,
9,
12,
17,
18,
23,
27). Whereas blood-stage
vaccines operate within the vertebrate host to prevent the clinical
manifestation of the disease, vaccines that target the vector stages
operate within the mosquitoes to block transmission of the parasite
from one vertebrate to another. Consequently, these vaccines are
referred to as transmission-blocking vaccines (TBVs)
(4). The success of a TBV
depends on its ability to alter mosquito vector competence and,
consequently, its vectorial capacity
(21). In laboratory
animals, membrane antigens of sexual stages, or more specifically those
of the ookinete (7,
13,
19,
26,
29,
30), induce
transmission-blocking immunity. Knockout of crucial genes that are
expressed in the ookinete stage of the parasite was found to block its
ability to develop as oocysts in the mosquito
(6,
30). However, such
efforts to understand ookinete biology present only one view of the
intimate parasite-vector interaction in the gut lumen. To completely
understand this interplay, the mosquito midgut proteins that are
recognized by Plasmodium ookinetes in the mosquito midgut must
be elucidated.
In this study, we used a mosquito midgut-specific
mouse monoclonal antibody (MAb), MG96, to partially characterize a
microvillus-associated, membrane-bound midgut glycoprotein in
Anopheles gambiae and Anopheles
stephensi. MG96 exhibited a dose-dependent blocking of the
Plasmodium yoelii ookinete-to-oocyst
transition.
|
MATERIALS AND
METHODS
|
|---|
Biological material.
The An. stephensi (Liston)
colony was maintained at the Biomedical Research Institute (Rockville,
Md.) under standard rearing conditions (27°C, 80%
relative humidity). An. gambiae (Giles) mosquitoes were raised
under standard conditions as described above and were a generous gift
from Nirbhay Kumar (Johns Hopkins University, Baltimore, Md.). Cohorts
of 5- to 7-day-old female mosquitoes were either fed on uninfected mice
or kept on a sugar diet. At 36 h after blood feeding, the
midguts were removed from both blood-fed females and age-matched
sugar-fed females and incubated in ice-cold phosphate-buffered saline
(PBS). The dissected guts were transferred into mammalian protein
extraction reagent (Pierce, Rockford, Ill.) with the addition of
2% ASCB8Ø (Calbiochem, San Diego, Calif.)
and a 1:100 dilution of protease inhibitor cocktail (Sigma, St. Louis,
Mo.). To extract protein, the sample was ground with a sterile pestle,
subjected to repeated freeze-thaw cycles, and then spun down at 14,000
x g at 4°C. Mosquito carcass
(minus midgut) protein extract was also prepared for the corresponding
time point. Protein extracts from An. stephensi fourth-instar
larvae, early-stage pupae, and 5- to 7-day-old males were prepared as
described above. The protein concentration was determined by the
bicinchoninic acid protein assay (Pierce). P. yoelii (17XNL,
nonlethal strain, clone 1.1) sporozoites were isolated from infected
An. stephensi mosquitoes (Naval Medical Research Center,
Silver Spring, Md.) by density gradient centrifugation as previously
described (20). The
isolated sporozoites were then used to infect CD1 mice (Charles River
Laboratories, Wilmington, Mass.). P. yoelii infection was
maintained by blood passage to naive mice every week for a maximum of
three passages.
Generation of
MAbs.
The MAb MG96
(immunoglobulin G1 [IgG1] subclass) was produced at the
Laboratory of Malaria and Vector Research, National Institute of
Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, Md. (I. Fields and M. Shahabuddin, unpublished data).
Briefly, 6-week-old female BALB/c mice (Charles River Laboratories)
were immunized by subcutaneous injection with Aedes aegypti
midgut extracts in RIBI adjuvant (RIBI Immunochem Research, Hamilton,
Mont.). A boost was given 21 days after the initial immunization, and a
final boost immunization was given intravenously 14 days after the
second boost. Generation of hybridoma cells was by the polyethylene
glycol method with selection in hypoxanthine-aminopterin-thymidine
medium (31). The
resulting hybridomas were screened for midgut-specific antibodies by
immunofluorescence assay with paraformaldehyde-fixed,
Triton X-100-permeabilized Ae. aegypti midgut sections. The
specificity for mosquito midgut was confirmed by Western blot analysis
with midgut extracts and extracts of total carcasses. The isotype of
MAb MG96 was determined with a mouse monoclonal typing kit (Pierce)
according to the instructions provided by the
manufacturer.
Immunoblotting.
