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INTRODUCTION |
Pattern recognition molecules serve as biosensors for detection of
invading pathogens in the innate immune systems of vertebrate and
invertebrate animals (1). They also play a crucial role in regulating
the adaptive immune reactions carried out by vertebrate lymphocytes
(2). Pattern recognition molecules bind to certain pathogen-associated
molecular patterns that are not found in the host, such as
lipopolysaccharide or peptidoglycan from bacterial cell walls and
1,3-glucan from fungal cell walls. Upon binding to the foreign
invaders, pattern recognition proteins trigger defense pathways such as
the complement system in vertebrates (2) and the prophenoloxidase
(PPO)1 activation pathway in
insects and other arthropods (3). The PPO activation pathway, like the
complement system, involves a protease cascade. Determining the
molecular mechanisms by which pattern recognition proteins
differentiate nonself from self and transduce signals that stimulate
defensive responses is a key to understanding the regulation of innate
immune systems.
Some pattern recognition proteins from mammals, such as the cellular
receptor CD14, the mannose-binding protein, the first component of
complement (C1q), and the macrophage mannose receptor and scavenger
receptor have been well characterized (4). Recent investigations of
pattern recognition molecules from insects have identified a group of
C-type lectins, which bind bacterial lipopolysaccharides and stimulate
antibacterial responses of hemocytes or activate the phenoloxidase
pathway (5-8). A peptidoglycan recognition protein has also been
discovered in the silkworm (9, 10). This 19-kDa hemolymph protein is
constitutively expressed in the fat body, epithelial cells, and
hemocytes of naive silkworms and can be induced by bacterial challenge.
Binding of peptidoglycan to peptidoglycan recognition protein can
trigger the PPO defense pathway (9). A cDNA which encodes a similar
bacteria-inducible peptidoglycan recognition protein has been cloned
from the moth, Trichoplusia ni (11). Furthermore, proteins
homologous to the insect peptidoglycan recognition proteins have been
identified in human and mouse and are expressed in bone marrow and
spleen (10, 11), indicating that this peptidoglycan recognition protein is conserved from insects to humans.
Fungal infections can be recognized by proteins which bind to cell wall
1,3-glucans, a molecular pattern specific for fungi. A group of
similar 1,3-glucan-binding proteins from crustaceans has been
isolated, and a cDNA for one of them was cloned from the crayfish,
Pacifastacus leniusculus (12-15). The crayfish protein can
induce spreading and degranulation of crayfish granular hemocytes when
bound to 1,3-glucan (16). An insect 1,3-glucan-recognition protein has been purified from the silkworm, Bombyx mori
(17). Binding of this 62-kDa protein with 1,3-glucan triggers the
PPO pathway in plasma. Several 1,3-glucan-binding proteins have been purified from cockroach species (18-20). These proteins also enhance PPO activation triggered by soluble 1,3-glucan. However, so far no
cDNA or amino acid sequence for an insect 1,3-glucan recognition protein has been reported.
In this paper, we describe the purification and cDNA cloning of a
1,3-glucan-recognition protein (GRP) from the tobacco hornworm, M. sexta. It is synthesized in the fat body and secreted
into hemolymph. The deduced amino acid sequence of GRP shows similarity with bacterial and sea urchin glucanases (21), clotting factor G
subunit 
from a horseshoe crab (22), earthworm CCF-1 (23), and also
with a Gram-negative bacteria-binding protein from the silkworm (24).
M. sexta GRP at physiological concentration caused aggregation of yeast and bacteria. Finally, we report that GRP can
trigger the activation of the PPO pathway upon binding to soluble
1,3-glucan.
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EXPERIMENTAL PROCEDURES |
Insects--
M. Sexta eggs were originally obtained
from Carolina Biological Supply. Larvae were reared on an artificial
diet as described by Dunn and Drake (25).
Affinity Purification of GRP for Antiserum
Production--
Hemolymph (100 ml) was collected from day 3 fifth
instar M. sexta larvae. Cell-free hemolymph (plasma) was
prepared by diluting the hemolymph 1:2 in anticoagulant buffer (4 mM NaCl, 40 mM KCl, 0.1% polyvinylpyrrolidone,
1.9 mM PIPES, 4.8 mM citric acid monohydrate, 13.6 mM sodium citrate, 4 mM EDTA, 5% sucrose,
pH 6.8) and centrifuging at 12,000 × g for 15 min to
remove hemocytes. The plasma (100 ml) was incubated with 0.5 g of
curdlan (an insoluble 1,3-glucan preparation, Sigma)
pre-equilibrated with PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, and 0.137 M sodium
chloride, pH 7.4, at 25 °C) at room temperature for 20 min with
mixing and then centrifuged (12,000 × g, 5 min). The
curdlan pellet was washed three times with 30 ml of PBS and then
incubated with 5 ml of PBS and 1 ml of 6× SDS sample buffer (0.35 M Tris, 10% (w/v) SDS, 30% (v/v) glycerol, 9.3% (v/v)
-mercaptoethanol, 0.012% bromphenol blue) at 95 °C for 10 min.
The sample was then centrifuged (12,000 × g, 5 min)
and the supernatant was analyzed by SDS-PAGE. The major band of 53 kDa
was cut from the SDS-PAGE gel and used as an antigen for the production
of a rabbit antiserum (Cocalico Biologicals, Reamstown, PA).
Western Blot Analysis--
For immunoblot analysis of proteins
after separation by SDS-PAGE, proteins were transferred to a
nitrocellulose membrane. The membrane was blocked with 3% dry skim
milk, and then incubated with rabbit antiserum to GRP (1:1000
dilution). Antibody binding was visualized by a color reaction
catalyzed by alkaline phosphatase conjugated to goat anti-rabbit IgG
(Bio-Rad). To estimate the concentration of GRP in hemolymph, 1-µl
samples of plasma were separated by SDS-PAGE followed by immunoblotting
as described above. The immunoreactive bands were digitized using a
Kodak DC120 digital camera, and band intensities were measured using
one-dimensional Image Analysis Software (Kodak Digital Science). Lanes
containing known concentrations of purified, native GRP were used to
produce a standard curve, for determining GRP concentration in plasma samples. Plasma from larvae of several insect species including the
Indian mealmoth Plodia interpunctella, the fruit fly
Drosophila melanogaster, and the tobacco budworm
Heliothis virescens (obtained from the Department of
Entomology, Kansas State University) was collected and analyzed by
SDS-PAGE and Western blotting, using antiserum to M. sexta GRP.
