From the Department of Basic Veterinary Science, the
United Graduate School of Veterinary Sciences, Gifu University,
Yanagito, Gifu 501-1193, Japan, § National Research Center
for Protozoan Diseases, Obihiro University of Agriculture and
Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan,
¶ National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba,
Ibaraki 305-0856, Japan, and ** Institute of Agriculture and
Forestry, Tsukuba University, Tennohdai,
Tsukuba, Ibaraki 305-0006, Japan
Received for publication, July 9, 2002, and in revised form, December 3, 2002
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ABSTRACT |
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A cDNA encoding tick chitinase was cloned
from a cDNA library of mRNA from Haemaphysalis
longicornis eggs and designated as CHT1 cDNA. The CHT1
cDNA contains an open reading frame of 2790 bp that codes for 930 amino acid residues with a coding capacity of 104 kDa. The deduced
amino acid sequence shows a 31% amino acid homology to Aedes
aegypti chitinase and a multidomain structure containing one
chitin binding peritrophin A domain and two glycosyl hydrolase family
18 chitin binding domains. The endogenous chitinase of H. longicornis was identified by a two-dimensional immunoblot analysis with mouse anti-rCHT1 serum and shown to have a molecular mass
of 108 kDa with a pI of 5.0. A recombinant baculovirus
AcMNPV·CHT1-expressed rCHT1 is glycosylated and able to degrade
chitin. Chitin degradation was ablated by allosamidin in a
dose-dependent manner. The optimal temperature and pH for
activity of the purified chitinase were 45 °C and pH 5-7. The CHT1
cDNA has an ELR motif for chemokine-mediated angiogenesis and
appears to be a chitinase of the chemokine family. Localization
analysis using mouse anti-rCHT1 serum revealed that native chitinase is
highly expressed in the epidermis and midgut of the tick. AcMNPV·CHT1
topically applied to H. longicornis ticks exhibited
replication. This is the first report of insect baculovirus infection
of ticks. The importance of AcMNPV·CHT1 as a novel bio-acaricide for
tick control is discussed.
Chitin, the Ticks are second only to mosquitoes as vectors of disease-causing
agents in humans and are the most important arthropod transmitting pathogens to domestic and wild animals, e.g.
Babesia and Theileria protozoa,
Borrelia bacteria, and hemorrhagic and encephalomyelitis virus (14, 15). The hard tick, Haemaphysalis longicornis, is
distributed mainly in East Asia and Australia, where it transmits these
pathogens (16, 17). A variety of methods have been employed to suppress
tick vector populations, including the application of biological
control agents and the heavy reliance on chemical acaricides. However,
the development of resistance to acaricides (18-20) and the increase
in legislation to combat detrimental effects of residues of acaricides
in the environment (21) emphasize the need to develop alternatives for
tick vector control. Recently, we have shown that troponin I (22) and
peroxiredoxin (23) from H. longicornis may be
candidates for control of ticks. Molting-associated molecules
responsible for protecting from invasion of pathogens, control of the
PM, and other necessary functions during blood feeding and molting in
ticks may be candidates for tick control. Chitinase is a selective
insect control protein and is safe as it is readily degradable in the
environment (24).
In the present study, we report the cDNA cloning, identification,
characterization, and expression of chitinase from the hard tick,
H. longicornis. In addition, we discuss the
possible application of tick chitinase in the control of tick vectors.
Tick--
The parthenogenetic Okayama strain of the hard tick
H. longicornis (16) has been maintained by feeding on
rabbits and mice in our laboratory since 1997.
DNA Probe for Screening of Chitinase cDNA--
Based on the
conserved regions of known chitinases of Manduca sexta (25),
Bombyx mori (26), and Drosophila melanogaster (2), two degenerate primers were designed for first PCR: sense primer,
5'-TGGKCVRTSTAYCGDC-3', and antisense primer,
5'-CCARTCRAKRTCNADNCCVT-3'. The following two degenerate primers were
designed for nested PCR: sense primer, 5'-TGYACBSAYHTVATBTAYKSSTTY-3',
and antisense primer, 5'-CCARTCVADRTCBADNCCRTCRAA-3'. Total RNA from
eggs of H. longicornis was isolated using the
thiocyanate-phenol-chloroform extraction method (27). Reverse
transcription-PCR was performed by standard techniques (28). The PCR
products were subjected to 1.5% agarose gel electrophoresis, and the
band of predictive size was purified and cloned into pBluescript SK(+)
vector by standard techniques (28).