Lysates were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to polyvinylidene difluoride membranes. Blots were probed
with MG96 antibodies from ascites at a 1:1,000 dilution. Bound
antibodies were detected with an alkaline phosphatase-conjugated goat
anti-mouse IgG antibody (Invitrogen, Carlsbad, Calif.), followed by a
chemiluminescent reaction with the addition of CDP-Star substrate
(Invitrogen). Protein loading and transfer efficiency were monitored by
Coomassie blue staining and the use of prestained molecular weight
markers (Pierce),
respectively.
Immunofluorescence
microscopy of midgut cryosections.
Whole mosquito midguts were dissected
in cold PBS, transferred into Tissue Tek OCT embedding medium (Miles,
Inc., Pittsburgh, Pa.), and frozen in a methyl-butane liquid nitrogen
bath. Several sequential 7-µm sections were fixed in absolute
methanol, air dried, and blocked with 10% bovine serum albumin.
Each section was probed with a 1:500 dilution of MG96 for 1 h
in a humidified chamber at 37°C. The slides were washed several
times with PBS and then incubated with a fluorescein isothiocyanate
(FITC)-labeled goat anti-mouse IgG secondary antibody (Kirkegaard &
Perry Laboratories, Inc., Gaithersburg, Md.) in 0.02% Evans blue
(as a general protein counterstain). After repeated washes, the slides
were mounted with DAPI (4',6'-diamidino-2-phenylindole)
(a nucleus stain) mounting medium (Vector Labs, Burlingame, Calif.),
and the staining patterns were assessed at magnifications of
x20 and x40 with a Nikon phase-contrast ellipse E500
microscope. Photomicrographs were taken with a SPOT RT camera and SPOT
RT software V3.2 (Diagnostic Instrument Inc., Sterling Heights, Mich.).
A slide with an FITC-labeled isotype-matched MAb of irrelevant
specificity was used as a
control.
Immunoelectron
microscopy.
The
localization of the midgut protein was examined by immunoelectron
microscopy by methods described previously, with some modifications
(10). Briefly, unfed
midguts from 8- to 10-day-old An. stephensi mosquitoes were
fixed in PBS containing 1% paraformaldehyde and 0.1%
glutaraldehyde for 24 h at room temperature. Ultrathin
sections of guts embedded in LR-White resin (Polyscience, Inc.,
Warrington, Pa.) were mounted on nickel grids and immunostained with
MG96. Antibody binding was visualized by using a 10-nm-colloidal
gold-conjugated goat anti-mouse IgG (Ted Pella, Redding, Calif.). The
grids were counterstained with uranyl acetate and examined with a JEOL
1200 EX transmission electron microscope. Controls for this experiment
included the use of isotype-matched, nonspecific antibodies as the
primary probe.
Characterization of the
epitope recognized by MG96.
Chemical deglycosylation was carried
out according to previously described protocols
(33,
34) with modifications.
Briefly, whole midgut lysates were electrophoretically separated on an
SDS-polyacrylamide gel and blotted onto a polyvinylidene difluoride
membrane. The membrane was divided into four identical sections and
incubated in 50 mM sodium acetate wash buffer (pH 4.5). Sections were
used as either control or experimental treatment groups. One
experimental strip was incubated with 20 mM sodium periodate in 50 mM
sodium acetate buffer (pH 4.5) at room temperature in the dark for
1 h. The other experimental strip was incubated in ice-cold 1
mM sodium periodate in the dark at 0°C on wet ice for
1 h. Controls strips were kept in wash buffer under the same
conditions without the addition of sodium periodate. The strips were
rinsed with three exchanges of wash buffer and then incubated with 50
mM sodium borohydride (in PBS) at room temperature for 30 min. The
membranes were washed three times with PBS, followed by the standard
detection of the antigens by MAbs as described for immunoblot analysis.
For enzymatic deglycosylation, midgut lysates were boiled at
95°C for 10 min and then treated with either 0.5 mU of peptide
N-glycosidase A (EC 3.5.1.52) or 1 mU of
neuraminidase (EC 3.2.1.18) and 1 mU of O-glycosidase (EC
3.2.1.97/3.2.1.110) (Roche Molecular Biochemicals, Indianapolis, Ind.).
The reaction proceeded overnight in deglycosylation buffer (10 mM Tris,
0.5% SDS, 2% 2-mercaptoethanol, 10 mM EDTA, 5%
NP-40) at 37°C. Deglycosylated proteins were run on
SDS-polyacrylamide gels and immunoblotted as described
above.