Purification of Native GRP--
One hundred and fifty ml of
hemolymph were collected as described above, and then fractionated by
ammonium sulfate precipitation. The 20-35% saturated ammonium sulfate
fraction of the plasma proteins was dissolved in 50 ml of 50 mM Tris, 20 mM NaCl, pH 7.6, and dialyzed
against the same buffer overnight at 4 °C with several changes of
buffer. Then the sample was centrifuged (10,000 × g, 15 min) before it was applied to a Q-Sepharose Fast Flow (Amersham Pharmacia Biotech) anion exchange column (3 × 10 cm). The column was washed with the same buffer until A280 was
less than 0.01 and then eluted at 2 ml/min with a gradient of 20 to 200 mM NaCl (200 ml total) in 50 mM Tris buffer, pH
7.6. Finally, the column was eluted with 100 ml of 200 mM
NaCl in 50 mM Tris buffer, pH 7.6. Fractions (5 ml) were
collected and analyzed for absorbance at 280 nm and by Western
blotting, using antibody to GRP. Fractions containing GRP were pooled
and passed through a concanavalin A (ConA) affinity column (3 × 5 cm). The flow-through fractions, containing GRP, were pooled and
applied to a hydroxyapatite column (3 × 3 cm) pre-equilibrated
with 10 mM sodium phosphate buffer, pH 6.8. The
hydroxyapatite column was washed with the same buffer until
A280 was less than 0.01 and then eluted at 0.1 ml/min with a gradient of 10 to 100 mM sodium phosphate, pH
6.8 (200 ml total). Finally, the column was eluted with 100 ml of 500 mM sodium phosphate buffer, pH 6.8. Fractions (5 ml)
containing GRP (detected by Western blot analysis) were pooled and
analyzed by gel filtration HPLC using a Bio-Sil SEC250 column (300 × 7.8 mm, Bio-Rad). The column was eluted with 50 mM
sodium phosphate, pH 6.8, 150 mM NaCl at 1 ml/min, and
fractions collected every 15 s were analyzed by SDS-PAGE and
silver staining. The column was calibrated by separating a mixture of
molecular mass standard proteins (Bio-Rad) containing thyroglobulin
(670 kDa), bovine 
-globulin (158 kDa), chicken ovalbumin (44 kDa),
equine myoglobin (17 kDa), and vitamin B12 (1.35 kDa) to produce a
standard curve for estimating the apparent mass of native GRP.
Amino-terminal and Internal Amino Acid Sequence
Determinations--
GRP (2 µg) purified by affinity to curdlan was
separated by SDS-PAGE, transferred to polyvinylidene difluoride
membrane (0.2 µm, Bio-Rad), and stained with Amido Black. The GRP
band was cut from the membrane and subjected to automated Edman
degradation in an Applied Biosystems Model 473 Pulse-liquid Sequencer.
To generate a peptide fragment used to obtain an internal sequence, purified GRP (100 µg) in 100 µl of 10 mM sodium
phosphate buffer was dried using a Speed-Vac and dissolved in 100 µl
of freshly prepared 70% formic acid (26). After incubation at 37 °C
for 48 h, the sample was dried using a Speed-Vac and dissolved in 20 µl of water. The hydrolyzed peptides were separated by
two-dimensional gel electrophoresis using the O'Farrell System (27),
transferred to polyvinylidene difluoride membrane, and stained with
Amido Black. One peptide spot with mass of 25 kDa was cut from the
membrane and subjected to automated Edman degradation.
Glycosylation and Mass Analysis--
GRP purified from plasma
(0.07 µg/µl, 100 µl) was mixed with 4 µl of 0.5 M
EDTA, 1 µl of 10% SDS, 1 µl of -mercaptoethanol and incubated
at 100 °C for 3 min, then cooled to room temperature. Then 10 µl
of 10% Triton X-100 and 10 µl of N-glycosidase F (0.2 units/µl, Roche Molecular Biochemicals) were added, and the mixture was incubated at 37 °C for 24 h. The digested GRP and the
untreated GRP were analyzed by SDS-PAGE and by matrix-assisted laser
desorption ionization mass spectrometry on a Lasermat 2000 instrument
(Finnigan MAT). Protein samples were mixed with cyano-4-hydroxycinnamic acid (1 mg/ml) as the matrix, and bovine serum albumin was used as an
internal standard.
cDNA Isolation and Sequencing--
Primers for PCR
amplification of a cDNA fragment encoding GRP were designed based
on amino acid sequences of the amino terminus and an internal fragment
(Fig. 3). Three sense-strand degenerate primers were synthesized: P1,
5'-GA(A/G)GTICCIGA(T/C)GC(T/C/A/G)AA-3'; P2,
5'-GA(A/G)GCIAT(T/C/A)TA(T/C)CCIAA(A/G)GG-3'; and P3,
5'-CGAGTATCCAT(A/C/TC)C(A/C/G/T)GA(C/T)GA-3', which encode LEVPDA,
EAIYPKG, and RVSIPDD, respectively. Two antisense primers were
synthesized: P6, 5'-GGTGGTACAAT(A/T)AT(A/C/G/T)GC(A/G)TC-3' and P7,
5'-TTGATTTTGGC(A/C/G/T)GT(A/C/G/T)AC(A/T)AT-3', which encode DAIIVPP
and IVTAKIN, respectively. Single-stranded cDNA was synthesized
from mRNA isolated from fat body of fifth instar larvae (collected
24 h after injection of 107 formalin-killed
Escherichia coli) using the antisense primer 008 (5'-GGAGTACTCTAGAAGC(T)17-3') (28). This cDNA template
was amplified with primers 009 (5'-GGAGTACTCTAGAAGCTT-3') and P1 (each at 1 µM) in 10 mM Tris-HCl, pH 8.3, 50 mM
KCl, 1.5 mM MgCl2, 200 µM dNTP, and 2 units
of Thermus aquaticus DNA polymerase (Fisher Scientific). PCR
was performed with denaturing at 94 °C for 30 s, annealing at
50 °C for 40 s, and extension at 72 °C for 1 min for 40 cycles, using a DNA Thermal Cycler 480 (Perkin Elmer). The PCR product
was used as a template to amplify again using primers P2 and P7 or P3
and P6 under the same conditions described above. A 700-base pair PCR
product amplified using primers P2 and P7 was cloned into plasmid
vector pGEM-T using a PCR cloning kit (Promega).
A M. sexta larval fat body cDNA library in 
Uni-Zap
XR vector (Stratagene) (28) was screened using the 700-base pair cloned cDNA PCR product, labeled with [-32P]dCTP
(multiprime DNA labeling system, Amersham Pharmacia Biotech) as a
probe. Plaques that hybridized to the probe were purified to
homogeneity and subcloned by in vivo excission of pBluescipt plasmids. The positive clone with the longest cDNA insertion (1.6 kilobases) was sequenced from both strands by the DNA Sequencing Facility at Iowa State University, using vector primers and primers designed from previously determined sequences.