Plaque Screening of a cDNA Expression
Library--
Construction of a cDNA expression library was
performed as described previously (22). The cDNA
expression library was screened with an alkaline phosphatase-labeled
DNA fragment amplified by PCR with degenerate primers. Plating phages
and plaque lifting were performed according to the manufacture's
instructions (Stratagene, La Jolla, CA). Labeling probes,
prehybridization, and hybridization were performed according to Alkphos
Direct manual (Amersham Biosciences). Positive plaques were rescreened
until 100% plaque purification was achieved. The cloned insert in the
plaque-purified cDNA Sequencing--
Restriction enzyme-generated fragments
for sequencing were subcloned into pBluescript SK(+) vectors. An insert
cDNA designated CHT1 cDNA was sequenced by the dideoxy
chain-termination method using M13 reverse and universal primers
(PerkinElmer Life Sciences). Sequence analysis was performed with the
computer program MacVector (Oxford Molecular, Madison, WI).
Expression of the CHT1 cDNA in Escherichia coli--
A
2790-bp PCR fragment from H. longicornis chitinase
containing an open reading frame was inserted into the EcoRI
site of pGEX-4T-3 to generate the recombinant plasmid pGEX-4T-CHT1.
Restriction enzyme analysis was performed to identify the construct
containing the insert in the correct orientation. Expression of the
CHT1 cDNA in E. coli was performed as described
previously (22). The recombinant CHT1 (rCHT1) was expressed as
glutathione S-transferase (GST) fusion protein and
designated GST-CHT1 protein.
Production of anti-rCHT1 Sera--
Female mice (BALB/c, 8 weeks
old) were immunized intraperitoneally three times at 2-week intervals
with 100 µg of the recombinant fusion protein in Freund's incomplete
adjuvant. Sera were collected from immunized mice 10 days after the
last immunization.
Two-dimensional Immunoblot--
Tick extracts were treated with
an equal volume of urea mixture consisting of 9 M urea, 4%
Nonidet P-40, 0.8% ampholine (pH 3.5-8; Amersham Biosciences), and
2% 2-mercaptoethanol and then subjected to two-dimensional PAGE.
Non-equilibrium pH gradient electrophoresis was performed (29) in the
first dimension using a rectangular gel electrophoresis apparatus
(AE-6050 A; ATTO, Tokyo, Japan). After electrophoresis at 400 V for
2 h, the gels were incubated in the equilibration buffer for 10 min on a shaker. Electrophoresis in the second dimension was performed
on 8% SDS-PAGE gels under reducing conditions. The proteins were
either stained using a silver staining kit (Dai-ichi Pure Chemical,
Tokyo, Japan) or transferred to nitrocellulose membranes. Immunoblot
analysis was carried out as previously described (30). Anti-mouse rCHT1 serum was used at a dilution of 1:500. The proteins bound to the secondary antibody were visualized with nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate.
Northern Blot Analysis--
Formaldehyde-denatured RNA (10 µg)
extracted from eggs of H. longicornis incubated at 25 °C
for 10 days was fractionated on 1.2% formaldehyde-agarose gel,
transferred to a nylon membrane (Hybond-N, Amersham Biosciences), and
hybridized with an alkaline phosphatase-labeled probe derived from the
CHT1 cDNA. The presence of probe was detected using CDP-Star
(Amersham Biosciences) by chemiluminescent technique. The hybridization
technique was performed as described in the Alkphos Direct manual
(Amersham Biosciences).
Cells and Viruses--
Spodographa frugiperda
(Sf9) cells were propagated at 27 °C in TC-100 and SF-900 II
serum-free insect cell culture medium obtained from Invitrogen.
Autographa californica multiple nuclear polyhidrosis virus
(AcMNPV) and recombinant viruses were grown in Sf9 cells.
Construction of Recombinant Baculovirus Expressing the H. longicornis CHT1 cDNA--
The 2790-bp PCR fragment from H. longicornis CHT1 cDNA containing the open reading frame was
inserted into the EcoRI site of an AcMNPV transfer vector,
pBlueBac4.5/V5-His (Invitrogen) to produce the recombinant transfer
plasmid pBlueBac4.5/V5-His-CHT1, and the recombinant protein was
expressed by recombinant baculovirus as a protein fused with a
COOH-terminal peptide (3 kDa) containing the V5 epitope and a
His6 tag. Restriction enzyme analysis was performed to
identify the construct containing the insert in the correct
orientation. Sf9 cells were cotransfected with
pBlueBac4.5/V5-His-CHT1 and linear AcMNPV DNA, Bac-N-Blue DNA
(Invitrogen), by using Cellfectin reagent (Invitrogen). After 4 days of
incubation at 27 °C, the culture supernatant containing recombinant
virus was harvested and subjected to plaque purification. The
expression of rCHT1 in the plaques was confirmed by indirect
immunofluorescent antibody test with anti-rCHT1 serum. Positive
plaques were selected, and after three cycles of purification a
recombinant virus, AcMNPV·CHT1 was obtained.