Transmission blocking
assay.
Six-week-old CD1
mice (Charles River Laboratories) were infected with P. yoelii
by inoculation with blood-stage parasites. At 5 days postinfection,
gametocytemic mice were exsanguinated and the cells were washed and
resuspended in RPMI 1640 plus 10% heat-inactivated mouse serum
at a 45% hematocrit. Protein G-purified MG96 at a final
concentration of 100, 50, 25, or 5 µg/ml was delivered in 1 ml
of infective blood into individual Parafilm-covered membrane feeders.
Six-day-old An. stephensi mosquitoes, which had been starved
for 36 h, were allowed to feed on the blood for 1
h. Infected blood without antibody treatment was used as a control. In
addition, the IgG MAb PvNSV against Plasmodium vivax
circumsporozoite protein, diluted to 100 µg/ml in infective
blood, was included as a second control. Treatment and control groups
were maintained at 22°C and 58% relative humidity.
Infectivity was assessed at 10 days after blood feeding by dissecting
midguts, staining with mercurochrome, and counting the number of
oocysts per mosquito for a minimum of 20 mosquitoes. For each run, only
those mosquitoes that showed egg maturation as a proxy for feeding to
repletion were used for oocyst counts. Therefore, only guts from fully
gravid females were included in the denominator in assessment of
percent oocyst infection. All assays were repeated, or treatments were
done in duplicates. The differences in the number of oocyst-infected
guts between experimental and control groups were analyzed by the
Mann-Whitney U test, and differences in the transmission-blocking
effect across different antibody doses were analyzed by the Friedman
test with STATVIEW 5.0 software (SAS Institute, Cary,
N.C.).
|
RESULTS
|
|---|
MG96
recognizes distinct midgut proteins in both blood-fed and unfed An.
stephensi and An. gambiae as determined by Western
immunoblotting.
Although the
hybridoma cell line MG96 was derived from mouse splenocytes that were
reactive to Ae. aegypti midgut plasma membrane antigens,
antibodies from this line were capable of binding to the midguts of
Anopheles mosquitoes. A reproducible banding pattern was
observed for midgut lysates from blood-fed and unfed female mosquito
midguts in Western immunoblotting (Fig.
1a). MG96 recognized three primary protein double bands from An.
gambiae and An. stephensi, at 
40, 80,
and 150 kDa, on reducing SDS-PAGE. On the other hand, only the
80- and 150-kDa double peptide bands were observed
from a midgut lysate of blood-fed Ae. aegypti (data not
shown). The banding pattern showing multiple secondary bands in
addition to the primary double bands strongly suggests that the midgut
protein is glycosylated. Separation of the protein products under
nonreducing conditions greatly reduced the presence of secondary bands
but maintained the three primary doublets (data not shown). The three
primary doublets observed for An. stephensi suggest that the
midgut protein is polymeric. A similar but not identical banding
pattern was observed for midgut lysates from An. gambiae-fed
and unfed females, with the addition of a 200-kDa protein
band. The presence of this band consistently differentiates the two
banding patterns of the two vector species. Recognition of the protein
doublets in unfed midguts indicates that expression is not induced by
the ingestion of a blood meal. To understand the recognition
specificity of MG96, a protein lysate from An. stephensi
carcasses minus midguts was compared to both fed and unfed midguts by
Western blotting. The results confirmed that the banding pattern is
found specifically in the midguts of anopheline mosquitoes (Fig.
1a). An isotype-matched
MAb control did not reveal any nonspecific recognition of midgut
antigens.
In addition, MG96 was capable of recognizing both the
150- and 80-kDa protein bands from whole-body protein
extracts from a mixed-sex population of fourth-instar larvae and pupae
of An. stephensi (Fig.
1b). Similarly, faint
bands corresponding to the 150- and 80-kDa molecules
were also recognized by MG96 in whole-body protein lysates from An.
stephensi males.
Localization of
midgut antigens by immunofluoresence microscopy.
To further assess the specificity of
MG96 for gut antigens, midgut cross-sections from 11-day-old unfed and
blood-fed Anopheles were immunostained with MG96. Intense
fluorescent staining was observed throughout the apical, luminal brush
border of the midgut epithelia from both fed (Fig.
2a) and unfed (data not shown) mosquitoes. The sucrose cryoprotection step
(14) was omitted from our
protocol, and this may have contributed in part to the shearing of the
microvilli, owing to the pattern of staining among particles within the
gut lumen itself. Immunofluorescence microscopy with FITC-labeled
isotype-matched IgG as a control showed insignificant background
fluorescence (Fig.