Northern Blot Analysis--
Total RNA from day 3 fifth instar
M. sexta larval fat body, hemocytes, or integument was
extracted using the Rapid Total RNA Isolation Kit (5 Prime
3 Prime, Inc.). Samples of total RNA (20 µg) from these tissues
were resolved by electrophoresis on an agarose gel containing
formaldehyde, transferred to a nitrocellulose membrane, and hybridized
with 32P-labeled GRP cDNA. To confirm equal mRNA
loading in different samples, a duplicate blot was hybridized with a
cDNA for M. sexta ribosomal protein S3 (29) as a
control. To test whether GRP mRNA level is affected by microbial
infection, M. sexta fifth instar day 3 larvae were
injected with a mixture of killed E. coli (107
cells) and yeast (107 cells). Control larvae were not
injected. Total RNA from fat body was extracted 24 h after
injection and analyzed by Northern blotting as described above. Band
intensities were measured using one-dimensional Image Analysis Software
(Kodak Digital Science), and the ratio of GRP mRNA to ribosomal
protein S3 mRNA band intensity was calculated.
Fat Body Culture--
Fat body from day 2 fifth instar M. sexta larvae was dissected, washed with PBS, and then incubated at
28 °C in 2 ml of EX-CELL 405 (JRH Biosciences) insect cell culture
medium containing penicillin (100 units/ml) and streptomycin (100 µg/ml) in wells of a 24-well plate with shaking at 100 rpm. At
different times of incubation (0, 7, 11, 29, 40, and 64 h), 20 µl of the medium was removed and stored at 
20 °C for later
analysis. These samples were then analyzed for the presence of GRP by
Western blotting using antibody to GRP as described above.
Computer Analysis of Sequence Data--
Preliminary sequence
editing and analysis was performed using IBI pustell programs. The
amino acid sequence deduced from the cDNA was used to search the
nonredundant peptide sequence data base and the expressed sequence tag
data base (National Center for Biotechnology Information) with the
blast program (30). The most similar protein sequences were retrieved
and aligned with the GRP sequence using the pileup program from the GCG
Sequence Analysis Software Package 7.3.1 (31).
Recombinant GRP--
PCR was used to generate a GRP cDNA
fragment encoding amino acid residues 1-468, which was cloned into the
NcoI and HindIII sites of E. coli
expression vector H6-pQE-60 (32) and used to transform E. coli strain XL1Blue. Expression of GRP in this vector yields a
protein with an amino-terminal sequence of
Met-(His)6-Ala-Met-Gly-Leu-Glu-Val. Maximum expression of
the recombinant protein (0.1 mg/ml E. coli culture) was
obtained from cultures in Luria-Bertani medium containing 100 µg/ml
ampicillin, with shaking for 20 h at 37 °C. The bacteria from
100 ml of culture were centrifuged at 10,000 × g for 5 min and then resuspended in 10 ml of 8 M urea, 100 mM sodium phosphate, pH 6.8, 200 mM sodium
chloride, 10 mM -mercaptoethanol (buffer B) to lyse the
bacteria. After centrifugation at 12,000 × g for 15 min, the supernatant was used for purification of recombinant GRP
(H6GRP) by Ni2+-affinity chromatography.
Approximately 5 mg of recombinant H6GRP was mixed with 5 ml
of Ni-agarose resin (Qiagen) for 12 h at room temperature. This mixture was then packed in a column and washed with 15 ml of buffer B. Then the column was washed with a 60-ml gradient from 8 M
urea in Buffer B to 0 M urea in 100 mM sodium
phosphate, pH 6.8, 200 mM NaCl, 2 mM
glutathione, 0.02 mM oxidized glutathione, 10% glycerol (buffer C) at 1 ml/min. Finally, the column was eluted with 100 mM imidazole in buffer C. Three-ml fractions were collected
and analyzed by SDS-PAGE. Fractions containing purified
H6GRP were stored at 4 °C. Immediately before use,
samples were passed through a Sephadex G-25 column (PD-10, Amersham
Pharmacia Biotech) to change the buffer to 50 mM Tris, pH
7.0, 50 mM NaCl, 10% glycerol.
Labeling H6GRP with Fluorescein Isothiocyanate
(FITC)--
Purified H6GRP (1.6 mg) in 4 ml of 0.1 M sodium carbonate, pH 9.0, was mixed with 14 mg of
fluorescein isothiocyanate (Isomer I, on Celite, Sigma), stirred at
room temperature for 4 h, and then centrifuged (10,000 × g, 1 min). The FITC-H6GRP conjugate in the
supernatant was separated from the unbound dye by gel filtration using
a Sephadex G-25 column (PD-10, Amersham Pharmacia Biotech). The purity
of the FITC-H6GRP was analyzed by SDS-PAGE and Coomassie Blue staining.
Binding of FITC-labeled H6GRP to Microbes--
An
aliquot of 50 µl of FITC-H6GRP (0.3 mg/ml) in 50 mM Tris, pH 7.0, 50 mM NaCl, and 10% glycerol
was mixed with Saccharomyces cerevisiae (5 × 107 cells), E. coli (5 × 107
cells), or Micrococcus lysodeikticus (5 × 107 cells) at room temperature for 8 h and then at
4 °C overnight. The cells were then washed with 1 ml of TBS (0.137 M NaCl, 0.003 M KCl, 0.025 M
Tris-HCl, pH 7.6) and resuspended in 1 ml of TBS. Binding of
FITC-H6GRP was measured by subjecting the washed cells to
flow cytometry analysis using a Becton Dickinson FACScan flow cytometer. For each sample, fluorescence of 10,000 cells was
determined. Untreated cells of each kind were used as controls to
measure background fluorescence. To test the concentration dependence of FITC-H6GRP binding to S. cerevisiae or
E. coli, aliquots of 20 µl of FITC-H6GRP (0 to
0.3 mg/ml) in 50 mM Tris, pH 7.0, 50 mM NaCl
were mixed with 20 µl of S. cerevisiae or E. coli (5 × 107 cells/ml) suspended in TBS. After
incubation at room temperature for 30 min, the cells were washed with
TBS, resuspended in 1 ml of TBS, and subjected to flow cytometry as
described above.
Aggregation of Microbes by H6GRP--
An aliquot of
10 µl of H6GRP (0.1 mg/ml) in 50 mM Tris, pH
7.0, 50 mM NaCl, and 10% glycerol was incubated with 10 µl of fluorescein-conjugated Staphylococcus aureus (2 × 109 cells/ml), E. coli (K-12 strain, 2 × 109 cells/ml), or S. cerevisiae (2 × 108 cells/ml) in TBS (all from Molecular Probes) at room
temperature for 30 min. Samples of the cells were then applied to
microscope slides, and the degree of aggregation of the cells was
observed using an Olympus BH-2 fluorescence microscope.