Purification of H. longicornis Chitinase--
Thirty ml of
Sf9 cells culture supernatant infected with the recombinant
virus (AcMNPV·CHT1) or as a control infected with the wild type virus
(AcMNPV·wt) were dialyzed against binding buffer (20 mM sodium phosphate, 500 mM NaCl, 5 mM imidazole (pH 7.4)) and concentrated by ultrafiltration
(Concentrator 10/20, exclusion size: 30 kDa). Two ml of 50% nickel
nitrilotriacetic acid slurry (Invitrogen) was centrifuged (5 min at
500 × g) and washed twice with binding buffer. After
incubation for 1 h at 4 °C under continuous shaking, the slurry
was centrifuged, and the supernatant was discarded. The pellet was
washed at 4 °C with 20 ml of washing buffer (20 mM
sodium phosphate, 500 mM NaCl (pH 6.0)) containing 50 mM imidazole. Elution was performed with 5 ml of elution
buffer (pH 6.0) containing 350 mM imidazole. The final
eluent containing the isolated protein was dialyzed against 20 mM sodium phosphate (pH 6.0) at 4 °C and stored at
In Vitro Chitinase Activity
Assay--
4-Methylumbelliferyl-N,
N',N ''-triacetyl- Glycosylation Studies--
Glycosylation studies with
tunicamycin were performed as described previously (24). Briefly,
tunicamycin (Sigma) was added at a concentration of 10 µg/ml to the
Sf9 cells 15 h after infection with AcMNPV·CHT1. Purified
rCHT1 (20 µg) was treated with N-glycosidase F under
denaturing conditions as recommended by the manufacturer (N-glycosidase F deglycosylation kit, Roche Molecular
Biochemicals) and analyzed by immunoblotting with mouse anti-rCHT1 serum.
Immunohistochemistry--
Immunohistochemistry was performed
with peroxidase-labeled goat anti-mouse IgG secondary antibody as
described previously (22). Molting nymphs 10 days after engorgement,
unfed females, and tick tissues were handled as described previously
(22).
Infection with AcMNPV·CHT1 and Detection of rCHT1 in H. longicornis Infected with AcMNPV·CHT1--
AcMNPV·CHT1 culture
medium (ACM) was applied topically to the dorsal surface of unfed
H. longicornis larvae and adults. Ticks were collected 10 days after application. The salivary glands of adult ticks were
dissected under a microscope (16). The dissected ticks were stored at
Nucleotide Sequence Accession Number--
The nucleotide
sequence data reported in this paper appears in the
DDBJ/EMBL/GenBankTM nucleotide sequence data base with the
accession number AB074977.
DNA Probe, cDNA Library Screening, and DNA Sequence
Analysis--
Four degenerate primers based on a conserved region in
the cDNAs of several chitinases were used to amplify the H. longicornis chitinase cDNA fragment. A PCR fragment of 291 bp
was inserted into pBluescript SK(+) vector and sequenced. The
nucleotide sequence of the PCR fragment from H. longicornis
had a putative conserved chitinase domain. The PCR fragment was used as
a probe for screening the entire chitinase cDNA from the cDNA
library of H. longicornis eggs. Five positive clones were
isolated from the H. longicornis egg cDNA library, and
no sequence heterogeneity was found among these clones. The clones
appeared to be about 2.9, 4.1, 4.3, 5.5, and 6.5 kilobase pairs in
size. One of the 5 cDNA clones of 6.5 kilobase pairs was designated
CHT1. DNA sequencing revealed that the CHT1 cDNA with 6439 bp had a
start codon at 571-573 bases and a stop codon at 3358-3360 bases
(Fig. 1). CHT1 cDNA has an open
reading frame extending from position 571 to position 3360 and codes
930 amino acid residues with a predicted molecular mass of about 104 kDa (Fig. 1). This deduced protein has six potential N-glycosylation sites (Fig. 1). The mRNA corresponding
to the cloned cDNA was confirmed by Northern blotting analysis of
total RNA extracted from eggs of H. longicornis ticks to be
about 7.5 kilobases in size (Fig. 2).
Expression of CHT1 in E. coli and Sf9 Cells--
As shown
in Fig. 3, the molecular mass of GST-CHT1
fusion protein was estimated as 130 kDa containing a GST protein of 26 kDa. The mouse anti-rCHT1 serum reacted with a 116-kDa protein from
recombinant baculovirus-infected Sf9 cell culture medium and
107- and 116-kDa proteins from the recombinant baculovirus-infected Sf9 cell extracts (see Fig. 5A, lanes 2 and 4).
Detection of Endogenous CHT1--
We performed two-dimensional
immunoblot analysis to identify endogenous CHT1 in ticks. Mouse
anti-rCHT1 serum strongly reacted with a protein having a molecular
mass of 108 kDa with a pI of 5.0 (Fig.