2b).
View larger version (20K):
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FIG. 2. (a)
Localization of midgut antigens by immunofluorescence staining of
midgut cryosections from an 11-day-old non-blood-fed An.
gambiae midgut cryosection probed with MAb MG96 demonstrates
staining along the microvilli (thick arrow) and glycocalyx (thin arrow)
on the luminal side of midgut epithelia. (b) An isotype-matched IgG was
used to stain an An. gambiae midgut cryosection as a control.
Evans blue (red) was used as a counterstain against general cellular
protein, and DAPI staining (blue) was used to identify nuclei. L, lumen
of the midgut. Magnification,
x40.
|
|
Subcellular
localization of midgut antigens by immunoelectron microscopy.
Immunoelectron microscopy was performed
to determine more precisely the subcellular localization of midgut
antigens recognized by MG96 in An. stephensi (Fig.
3a). Gold particle labeling showed that MG96 was preferentially associated
with the apical microvilli and the microvillus network in
cross-sections of the gut epithelial cell. Labeling of the cell
cytoplasm was also observed and is presumed to be evidence of antigen
transport to the cell surface. Immunostaining with an isotype-matched
control antibody to a serial cross-section of the midgut showed no
background labeling of either extracellular or intracellular components
(Fig.
3b).
View larger version (102K):
[in this window]
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|
FIG. 3. Immunoelectron
microscopy of unfed An. stephensi midguts. (a) Cross-section
of the midgut showing the association of MG96 with the extracellular
microvilli (arrowhead) and glycocalyx (thin arrow) on the apical end of
the midgut epithelial cell. (b) Cross-section of the midgut following
staining with an isotype-matched control MAb. MVN, microvillus network;
MV, microvilli; E, epithelial lining; L, lumens Bars, 1
µm. Magnification,
x15,000.
|
|
Effect
of MG96 on P. yoelii development in An. stephensi
mosquitoes.
In the initial
series of experimental trials, mosquitoes were provided an infective
blood meal that contained a 1:500 dilution of MG96 from ascites.
Transmission-blocking activity of MG96 was assessed as the number of
infected mosquitoes (i.e., mosquitoes having oocysts in the midguts at
10 days postinfection) among treatment groups. The results showed a
reduction in the number of P. yoelii-infected midguts at 10
days postinfection, with 8% of mosquitoes remaining infected
among treatment groups (n = 75) compared to 40%
remaining infected among controls (n = 43). This
difference was found to be statistically significant by using the
Mann-Whitney U test at 
= 0.05 (P =
0.03). To determine the spectrum of transmission blocking induced by
MG96, serial dilutions of purified antibody were made and added to
infective blood meals. We achieved complete elimination of oocyst
development at a 100-µg/ml concentration of MG96 (P
< 0.05). In addition, the trend in the blocking effect was
observed to be dose dependent (Table
1). However, the difference in treatment effect between any two doses was
not statistically significant (Friedman test, P >
0.05). These assay results were reproducible across three
trials.
The epitope recognized by MG96 has
a carbohydrate component.
To
understand the banding pattern observed on Western immunoblots, a
partial characterization of the epitope was ascertained through a
combination of enzyme and chemical deglycosylation assays. Treatment of
mosquito midgut lysates with PNGase A, an N-linked
oligosaccharide-Asn-specific exoglycosidase, slightly altered the MG96
recognition pattern. A mobility shift was expressly observed for the
40-kDa double band. However, there was no modification in the
MG96 recognition of the two higher-molecular-mass bands (Fig.
4a). Initial treatment with a nonspecific neuraminidase to remove terminal
neuraminic acid, followed by treatment with O-glycosidase, an
O-linked Galß(1,
3)GalNAc--Ser/Thr-specific endoglycosidase, completely altered
the staining profile of the 40-kDa band (Fig.
4a). As with treatment
with PNGase A, staining of the 150- and 80-kDa bands
remained unaffected. This observation may have been due to presumably
incomplete digestion, as the enzyme rate of hydrolysis may be much
lower for larger glycoproteins, thereby requiring a more protracted
incubation period in a denaturing reaction buffer. Incomplete
denaturing of the protein secondary structures may not have revealed
masked or obscured glycosidase recognition sites. Treatment with 1 mM
sodium periodate at 0°C preferentially cleaves terminal
neuraminic acid on both N- and O-linked branching oligosaccharides,
whereas treatment with 20 mM sodium periodate cleaves off all
oligosaccharides from the protein backbone. The MG96 recognition
profile was unaffected following treatment with 1 mM sodium periodate;
however, treatment with 20 mM sodium periodate completely eliminated
recognition by MG96 on a Western blot (Fig.