Activation of the PPO Pathway by H6GRP in the
Presence of Laminarin--
Twenty µl of H6GRP (0.2 mg/ml
in 50 mM Tris, pH 7.0, 50 mM NaCl, and 10%
glycerol) was mixed with 20 µl of fresh plasma from day 3 M. sexta larvae (diluted 1:2 in anticoagulant buffer) and 5 µl of laminarin (10 mg/ml or 1 mg/ml in TBS) in wells of a 96-well microplate. After incubation for 20 min at room temperature, 20 µl of
dopamine (16 mM in distilled water) as a phenoloxidase
substrate, was added to each well. Phenoloxidase activity was
determined by measuring the absorbance at 492 nm at 1-min intervals
using a PowerWave X 340 microplate scanning spectrophotometer (Bio-Tek Instruments, Inc.). Control samples lacked either H6GRP,
laminarin, or both, which were replaced with appropriate buffers.
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RESULTS |
Identification of a 1,3-Glucan-binding Protein from M. sexta
Larval Hemolymph--
To identify hemolymph proteins that can bind to
1,3-glucan, curdlan (an insoluble 1,3-glucan preparation) was
used as an affinity matrix to purify proteins from M. sexta
larval plasma. After incubating curdlan with plasma and washing the
curdlan with buffer, the bound proteins were eluted by treatment with
SDS and -mercaptoethanol at 95 °C for 10 min. When this sample
was analyzed by SDS-PAGE, a major protein with a molecular mass of
approximately 53 kDa was observed (Fig.
1A). This protein was later
named 1,3-glucan-recognition protein (GRP). GRP accounted for more
than 50% of the total protein eluted from curdlan. Several other
weaker bands were also observed, including a band at ~17 kDa. GRP
bound so tightly to curdlan that it could not be eluted without
denaturing in SDS. Treatments with acid (pH 1.5), high salt
concentration (2 M NaCl), or 8 M urea did not
elute GRP from curdlan. Approximately 3 mg of denatured GRP was
obtained from 100 ml of hemolymph by this affinity method.
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Fig. 1.
Analysis of GRP by SDS-PAGE and Western
blot. A, SDS-PAGE and Commassie Blue staining of purified GRP.
Lane M, molecular weight markers; lane 1, GRP
purified from plasma by binding to curdlan; lane 2, GRP
purified from the plasma by ion exchange, lectin affinity, and
hydroxyapatite chromatography; lane 3, recombinant
H6GRP purified by Ni2+ affinity chromatography.
The arrows indicate the 53-kDa GRP bands and a 17-kDa GRP
fragment. B, Western blot analysis of GRP. Lanes 1 and
3, total protein from 1 µl of larval plasma; lanes
2 and 4, 0.7 µg of GRP purified under nondenaturing
conditions. Lanes 1 and 2 are stained with
Coomassie Blue; lanes 3 and 4 are detected by
Western blotting using antibody to GRP.
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GRP eluted from curdlan was further purified by SDS-PAGE, and the
53-kDa band on the gel was excised and injected into a rabbit to raise
an antiserum. This antiserum recognized a single protein band of 53 kDa
in Western blot analysis of M. sexta plasma (Fig. 1B). The sequence of the amino-terminal 23 residues of this
protein was determined by Edman degradation (Fig. 3). The
amino-terminal sequence (19 residues) of the protein in the 17-kDa band
that bound to curdlan also was determined and found to be identical to
that of the 53-kDa GRP. The 17-kDa protein thus appears to be an
amino-terminal fragment of GRP. When plasma proteins bound to curdlan
were eluted by SDS treatment in the absence of -mercaptoethanol, the
17-kDa amino-terminal fragment of GRP was still observed by SDS-PAGE
analysis (data not shown), suggesting that this fragment bound to
curdlan by itself rather than through a disulfide link to the
carboxyl-terminal fragment of GRP.
Purification and Analysis of GRP--
To obtain nondenatured GRP
for functional studies, GRP was purified from fifth instar larval
plasma by a series of chromatographic steps that did not involve
binding to curdlan. The purification procedure consisted of ammonium
sulfate fractionation, followed by column chromatographic separations
using Q-Sepharose, ConA, and hydroxyapatite (Fig.
2). The 20-35% ammonium sulfate
fraction contained about 50% of the total GRP in plasma. This step
removed most of the storage proteins, which are present at high
concentration in the plasma. GRP bound to Q-Sepharose at pH 7.6 in a
buffer containing 50 mM Tris and 20 mM NaCl.
When the column was eluted with a sodium chloride gradient from 20 to
200 mM at pH 7.6, GRP eluted at the end of the gradient
(Fig. 2A). No additional GRP was eluted when the column was
washed with 1 M sodium chloride. Fractions from the
Q-Sepharose column containing GRP were combined and applied to a ConA
column. GRP did not bind to the ConA column and was present in the
flow-through fractions. However, other glycoproteins that did bind to
ConA were separated from GRP in this step. Pooled fractions from the
ConA column were applied to a hydroxyapatite column pre-equilibrated
with 10 mM sodium phosphate buffer, pH 6.8. GRP eluted near
the end of a sodium phosphate gradient from 10 to 100 mM.
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Fig. 2.
Purification of
1,3-glucan recognition protein from plasma under
nondenaturing conditions. Conditions for each chromatographic step
are described under "Experimental Procedures." Solid
lines show the absorbance at 280 nm; broken lines show
the gradient of elution buffers with a scale on the right
axis. The bars represent the fractions that contained
GRP (detected by Western blot analysis) which were pooled and subjected
to next purification step. A, Q-Sepharose Fast Flow column
chromatography of a 20-35% saturated ammonium sulfate fraction of
larval plasma. B, concanavalin A-agarose column
chromatography. The column was pre-equilibrated with 50 mM
Tris, 20 mM NaCl, pH 7.6. After washing with the same
buffer, the column was eluted with 0.5 M
methyl--D-mannopyranoside. C, hydroxyapatite
column chromatography of the pooled fraction from the ConA column.
D, gel filtration HPLC of purified GRP. The indicated
elution times of standard proteins were used to calculate an apparent
native mass of 73 kDa for GRP. Arrows indicate peak
positions of standard proteins and GRP.