4A), confirming that it
corresponded to the predicted size of the putative mature protein (104 kDa) calculated from the CHT1 amino acid sequence except for a signal
peptide. In addition, an endogenous CHT1 was identified on
silver-stained two-dimensional gels on which more than 200 visible
protein spots appeared (Fig. 4B). To determine the internal
amino acid residues, protein spots of interest were excised from tick
extracts using the two-dimensional gels and processed as described
previously (33). The spots were pooled from 20 gels, washed with a
solution of 2% citrate, and subjected to lysyl endopeptidase digestion
at 35 °C for overnight (34). After in-gel digestion of the protein,
peptides were collected by reverse phase high pressure liquid
chromatography, and several peptides was analyzed for internal amino
acid sequence in Procise 494 cLC protein sequencing system (Applied
Biosystems, Foster, CA) (35, 36). The resultant amino acid sequences of
four major reverse phase high pressure liquid chromatography purified
peaks were identical to those of the deduced amino acid sequences
encoded by CHT1 cDNA.
The H. longicornis Chitinase Secreted into the Medium of Sf9
Cells Culture Is Glycosylated--
Expression of chitinase in
AcMNPV·CHT1-infected Sf9 cells in the presence or absence of
tunicamycin, an inhibitor of protein N-linked glycosylation,
is shown in Fig. 5A. In the
absence of tunicamycin, immunoreactive chitinase was released into the
medium as a 116-kDa protein (Fig. 5A, lane 4).
However, tunicamycin inhibited the secretion of this protein into the
medium (Fig. 5A, lane 3). In extracts of cells
not treated with tunicamycin, the anti-rCHT1 serum reacted with major
proteins of ~107 and 116 kDa. Cells treated with tunicamycin
exhibited an immunoreactive protein with an apparent molecular mass of
about 107 kDa (Fig. 5A, lane 1).
N-Glycosidase-treated rCHT1 protein exhibited a shift to a
lower apparent molecular mass of about 107 kDa (Fig. 5B,
lane 2), indicating the existence of N-linked
glycan chains.
Purification of Recombinant H. longicornis CHT1 and in Vitro
Chitinase Activity Assay of Purified rCHT1--
The His-tagged
H. longicornis rCHT1 was separated from the endogenous viral
chitinases using affinity chromatography. In vitro chitinase
activity assay using the purified rCHT1 showed a strong hydrolysis of
4MU-(GlcNAc)3 substrate (Fig.
6). In vitro inhibition assay
of chitinase activity using 0.05 µM (final concentration) allosamidin with purified rCHT1 showed a strong inhibition of the
chitinase activity (Fig. 6). The specific activity (fluorescence unit)
of His6-tagged H. longicornis rCHT1 against
4MU-(GlcNAc)3 was optimal at pH 5-7 but was inactive under
acidic (pH < 3) or alkaline (pH > 10) conditions (Fig.
7A). The enzymatic activities increased gradually as the temperature was raised from 0 to
45-50 °C (Fig. 7B). The maximum activity was exhibited
at 45 °C (Fig. 7B). The enzymatic activities started to
decrease dramatically when the temperature was above 50 °C (Fig.
7B). The heat stability of the enzyme was tested after
preincubation in 20 mM sodium phosphate buffer (pH 6.0) at
4, 15, 27, 37, 45, 50, 60, and 70 °C for 60 min. The results are
shown in Fig. 7C. The rCHT1 protein was stable up to
45 °C and completely inactivated at 60 °C and higher
temperatures.
Localization of Native CHT1--
The location of native CHT1 in
molting nymphal ticks was determined by immunohistochemistry using
mouse anti-rCHT1 serum. The immunohistochemistry analysis showed
chitinase of H. longicornis in epidermis and midgut of
molting nymphs after engorgement (Fig. 8A) and non-engorged females
(Fig. 8B). Reaction was strongly detected between old and
new cuticle in molting nymphs (Fig. 8A). No reaction was
detectable using preimmune mouse serum (Fig. 8, panels
a'-d') in molting nymphs after engorgement and
non-engorged females.
Detection of rCHT1 in H. longicornis Infected with
AcMNPV·CHT1--
The detection of rCHT1 expressed in H. longicornis infected with AcMNPV·CHT1 was performed with mouse
anti-rCHT1 serum. H. longicornis adult females treated
topically with ACM were dissected. A flat section of salivary gland was
reacted with mouse anti-rCHT1 serum by immunofluorescence assay. Red
fluorescence was present on the flat section treated with ACM (Fig.
9A). Existence of rCHT1 in
infected H. longicornis larvae was also confirmed by
immunoblot analysis with anti-V5-AP antibody (Fig. 9B).
H. longicornis larvae lysate treated with ACM reacted with
the 107-kDa band.