4b).
|
DISCUSSION
|
|---|
MAb
MG96, generated against the Ae. aegypti midgut, shows
cross-reactivity with midgut antigens from An. stephensi and
An. gambiae. This phenomenon is, in itself, particularly
interesting within an evolutionary context, especially since it is
believed that Culicinae and Anophelinae diverged over 200 million years
ago (3). However, from a
functional perspective, the most intriguing observation is that the
antibody is capable of inducing transmission blocking in An.
stephensi when ingested with an infective blood meal. At a
concentration of 100 µg/ml, complete nullification of both
oocyst development and salivary gland infection was achieved. While we
saw a decreasing trend in the transmission-blocking effect with
increasing dilutions of the antibody, the difference
between the proportions of mosquito guts that remained
infected at different doses was not statistically significant. This
highlights the efficiency of the blocking effect of MG96, since low
concentrations of this MAb can still effectively block parasite
development in the mosquito midgut. The mechanism by which this
antibody obstructs the natural progress of infection by
Plasmodium is not yet understood. However, it is evident that
MG96 recognizes midgut antigens that are specifically located along the
brush border that lines the apical side of gut epithelial cells facing
the lumen. Since the peritrophic matrix, which had enclosed the blood
meal, had been completely removed to ensure a clean specimen, the
presence of this glycoprotein on the luminal side of the peritrophic
matrix (24,
31,
35,
36) could not be
completely ascertained by our assays. The staining pattern demonstrated
by indirect fluorescent-antibody assay and immunoelectron microscopy
does suggest that the glycoprotein is present in the glycocalyx network
attached to the microvilli. The apical side of gut epithelial cells is
the first to confront invading ookinetes. The disruption of the
ookinete-to-oocyst transition may therefore occur at either the level
of attachment to the glycocalyx or to the microvillus itself
(36) or at the level of a
more downstream event following invasion, e.g., interference of cell
signaling and/or cell functioning.
The distinct multiple-banding
profile observed on Western blots was found to be present in the
midguts of both unfed and blood-fed adult female mosquitoes. This
multiple-banding profile was also observed in protein lysates from
males, mixed-sex pupae, and fourth-instar larvae. However, the banding
patterns were not identical. Males and aquatic life stages do not
engage in blood feeding. This implies that the midgut proteins
recognized by MG96 may not have anything to do with blood feeding and
may in fact be found in other tissues outside the midgut. A comparison
of unfed adult female mosquito carcasses with the midguts removed to
unfed midguts alone confirmed the tissue specificity of the observed
banding pattern. Moreover, whether these antigens are present only in
the guts of larvae and pupae remains to be seen. The fact that these
proteins are present across different life stages, sexes, and tissues
strongly suggests an integral, albeit poorly understood, functional
role in mosquito development and survival.
The detection of
multiple protein bands by Western analysis is not uncommon
(11,
28,
31,
34). Posttranslational
modification of proteins, through the addition of carbohydrate
moieties, has been shown to affect the apparent molecular weight of
glycoproteins analyzed by SDS-PAGE. Glycosylation contributes as much
as 50 to 80% of the molecular weight of mature glycoproteins in
mammals (5,
8,
28) and up to 40%
in invertebrates (5,
16). In addition, it has
been shown that the glycosylation profile can influence MAb binding to
glycoproteins (5,
28). Enzyme and chemical
deglycosylation assays revealed that MG96 recognizes a midgut epitope
in which carbohydrates are a major component. MG96 recognition of the
gut epitope is influenced by N-linked oligosaccharides, as prolonged
treatment with PNGase A modified the antibody recognition profile.
PNGase A is an N-glycosidase that hydrolyzes an
N4-(acetyl-ß-D-glucosaminyl)
asparagine (Asn) residue
(11). It can also cleave
fucose linked to Asn-N-acetylglucosamine
(GlcNAc). In contrast to N-glycosidases,
O-glycosidases specifically cleave the Gal(ß1,3)GalNAc
disaccharide attached to serine and threonine residues
(11). Treatment of midgut
lysate with this enzyme eliminated MG96 recognition of the
40-kDa band, suggesting that an O-linked carbohydrate may be a
component of the MG96 epitope. Periodate treatment of An.
stephensi midgut lysates completely abolished recognition by MG96,
thereby confirming the importance of carbohydrates to the MG96 epitope
structure.