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When the fractions containing GRP were analyzed by SDS-PAGE, GRP
appeared as a single band with an apparent molecular mass of 53 kDa
(Fig. 1A, lane 2). Analysis by MALDI mass spectrometry gave
a mass of 53,583 Da for GRP. Approximately 0.3 mg of pure GRP was
obtained from 100 ml of plasma. When this purified GRP was analyzed by
gel filtration HPLC, it eluted as a single peak, with an elution volume
consistent with a native molecular mass of about 70 kDa. This result
suggests that GRP exists as a monomer in solution.
The amino-terminal sequence of GRP isolated as described above was
identical to that obtained from the protein eluted from curdlan. To
obtain sequence information from an internal peptide fragment, GRP was
treated with formic acid to hydrolyze between Asp and Pro residues. The
resulting peptide fragments were separated by two-dimensional gel
electrophoresis and transferred to a polyvinylidene difluoride
membrane. From one of the peptide spots on the membrane with a mass of
approximately 25 kDa, an amino acid sequence of 17 residues was
determined by Edman degradation (Fig.
3).
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Fig. 3.
Nucleotide and deduced amino acid sequences
of GRP. The deduced amino acid sequence is shown below the
cDNA sequence. Amino acid residues in the mature protein are
assigned positive numbers, and those in the signal peptide are assigned
negative numbers. Two potential N-linked glycosylation sites
are marked with a  . A single underline represents
sequences of the amino-terminal of mature GRP and the isolated peptide
determined by Edman degradation. In the cDNA sequence, the
polyadenylation sequence AATAAA is double underlined, and
the termination codon TGA was marked with an asterisk.
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cDNA Cloning and Deduced Protein Sequence--
The
amino-terminal sequence of GRP and the internal amino acid sequence of
the fragment derived from formic acid hydrolysis were used to design
degenerate oligonucleotide primers for PCR, to amplify a fragment of a
GRP cDNA. PCR was carried out as described under "Experimental
Procedures" using single-stranded cDNA synthesized from fat body
mRNA as a template. A 700-base pair PCR product was obtained and
cloned into a plasmid vector, and this cloned fragment was used as a
hybridization probe to screen a larval fat body cDNA library.
Forty-eight positive clones were isolated from a screen of
approximately 3 × 105 plaques. The GRP cDNA clone
(pGRP9) containing the longest insert (approximately 1.6 kilobases) was sequenced.
The sequence of pGRP9 contains a 47-nucleotide 5' noncoding region, an
open reading frame of 1461 nucleotides, and a 3'-untranslated sequence
of 56 nucleotides (Fig. 3). The open reading frame encodes 487 amino
acid residues. The first 19 amino acid residues make up a secretion
signal peptide, and the amino-terminal sequence of the mature protein
begins at residue 20. The internal amino acid sequence of 17 residues
determined from a peptide obtained after hydrolysis of GRP with formic
acid was consistent with the deduced sequence between residues 240 and
256 (Fig. 3). The calculated molecular mass of the 468-residue mature
protein is 52,335 Da, which is 1,247 Da less than the mass determined
by mass spectrometry. There are two putative N-linked
glycosylation sites in the carboxyl-terminal region of the protein.
After GRP was treated with N-glycosidase F, it migrated
slightly faster than untreated GRP in SDS-PAGE analysis (Fig.
4), indicating that this glycosidase
removed approximately 1 kDa of N-linked carbohydrate from
the protein. There are five Cys residues in the mature protein.
Therefore, GRP must contain at least one free Cys that is not involved
in a disulfide bond. The calculated pI of the mature GRP is 5.0. This
is in agreement with the results of two-dimensional gel
electrophoresis, in which the pI of GRP was estimated to be
approximately 5.5 (data not shown).
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Fig. 4.
Deglycosylation of GRP by treatment with
N-glycosidase F. Lane M, molecular
weight markers; lane 1, GRP purified from plasma; lane
2, purified GRP after treatment with N-glycosidase
F.
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Sequence Comparisons--
Searching of the GenBank sequence data
bases indicated that the GRP open reading frame has significant
sequence similarity with bacterial -glucanases and with several
invertebrate proteins that contain glucanase-like regions. The
carboxyl-terminal region (residues 174-442) was similar to a group of
1,3-glucanases, with the highest degree of similarity among these to
enzymes from a sea urchin and from Bacillus circulans (Fig.
5). The amino-terminal region was similar
to several proteins from invertebrates, which also contain a
carboxyl-terminal glucanase-like sequence. GRP was similar throughout
its sequence to a Gram-negative bacteria-binding protein (GNBP) from
the silkworm, B. mori (24) (42% identity), and to
homologous putative GNBPs from the fall webworm, Hyphantria cunea (33) (39% identity), and a mosquito Anopheles
gambiae (34) (38% identity). Partial amino acid sequences
available from cDNAs corresponding to expressed sequence tags (EST)
from B. mori and from Drosophila melanogaster
also were quite similar to GRP. M. sexta GRP is 59%
identical to the protein encoded by the B. mori EST
(accession number AU004243) but only 37% identical to the
corresponding amino-terminal region of B. mori GNBP, which suggests that this EST, rather than GNBP, is the silkworm ortholog of
M. sexta GRP. In addition to these insect proteins, GRP is similar to sequences of coelomic cytolytic factor 1 (CCF-1), which is a
glucan-binding protein from an earthworm, Eisenia foetida (23) (34% identity) and to a 1,3-glucanase from a sea urchin, Strongylocentrotus purpuratus (21) (34% identity) (Fig. 5). These proteins are similar to GRP in both the glucanase domain and the
amino-terminal region, although the amino-terminal extension is much
shorter in CCF-1 than in the sea urchin glucanase or the insect
proteins.
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Fig. 5.
Alignment of the amino acid sequence of GRP
with those of other similar invertebrate proteins and with bacterial
glucanases. The PILEUP program (31) was used to align the sequence
of GRP with the most similar proteins identified by searching the
GenBank data base with the BLAST program. These include a group of
proteins from invertebrates, all of which contain a carboxyl-terminal
regions with similarity to bacterial glucanases. Ms-GRP,
M. sexta 1,3-glucan-recognition protein (AF177982);
Bm-EST, an expressed sequence tag from B. mori
(AU004243); Bm-GNBP, B. mori Gram-negative
bacteria-binding protein (L38591); Hc-GNBP, Hyphantria
cunea putative Gram-negative bacteria-binding protein (AF023916);
Dm-EST, two expressed sequence tags (5' and 3' ends) from
D. melanogaster (AI257586, AI109637); Ag-GNBP,
Anopheles gambiae putative Gram-negative bacteria-binding
protein (AJ001042); Sp-GLUC, Strongylocentrotus
purpuratus 1,3-glucanase (U49711); Ef-CCF-1,
E. foetida coelomic cytolytic factor 1 (AF030028). Two
bacterial glucanases are also shown in this alignment:
Bc-GLUC, B. circulans -1,3-glucanase A1
(P23903), the bacterial glucanse most similar to M. sexta
GRP; Bm-GLUC, Bacillus macerans
endo-1,3-1,4--glucanase (P23904), a related glucanase whose
structure has been determined by x-ray crystallography (35). Residues
conserved in all of the invertebrate sequences are marked with *, and
residues identical in at least 4 of the invertebrate sequences are
marked with +. Residues corresponding to the active site of the
B. macerans glucanase are marked with #. Positions in
M. sexta GRP which are identical to at least one of the
bacterial glucanases are underlined. Numbering corresponds
to the M. sexta GRP sequence as shown in Fig. 3. In the
sequences derived from expressed sequence tags, regions that have not
yet been sequenced are shown with ~ symbols. A  marks the
approximate site of proteolytic cleavage of GRP to produce the 17-kDa
fragment shown in Fig. 1.