Chitinases are extremely important for the hydrolytic cleavage of
the The complete sequence of a chitinase cDNA, CHT1, was obtained from
a cDNA library of H. longicornis eggs. The open reading frame codes a protein with a predicted molecular mass of 104 kDa. The
NH2-terminal sequences of the encoded protein contain
numerous hydrophobic residues, characteristic of a leader peptide (Fig. 1). The putative cleavage site of the signal peptide was between residues 22 and 23 as determined by signalP prediction (39). Expression
of the CHT1 cDNA in S. frugiperda cells infected
with a recombinant baculovirus under the control of a polyhedrin
promoter produced and secreted an enzymatically active protein with a
molecular mass of 116 kDa. These results indicate the presence of a
functional signal peptide.
H. longicornis CHT1 possesses potential
N-glycosylation sites at the amino acid residues 115-118
(NPSL), 257-260 (NETT), 479-482 (NWSA), 729-732 (NLTD), 745-748
(NYTG) and 909-912 (NESV). Studies with tunicamycin indicated the
H. longicornis rCHT1 is glycosylated (Fig. 5A).
The open reading frame of the CHT1 cDNA in the recombinant virus
directs the synthesis of a 116-kDa protein larger than the predicted
molecular mass of 104 kDa, implying that the H. longicornis rCHT1 undergoes posttranslational modification. Consistent with this
are the observations that the mouse anti-rCHT1 serum in the presence of
tunicamycin did not react with major constituent protein bands of 116 kDa in the media or the cells. Instead, mouse anti-rCHT1 serum detected
a 107-kDa protein accumulating in the cells. Use of the enzyme
N-glycosidase F is the most effective method of removing
virtually all N-linked oligosaccharides from glycoproteins (40). Fig. 5B of N-glycosidase-treated CHT1
protein provides evidence that the CHT1 protein from the
tunicamycin-treated cells of Fig. 5A is deglycosylated.
These results verify that H. longicornis rCHT1 with
N-glycosylation sites is glycosylated.
H. longicornis CHT1 possesses a chitin binding peritrophin A
domain at amino acid residues 868-922 and 2 glycosyl hydrolase family
18 chitin binding domains at amino acid residues 44-389 and 471-817.
These domains strongly suggest H. longicornis CHT1 plays an
important role as a chitin hydrolase in the life cycle of ticks.
The deduced amino acid sequence showed low homology to other reported
chitinase sequences, such as Aedes aegypti
(GenBankTM accession number T14075; 31% amino acid
homology) by BLASTN (prediction of protein localization site) and
MacVector Ver. 6.5 programs (sequence analysis software). Comparison of
conserved regions in the amino acid sequences of chitinases revealed
H. longicornis CHT1 has two pairs of conserved domains
similar to those of A. aegypti chitinase (Fig.
10). The low homology in chitinase sequences indicates a strong difference between species. Only the
sequence of the actual catalytic center, region II (Fig. 10), which
includes the amino acids Asp-164 and Asp-592 and Glu-168 and Glu-596 in
H. longicornis CHT1, are highly conserved in all family 18 chitinases characterized so far. This catalytic center is indispensable
for enzyme activity (41) and implies that H. longicornis
CHT1 is a member of the family 18 chitinases.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-1,4-linked homopolymer of
N-acetyl-D-glucosamine, is an insoluble
structural polysaccharide that is important as a supporting element in
extracellular structures. It is seen in the exoskeleton of arthropods
where the chitin microfibrils complex with proteins to form the
chitinous structures of the cuticle and the peritrophic membrane
(PM)1 lining the gut (1, 2).
The PM extracellularly surrounds the food bolus in the guts of most
arthropods (3, 4). It has been shown to be important in preventing
damage or clogging of microvilli by the luminal contents (5, 6) and
compartmentalization of digestive events by acting as a permeability
barrier for digestive enzymes (7) and to have a novel role for
protecting Leishmania from the hydrolytic activities of the
sand fly midgut (8). However, arthropods must be able to hydrolyze the
chitin to allow degradation of the cuticle and development during the
immature stages. Events during the molting cycle have been investigated biochemically and morphologically (9), and chitinase has been shown to
be an essential component in the hydrolysis of chitin. Ticks are
hemimetabolous in nature, implying they also require chitinolytic enzymes to remove old cuticle and allow synthesis of new
cuticle for continued growth and development. Chitinase is induced by
ecdysteroids to degrade the older chitin at the time of molting (10,
11). The chitinase inhibitor, allosamidin, isolated from culture broth
of Streptomyces sp. (12), has been used as an important tool
to elucidate several essential features of insect chitinases (13).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phage was subcloned into pBluescript SK(+)
(Stratagene) by using the in vivo excision capabilities of
ZAP II.
80 °C until further analysis.