These results suggest that the epitope is complex and
may be composed primarily of O-linked carbohydrates. However, N-linked
carbohydrates and the peptide backbone may also contribute to a lesser
degree. Sustained recognition by MG96 of the 40-kDa band
following PNGase A treatment may be due to the presence of O-linked
oligosaccharides that partially maintain the integrity of the epitope.
Inefficient enzyme digestion of proximal N- and O-linked
oligosaccharides may have contributed to sustained recognition of the
two higher-molecular-weight molecules. Another possibility would be
that the epitope is composed of both carbohydrate and protein. This is
not an uncommon phenomenon, as there are several examples of antibodies
that recognize epitopes consisting of protein carbohydrate linkages,
e.g., antibodies to M and N glycophorins on erythrocyte membranes
(5,
28), or a combination of
peptides and carbohydrates
(28). For example, in the
development of antibodies to human epithelial mucin MUC1, the
specificities of several MAbs were shown to be contingent upon the
addition of a Gal(ß1,3)GalNAc disaccharide
(13). It is difficult to
ascertain from these assays which of these explanations is the most
reasonable. Only an in-depth analysis of sequentially detached
carbohydrates would allow us to gain insight into the essential
carbohydrate moieties that contribute to the complexity of this midgut
epitope.
The recognition of a carbohydrate epitope helps clarify
our observations of multiple bands on SDS-PAGE. One possible
explanation is that the midgut protein is multimeric. Another
possibility is that the 40-kDa doublet represents a
polypeptide heterodimer captured in its nascent form as it cycles
through the Golgi compartments. For both of these possibilities, a
common denominator is requisite. MG96 may therefore recognize a
carbohydrate epitope, a result of distinct branching structures of
several oligosaccharide side chains, which is shared among different
multimers of the same protein or among various glycoproteins. Several
studies have shown that insect midgut proteins in particular are
heavily O glycosylated
(15,
16,
32,
33). Although several
different glycoproteins are present in the midgut of
Anopheles, only a limited repertoire of carbohydrate moieties
are added onto proteins as they cycle through the Golgi apparatus
(33). This limited
repertoire is presumably due to the nature of the mosquito gut
environment. In mammals, specific tissues and organs display distinct
glycan patterns in accordance with their primary function. For example,
heavily O-glycosylated proteins such as intestinal mucins have
characteristic GalNAc-based glycan structures that protect the gut
epithelial cells from the harsh proteolytic environment
(11). O-linked
glycoproteins that harbor similar GalNAc oligosaccharide chains
covalently attached to serine and threonine repeat residues are
classified as mucin-type glycoproteins
(11). The AgMUC1 (from
An. gambiae)
(25) and AeIMUC1 (from
Ae. aegypti)
(22) mucin-like proteins
have been described to be midgut-specific O-glycosylated proteins that
may be involved in parasite recognition of the midgut. It remains to be
seen whether MG96 can recognize O-linked carbohydrate epitopes on these
and other mucin-like proteins that may be conserved across different
arthropod vectors. If so, we can then determine whether or not the
transmission-blocking effect of anticarbohydrate antibodies can indeed
translate to other insect vector-pathogen systems.
We have
described a potential TBV target carbohydrate receptor on midgut
glycoproteins that may serve as an attachment site for ookinetes.
Experiments are currently under way to identify the gene(s) encoding
these glycoproteins. More importantly, future research will focus on
characterizing the carbohydrate epitope and determining its role in the
ookinete-to-oocyst transition in the mosquito
midgut.
|
ACKNOWLEDGMENTS
|
|---|
We are grateful to Tom
Mitchell for generously providing the An. stephensi
mosquitoes, to Joan Buenconsejo for assistance with statistical
analysis, and to Albert Mulenga and Patricia Strickler for reviews of
the manuscript.
This work was supported by grants from
NIAID/NIH.
|
FOOTNOTES
|
|---|
* Corresponding
author. Mailing address: Department of Microbiology and Immunology,
School of Medicine, University of Maryland, 655 West Baltimore St.,
Baltimore, MD 21201. Phone: (410) 706-7066. Fax: (410) 706-0282.
E-mail:
rding001{at}umaryland.edu. 
Editor:
B. B. Finlay
|
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Infection and Immunity, December 2003, p. 6995-7001, Vol. 71, No. 12
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.12.6995-7001.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
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Top
Abstract
Introduction