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The most conserved region of the glucanase-like domain of GRP and the
similar invertebrate proteins is in a sequence that aligns with a
region near the active site of bacterial 1,3-glucanases and
1,3-1,4-glucanases (Fig. 5). However, GRP lacks four conserved residues that line the active site of such enzymes, and in particular has nonconservative replacements of two Glu residues that are believed
to act as the catalytic residues responsible for cleaving 1,3- or
1,4-glycosidic bonds in the bacterial glucanases (35). This
observation is consistent with the fact that we have not detected any
1,3-glucanase activity associated with GRP (data not shown).
In addition to the sequences discussed above, BLAST searches using the
GRP sequence identified a number of sequences with lower but still
significant similarity to the conserved region near the glucanase
active site. These include 1,3-glucanases from bacteria and plants,
an expressed sequence tag from the solitary ascidian, Ciona
intestinalis, and a 1,3-glucanase-like domain from the
-chain of coagulation factor G from the horseshoe crab, Tachypleus tridentatus (22) (Fig.
6). In all of these sequences, the
putative glucanase active site residues are conserved, in contrast to
GRP and the other proteins from lepidopteran insects. The glucanase
domain, widespread in evolution, appears to have evolved in the insects
to function as a glucan-binding protein and has lost its hydrolase
activity and function.
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Fig. 6.
Alignment of a region in the
carboxyl-terminal of GRP with a conserved region from other
glucanases. Ms-GRP, M. sexta GRP (AF177982);
Bc-GLCNA1, B. circulans 1,3-glucanase A1
(P23903); Sp-GLCN, S. purpuratus 1,3-glucanase
(U49711); Ci-EST, C. intestinalis expressed
sequence tag (AJ227713); Ct-GLCN, Clostridium
thermocellum endo-1,3(4)--glucanase (CAA61884); Rm-GLCN,
Rhodothermus marinus endo-1,3-1,4 glucanase (P45798);
Tm-LMN, Thermotoga neapolitana laminarinase
(CAA88008); Tt-FCTG, Tachypleus tridentatus
clotting factor G subunit precursor (BAA04004); Cc-GLCN,
Cochliobolus carbonum mixed-linked glucanase precursor
(AAC49904); Zm-HMLG, Zea mays xyloglucan
endo-transglycosylase homolog (AAC49012). Residues conserved in at
least 6 of the sequences are marked with *, and residues corresponding
to the active site of the B. macerans glucanase are marked
with #.
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Expression of GRP in Vivo--
In Northern blot analysis, the GRP
cDNA hybridized to a 1.5-kilobase nucleotide band in RNA samples
from larval fat body, consistent with the size expected from the cloned
cDNA. No band was detected in RNA from hemocytes or integument
(Fig. 7A). These results
indicate that fat body is the primary site of GRP synthesis, as is true
for most insect hemolymph proteins. Furthermore, when fat body was
cultured in vivo, GRP was first detected in the culture medium after 7 h and its concentration gradually increased up to
64 h in culture (data not shown). This result is also consistent with synthesis of GRP by fat body and secretion into hemolymph. The
level of GRP mRNA in fat body did not increase significantly after
larvae were injected with bacteria and yeast (Fig. 7B). This
lack of inducibility distinguishes GRP expression from that of three
similar proteins from insects, B. mori GNBP and homologous proteins from H. cunea and A. gambiae, which are
synthesized in response to exposure to microbial elicitors (24, 33,
34).
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Fig. 7.
Northern blot analysis of GRP mRNA
levels. A, RNA samples (20 µg) from
Manduca fifth instar larval integument (I), fat
body (F), and hemocytes (H) were separated by
electrophoresis on a 1% agarose gel containing formaldehyde. The RNA
was transferred to a nitrocellulose membrane and then hybridized with
32P-labeled M. sexta GRP cDNA. Approximately
equal RNA loading of each lane was confirmed by probing a duplicate
blot with 32P-labeled cDNA for M. sexta
ribosomal protein S3 (29). The approximate size of the mRNA was
derived from RNA standards. B, RNA samples from fat body of
fifth instar larvae injected with a mixture of E. coli and
yeast 24 h earlier were analyzed by Northern blotting as described
above. Control larvae were injected with PBS. The bands intensities
were quantified using one-dimensional Image Analysis Software (Kodak
Digital Science), and the ratio of GRP mRNA to ribosomal protein S3
mRNA was calculated. Bars represent the mean ± S.E. (n = 5).
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Production of Recombinant GRP--
To obtain sufficient amounts of
GRP for functional studies, GRP was expressed as a recombinant protein
in E. coli. Recombinant GRP (H6GRP), which
contains an amino-terminal 6-histidine tag, was insoluble in the buffer
extract obtained after lysis of E. coli by sonication. It
could be dissolved in 8 M urea and was purified by affinity
chromatography. After H6GRP bound to the Ni2+
affinity column, it was renatured using a linear gradient of urea from
8 to 0 M, and then eluted by washing with 100 mM imidazole. This H6GRP preparation was
soluble and contained a single band at 53 kDa when analyzed by SDS-PAGE
and Coomassie Blue staining (Fig. 1A, lane 3). The renatured
H6GRP remained stable at 200 µg/ml and at 4 °C in
buffer C containing 10% glycerol for at least 1 month.
Binding of H6GRP to Microorganisms--
To investigate
potential functions of GRP, experiments were conducted to test whether
the recombinant protein can bind to microorganisms. H6GRP
labeled with fluorescein isothiocyanate (FITC-H6GRP) was
incubated with S. cerevisiae, E. coli, and
M. lysodeikticus as examples of yeast, Gram-negative, and
Gram-positive bacteria. After the incubation period, the microbial
particles were washed, and then the fluorescence due to bound
FITC-H6GRP was measured by flow cytometry. We observed
binding of FITC-H6GRP to yeast (whose cell walls contain
1,3-glucan) and to bacteria (Fig.