-chitotrioside (Sigma)
(4MU-(GlcNAc)3) was used for chitinase activity assay at a
concentration of 0.8 mM in distilled water. Kinetic studies of chitinase were performed in a fluorescence spectrophotometer (excitation 350 nm, emission 450 nm) (Japan spectrophotometer FP-770,
Tokyo, Japan) by continuously recording changes in fluorescence. Inhibition of chitinase activity was studied by using allosamidin at a
final concentration of 0.01 and 0.05 µM in the reaction
mixture. Allosamidin was obtained from S. Sakuda (12). Reactions were started by the addition of enzyme (1 µg). The enzymatic activity of
the rCHT1 was quantified using 4MU-(GlcNAc)3 (31). The
enzyme solutions (0.16 µg) were incubated with substrate
(4MU-(GlcNAc)3) in a wide range buffer (pH 2.0-12.0) (32)
at 37 °C for 15 min (final concentration 3.8 µM).
Reactions were terminated by the addition of 1.2 ml of 1 M
glycine buffer. In the chitinase activity assay, the enzyme solutions
(0.16 µg) were incubated with substrate (4MU-(GlcNAc)3)
in 20 mM sodium phosphate (pH 6.0) at 4, 15, 27, 37, 45, 50, 60, and 70 °C for 15 min (final concentration 3.8 µM). Reactions were terminated by addition of 1.2 ml of 1 M glycine buffer.
80 °C until use. Salivary glands were embedded in embedding medium
(Tissue-Tek®, Sakura, Tokyo, Japan) and fixed using
standard methods. Thin transverse sections of the ticks ~7 µm thick
were then prepared using a microtome. The slides were blocked for
1 h with 3% skim milk (Wako, Tokyo, Japan) in PBS and then
incubated for 4 h at room temperature with mouse anti-rCHT1 serum
diluted to 1:200 with PBS. The slides were rinsed thoroughly with PBS
and reacted 1 h with red fluorescence-labeled mouse IgG secondary
antibody (Alexa Flouor 594® goat anti-mouse IgG (H+L),
Molecular Probes, Eugene, OR). The slides were then rinsed thoroughly
with PBS, covered with glass slips, and observed under a fluorescence
microscope (ECLIPSE E600, Nikon, Tokyo, Japan). Larvae ticks topically
treated with ACM were washed 3 times with PBS containing 0.05% Tween
20, homogenized, and used for immunoblots as described previously (22).
The membranes were incubated for 1 h with anti-V5-AP antibody
(Invitrogen) at a dilution of 1:500.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide sequence and deduced amino acid
sequence of H. longicornis CHT1
cDNA. The initial codon ATG and termination codon TAG are
indicated in bold, and the putative signal peptide of the
deduced amino acid sequence is underlined.
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Fig. 2.
Northern blot of mRNA of the CHT1
cDNA. Total RNA isolated from adult female H. longicornis ticks was resolved on an 1% agarose gel containing
formaldehyde and transferred to a nylon membrane (Hybond
N+; Amersham Biosciences).
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Fig. 3.
SDS-PAGE analysis of recombinant CHT1
expressed in E. coli. The proteins expressed by
pGEX-4T-3/CHT1 (lane 2) or pGEX-4T-3 vector without insert
(lane 3) were detected by CBB staining. Lane 1 is
molecular mass marker.
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Fig. 4.
Identification of H. longicornis endogenous CHT1. Fifty micrograms of
tick-extract protein was separated by two-dimensional pH gradient gel
electrophoresis, and the proteins were then either transferred to a
nitrocellulose membrane (A) or stained with a
two-dimensional silver stain kit (B). The fact that
endogenous CHT1 was bound to the anti-rCHT1 serum was found by
alignment between the stained gel and immunoblot membrane. The
arrow shows endogenous CHT1, corresponding to the
immunoreactive spot at panel A. IEF, isoelectric
focusing.
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Fig. 5.
Immunoblot detection of glycosylation in
H. longicornis rCHT1. A: lane
1, lysate from cells with tunicamycin treatment; lane
2, lysate from cells without tunicamycin treatment; lane
3, medium from cells with tunicamycin treatment; lane
4, medium from cells without tunicamycin treatment in Sf9
cells. B: lane 1, lysate from cells with
tunicamycin treatment; lane +, rCHT1 treated with
N-glycosidase F (PNGase F; lane ,
rCHT1 untreated with N-glycosidase F (control).
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Fig. 6.
Inhibition by allosamidin of purified rCHT1
activity expressed by AcMNPV-CHT1. , no addition of
allosamidin;
, with 0.01 µM allosamidin;
, with
0.05 µM allosamidin.
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Fig. 7.
Effect of pH (A) and
temperature (B) on H. longicornis rCHT1 activity and enzyme stability
(C) after preincubation at different temperature
values. Enzyme activity was assayed using
4MU-(GlcNAc)3 as the substrate as described under
"Experimental Procedures." Data are presented as fluorescence
units. Mean ± S.D. (n = 3).
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Fig. 8.