8A). When we normalized the
fluorescence intensity per cell to obtain fluorescence intensity per
µm2 of cell surface area, we found that GRP bound to all
three types of microorganisms to about the same degree (Fig.
8B). The binding of FITC-H6GRP to yeast and to
E. coli was concentration-dependent and
saturable (Fig. 8, C and D), with a
Kd of 77 µg/ml (1.5 µM) for binding
to yeast and a Kd of 65 µg/ml (1.2 µM) for E. coli. The concentration
of GRP in plasma is approximately 30 µg/ml (determined by
densitometry analysis of Western blot samples as described under
"Experimental Procedures"). These results indicate that such
binding would occur at physiological GRP concentrations.
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Fig. 8.
Binding of H6GRP to
microorganisms. FITC-labeled recombinant GRP
(FITC-H6GRP) was used to assay binding of GRP to
microorganisms in a flow cytometry assay. After incubation of
FITC-H6GRP with 107 killed cells as described
under "Experimental Procedures," the bacteria (E. coli
or M. lysodeikticus) or yeast (S. cerevisiae)
were washed, and then the fluorescence of individual cells from each
sample was measured by flow cytometry. The fluorescence intensity shown
is the average of 10,000 cells. The standard error of each sample was
too small to be visible as an error bar. A,
FITC-H6GRP (0.3 mg/ml) binding to S. cerevisiae,
E. coli, or M. lysodeikticus. The binding time
was 8 h. The bars represent the mean fluorescence
intensities/cell of 10,000 cells. B, the same data as
A, normalized to show mean fluorescence intensity per
µm2 of cell surface area. C, concentration
dependence of FITC-H6GRP binding to S. cerevisiae. The binding time was 30 min. Each point is the mean
fluorescence intensity/cell of 10,000 cells. The binding curves
represent a one site model with R2 = 0.99 (Kd = 77 ± 10 µg/ml). D,
concentration dependence of FITC-H6GRP binding to E. coli. The procedure was the same as in C but using E. coli strain XL1Blue to bind to FITC-H6GRP. The binding
curve represents a one site model with R2 = 0.98 (Kd = 65 ± 19 µg/ml).
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Because H6GRP was able to bind to bacteria and yeast, we
tested whether H6GRP can aggregate these microorganisms.
The presence of H6GRP at 50 µg/ml caused significant
aggregation of S. cerevisiae, E. coli, and
S. aureus, whereas bovine serum albumin used as a control at
the same concentration did not lead to aggregation of these organisms
(Fig. 9). These results suggest that one
function of GRP may be to aggregate invading microorganisms, leading to more efficient clearance by hemocytes.
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Fig. 9.
Aggregation of microorganisms by GRP.
H6GRP (0.05 mg/ml) was incubated with FITC-labeled S. aureus, E. coli, or S. cerevisiae at room temperature
for 30 min. The microorganisms were incubated similarly with bovine
serum albumin (BSA) (0.05 mg/ml) as a control. The cells
were photographed using fluorescence microscopy.
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Activation of the PPO Pathway by GRP in the Presence of
Laminarin--
Activation of PPO in insect plasma can be triggered by
1,3-glucan (17, 36, 37). We performed experiments to determine whether interaction of GRP with 1,3-glucan might participate in
activation of this pathway. M. sexta larval plasma was
diluted 1:2 in anticoagulant buffer, which decreased the endogenous GRP concentration from 30 to 10 µg/ml. This diluted plasma was incubated with different amounts of the 1,3-glucan laminarin and 100 µg/ml H6GRP at room temperature for 20 min, and then the
phenoloxidase activity in the plasma was determined (Fig.
10). Laminarin alone (1 µg/µl) did
not trigger the PPO pathway within 20 min. However, when laminarin (1 or 0.1 µg/µl) combined with 100 µg/ml H6GRP was added
to plasma, PPO activity significantly increased. H6GRP alone stimulated a much smaller degree of PPO activation. These results
suggest that GRP serves as a biosensor for 1,3-glucan in insect
defense and that binding of GRP to fungi may lead to activation of a
proteinase in the PPO activation cascade.
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Fig. 10.
Activation of the PPO pathway by GRP and
laminarin. Laminarin and H6GRP were incubated with
diluted plasma, and after 20 min, phenoloxidase activity was detected
using dopamine as a substrate as described under "Experimental
Procedures." Samples containing GRP were at 0.10 mg/ml (2 µM) final concentration. Bars represent the
mean ± S.E. (n = 8).
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DISCUSSION |
We have purified from hemolymph of the tobacco hornworm, M. sexta, a 53-kDa protein that binds very tightly to 1,3-glucans. We have named this protein GRP, which is the name used for a very similar protein from hemolymph of silkworms (17). We first isolated GRP
by taking advantage of its affinity to curdlan, an insoluble 1,3-glucan, but this protein bound so tightly to curdlan that it
could only be eluted by treatment with SDS. We then developed a method
to purify GRP that did not require denaturing conditions. GRP is
present in hemolymph at all developmental stages of M. sexta
at approximately 30 µg/ml (0.6 µM). In immunoblot
analysis using antiserum to M. sexta GRP, we have
detected an immunoreactive band at ~50 kDa in plasma from other
insect species, including two lepidopterans: P. interpunctella and H. virescens; and a dipteran: D. melanogaster,2
suggesting that the presence of a similar GRP in plasma may be expected
in other insects. M. sexta GRP has a sequence very similar to that of a 1,3-glucan-recognition protein isolated from hemolymph of the silkworm, B. mori (17), which is a 62-kDa protein
that binds very strongly to curdlan and is involved in phenoloxidase activation.3
We isolated a cDNA clone that encodes GRP from a larval fat body
library. The amino acid sequence of GRP, deduced from the nucleotide
sequence of the cloned cDNA, indicated that the protein is composed
of two regions, which appear to represent amino-terminal and
carboxyl-terminal domains. The carboxyl-terminal domain is similar in
sequence to 1,3- and 1,3-1,4-glucanases from bacteria (38) and to
a 1,3-glucanase from a sea urchin (21), but GRP had no detectable
1,3-glucanase activity. Two catalytic Glu residues, conserved in the
active site of the bacterial glucanases (35) are replaced with Leu and
Cys in Manduca GRP, which explains the absence of glucanase
activity (Fig. 4). Thus, GRP may be an evolutionary descendent of a
glucanase that has lost its catalytic activity but has maintained the
ability to bind to 1,3-glucans. Clotting factor G from a horseshoe
crab, Tachypleus tridentatus, also contains a region with
similarity to these glucanases (Fig. 6), and it is possible that
binding of 1,3-glucans to this domain triggers the activation of the
enzyme's proteolytic subunit (22). Although no sequences similar to
the glucanase-like domain of GRP appear to have been found so far in
vertebrates, the discovery of a homologous sequence in an ascidian (a
primitive chordate) (39) suggests that such glucan-binding proteins may
exist in vertebrates as well.