Immunohistochemical localization of CHT1 in
engorged nymphs (A) and non-engorged females
(B) of H. longicornis with mouse
anti-rCHT1 serum. Ticks were fixed in paraformaldehyde and
embedded in paraffin as described under "Experimental Procedures."
Flat sections of whole molting nymphs 10 days after engorgement
(a, a') and unfed females (b,
b') were exposed to either mouse anti-rCHT1 serum
(panels a-d) or preimmune mouse serum (panels
a'-d'). Panels c-c' and
d-d', more highly magnified sections of the
epidermis and midgut of whole molting nymphs 10 days after engorgement
and unfed females, respectively. mg, midgut; ed,
epidermis; nc, new cuticle; oc, old cuticle;
cu, cuticle.
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[in a new window]
Fig. 9.
Detection of rCHT1. A, detection
of rCHT1 expressed in adult H. longicornis salivary glands
with mouse anti-rCHT1 serum by immunofluorescence assay.
AcMNPV·CHT1 was dropped as described under "Experimental
Procedures." The picture on the left was
detected with mouse anti-rCHT1 fusion protein serum. The
picture on the right was detected with mouse
anti-GST serum. B, immunoblot analysis with mouse anti-V5-AP
antibody. Lane 1 is larval H. longicornis lysate
treated with ACM as described under "Experimental Procedures."
Lane 2 is normal larval H. longicornis
lysate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycosidic linkages between GlcNAc residues of chitin to allow
molting and growth in the arthropods. In general this hydrolysis can
occur in one of two ways, either with retention of anomeric
configuration in the product or with inversion (37). Several arthropod
chitinases have been identified (25, 26, 38), but this is the first
report for identification of chitinase in the arachnids, including ticks.
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[in a new window]
Fig. 10.
Comparison of conserved regions in amino
acid sequences of H. longicornis CHT1 with
other chitinases. The chitinase sequences listed are H. longicornis (this work), B. mori (GenBankTM
accession number U86876), M. sexta (GenBankTM
accession number U02270), A. aegypti (GenBankTM
accession number T14075), Spodoptera litura
(GenBankTM accession number AB032107), Chelonus
sp. (GenBankTM accession number U10422), Brugia
malayi (GenBankTM accession number M73689),
Aphanocladium album (GenBankTM accession number
X64104). Numbers in parentheses list position in
the amino sequence. Asterisks indicate highly conserved
residues.
In vitro chitinase activity assay using the purified baculovirus-expressed rCHT1 showed chitin hydrolysis, indicating rCHT1 is a H. longicornis chitinase (Fig. 6). All family 18 chitinases are inhibited by allosamidin (13), and the activity of rCHT1 was inhibited by allosamidin (Fig. 6), indicating H. longicornis chitinase is a family 18 chitinase. Eosinophil chemotactic cytokine (ECF-L) possesses the CXC consensus sequence near the NH2 terminus. This is typical of CXC chemokines, and a comprehensive GenBankTM data base search revealed the ECF-L is a chitinase family protein (42). In addition the functional role for the ELR motif in CXC chemokine-mediated angiogenesis has been observed (43). H. longicornis CHT1 has ELR motifs at amino acid residues 185-187 and 613-615. The purified rCHT1 showed a maximum activity at 45 °C and pH 5-7 (Fig. 7), such as seen in the chitinases of some insects (38, 44). These results suggest that H. longicornis chitinase is a member of the chemokine family.
Chitinase is produced in molting fluid and gut tissues subsequent to feeding at the end of a larval instar in preparation for a molt (25, 45). Examination of flat sections showed a strong reactivity in the cuticle epidermis and midgut of H. longicornis, illustrating the H. longicornis chitinase is abundantly expressed there (Fig. 8). The PM formed in the midgut lumen of blood-sucking arthropods after a blood meal (3, 5) may act as a barrier for invasion of ingested microorganisms (46). Chitin is a key structural component of the PM (3, 47). In support of this, gut specific chitinases have been found, and PM shown to be stronger and more persistent in the guts of mosquitoes fed chitinase inhibitors (47, 48). The existence of PM in ticks has been demonstrated morphologically with Babesia microti passing through it in Ixodes ticks (49). The appearance of H. longicornis chitinase in the midgut (Fig. 8) suggests this chitinase may be involved in controlling turnover and porosity of the chitin-containing PM. The appearance of H. longicornis chitinase in the cuticle epidermis (Fig. 8) implies it acts as a molting enzyme. These results indicate H. longicornis chitinase is a very important enzyme for molting and control of turnover and porosity of the PM in ticks and, thus, is a major candidate as a bio-acaricide.