A group of proteins from insects and from an earthworm are also similar
in sequence to GRP. They contain the carboxyl-terminal glucanase-like
domain and also a unique amino-terminal domain. These include a GNBP
from hemolymph of the silkworm, B. mori. (24). GNBP-like
sequences have also been identified in the fall webworm, H. cunea (33), and a mosquito, A. gambiae (34), although these two proteins are known so far only from cDNA sequences and have not been tested for function. A partial B. mori
sequence encoded by an expressed sequence tag cDNA is more similar
to M. sexta GRP than is B. mori, GNBP (Fig.
5), suggesting that this cDNA may encode the silkworm GRP.
A protein called CCF-1 from an earthworm, E. foetida, also
has a sequence similar to that of GRP (23). CCF-1 binds to
1,3-glucan and bacterial lipopolysaccharide and thus may function in
a manner similar to GRP. Although the glucanase active site residues
are conserved in CCF-1 (Fig. 5), it also lacks apparent glucohydrolase activity (23). The catalytic Glu residues are also conserved in
A. gambiae GNBP (Fig. 5), but whether it can function as a glucanase is unknown.
The insect proteins related to GRP as well as the earthworm CCF-1 and
the sea urchin 1,3-glucanase contain an amino-terminal domain that
lacks similarity to glucanases or to any other sequences currently in
the Genbank data base. This amino-terminal domain (residues 1-173 in
M. sexta GRP) has a molecular mass of approximately 17 kDa
in M. sexta GRP and in the related invertebrate proteins, except for CCF-1 and the A. gambiae protein, in which this
region is somewhat smaller (Fig. 5). We identified a 17-kDa polypeptide from M. sexta plasma that bound to curdlan and had an
amino-terminal sequence identical to that of GRP (Figs. 1 and 5). This
polypeptide appears to represent the amino-terminal domain of GRP and
perhaps is a product of endogenous proteolytic activity in plasma. We observed that this species accumulated upon storage of plasma, which is
consistent with the hypothesis that it is a degradation product of GRP
and that the two domains are linked by a region that is sensitive to
proteolysis. Although the function of this domain is not known, it
appears that it can bind to curdlan independently from the
carboxyl-terminal glucanase-like domain and thus may have its own
glucan-binding site.
Northern blot analysis indicated that GRP mRNA is present in fat
body (a tissue analogous to mammalian liver) and not in other tissues
tested, including hemocytes. Cultured fat body released GRP into the
medium, which is consistent with the hypothesis that fat body is the
primary source of GRP in plasma. A significant difference between
M. sexta GRP and the insect GNBPs is that synthesis of the
GNBPs is strongly induced by microbial infection (24, 33, 34), whereas
GRP is constitutively expressed and not induced as an acute phase
response protein.
GRP bound to the surface of yeast cells, which contain 1,3-glucans
in their cell walls, and to Gram-negative and Gram-positive bacteria.
E. foetida CCF-1 also was shown to bind to 1,3-glucan and
bacterial lipopolysaccharide (23). The ability of GRP to bind to
microbial surfaces suggests that it may function as a pattern
recognition molecule for detection of bacterial or fungal pathogens.
Incubation of GRP with yeast or bacteria caused them to aggregate,
which could improve the efficiency of clearance of these microorganisms
by hemocytes through phagocytosis or nodule formation (40).
Activation of prophenoloxidase in hemolymph is a commonly observed
response to microbial infection in insects and many other invertebrates
(3). When GRP preincubated with the soluble 1,3-glucan, laminarin,
was added to diluted plasma, the prophenoloxidase activation pathway,
which involves a serine proteinase cascade (3) was triggered, and
active phenoloxidase accumulated in the plasma. This activation
occurred to a much lesser degree and took a significantly longer time
when laminarin or GRP was added to plasma separately. This result
suggests that a complex of GRP with laminarin stimulated activation of
the phenoloxidase cascade. Similar results have been observed with
B. mori GRP (17) and support the conclusion that these
proteins act as pattern recognition proteins as they bind to microbial
surfaces and stimulate a defensive response.
Proteins that bind to 1,3-glucans and stimulate phenoloxidase
activation have been identified in other invertebrate species and
appear to fall into several classes. A 100-kDa 1,3-glucan-binding protein, which enhances activation of phenoloxidase has been isolated from a crayfish, P. leniusculus (12). Proteins very similar to the crayfish glucan-binding protein have also been identified in
shrimp (13, 14). These 100-kDa proteins from crustaceans appear to
represent one family of invertebrate 1,3-glucan-binding proteins
that function as pattern recognition molecules. Two different types of
1,3-glucan-binding proteins have been isolated from hemolymph of
cockroaches from the genus Blaberus. An approximately 90-kDa
protein isolated from Blaberus craniifer, with subunits of
63 and 52 kDa, binds to laminarin and enhances activation of proPO from
a hemocyte lysate (18). A different type of 1,3-glucan-specific lectin isolated from Blaberus discoidalis is a hexamer of
80- and 82-kDa subunits, and appears to be a member of the hexamerin family of arthropod hemolymph proteins (20). Similar proteins have been
isolated from plasma of a group of different cockroach species (19).
These cockroach 1,3-glucan-specific lectins, in combination with
laminarin, are also able to activate the phenoloxidase system (20).
M. sexta GRP, B. mori GRP, and the earthworm protein CCF1
are members of another family of circulating proteins that bind to
1,3-glucans and lead to activation of phenoloxidase. The mechanism by which these pattern recognition molecules function to stimulate activation of the proteolytic cascade is not yet known. Perhaps binding
to the polysaccharide produces a conformational change in GRP, which
confers an ability to interact with a proteinase zymogen. Such an
interaction may stimulate autoactivation of the proteinase, as is
thought to occur in the lectin-mediated pathway for complement
activation (41). Further study of these 1,3-glucan-recognition proteins in arthropods should lead to a better understanding of the
function and evolution of pattern recognition molecules that bind to
polysaccharides on the surface of microbial pathogens and stimulate
innate immune responses.
1,3-Glucan Recognition Protein from an Insect, Manduca
sexta, Agglutinates Microorganisms and Activates the
Phenoloxidase Cascade*
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Dept. of Biochemistry,
Kansas State University, Manhattan, KS 66506. Tel.: 785-532-6964; Fax:
785-532-7278; E-mail: kanost@ksu.edu.