Baculoviruses have long been attractive biological agents for control of crop pest insect. However, limitations of their use have been a very narrow host range and slow killing rate, resulting in significant crop damage. Recently, viruses with increased host range and improved virulence are being engineered (50, 51). S. frugiperda larvae injected with a transformed AcMNPV expressing M. sexta chitinase died more quickly than those injected with a wild type virus (24). In this study, H. longicornis became infected with AcMNPV·CHT1 (Fig. 9), and rCHT1 was replicated. This is the first report of infection with recombinant insect-baculoviruses in ticks. H. longicornis adults treated with high concentration of ACM died (data not shown) as with M. sexta chitinase (24). Therefore, AcMNPV·CHT1 is a candidate for a potential bio-acaricide. However, to facilitate the use of chitinases in an integrated pest management system as a new and highly selective approach to tick control we must gain much more knowledge.
This study represents the first report of the cloning, identification,
and characterization of a tick chitinase, expression of the cDNA in
an insect baculovirus, demonstration of the ability of the purified
baculovirus expressed rCHT1 to degrade chitin, localization of
chitinase in the epidermis and midgut of ticks, and infection of ticks
with a recombinant insect-baculovirus. Further studies exploring the
usefulness of H. longicornis chitinase as a vaccine and
novel bio-acaricide against ticks need to be investigated extensively.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Y. Ando of National Institute of Animal Health for excellent technical assistance. We thank Dr. S. Sakuda (Tokyo University) for providing allosamidin.
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FOOTNOTES |
---|
* This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB074977.
Supported by the program for Promotion of Basic Research
Activities for Innovative for Bioscience.
Supported by the 21st Center of Excellence program of
the Scientific Research from the Ministry of Education, Culture,
Sports, Science, and Technology. To whom correspondence should be
addressed. Tel.: 81-155-49-5646; Fax: 81-155-49-5643; E-mail:
fujisaki@obihiro.ac.jp.
Published, JBC Papers in Press, December 26, 2002, DOI 10.1074/jbc.M206831200
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ABBREVIATIONS |
---|
The abbreviations used are:
PM, peritrophic
membrane;
GST, glutathione S-transferase;
AcMNPV, A.
californica multiple nuclear polyhedrosis virus;
4MU-(GlcNAc)3, 4-methylumbelliferyl-N,
N',N''-triacetyl--chitotrioside;
ACM, AcMNPV·CHT1 culture medium;
PBS, phosphate-buffered saline.
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REFERENCES |
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---|
1. | Shao, L., Devenport, M., and Jacobs-lorena, M. (2001) Arch. Insect Biochem. Physiol. 47, 119-125[CrossRef][Medline] [Order article via Infotrieve] |
2. | de la Vega, H., Specht, C. A., Liu, Y., and Robbins, P. W. (1998) Insect Mol. Biol. 7, 233-239[Medline] [Order article via Infotrieve] |
3. | Peters, W. (1992) in Zoophysiology: Peritrophic Membranes (Bradshaw, S. D. , Burggren, W. , Heller, H. C. , Ishii, S. , Langer, H. , Neuweiler, G. , and Randall, D. J., eds), Vol. 130 , Springer-Verlag, Berlin |
4. | Lehane, M. J. (1997) Annu. Rev. Entomol. 42, 525-550[CrossRef] |
5. | Richards, A. G., and Richards, P. A. (1977) Annu. Rev. Entomol. 22, 219-240[Medline] [Order article via Infotrieve] |
6. | Burner, R., Rudin, W., and Hecker, H. (1983) J. Ultrastruct. Res. 83, 195-204[Medline] [Order article via Infotrieve] |
7. | Terra, W. R. (1990) Annu. Rev. Entomol. 35, 181-200[CrossRef] |
8. | Pimenta, P. F. P., Modi, G. B., Pereira, S. T., Shahabuddin, M., and Sacks, D. L. (1997) Parasitology 115, 359-369[CrossRef][Medline] [Order article via Infotrieve] |
9. | Reynolds, S. E., and Samuels, R. I. (1996) Adv. Insect Physiol. 26, 157-232 |
10. | Kimura, S. (1973) J. Insect Physiol. 19, 115-123[CrossRef] |
11. | Koga, D., Fujimoto, H., Funakoshi, T., Mizuki, Ide, A., Kramer, K. J., Zen, K. C., Choi, H., and Muthukrishnan, S. (1992) Insect Biochem. Mol. Biol. 22, 305-311[CrossRef] |
12. | Sakuda, S., Isogai, A., Matsumoto, A., Suzuki, A., and Koseki, K. (1986) Tetrahedron Lett. 27, 2475-2478[CrossRef] |
13. | Spindler, K. D., and Spindler-Barth, M. (1999) in Chitin and Chitinases (Jollès, P. , and Muzzarelli, R. A. A., eds) , pp. 201-209, Birkhaeuser Verlag, Basel, Switzerland |
14. | Balashov, Y. S. (1972) Misc. Publ. Entomol. Soc. Am. 8, 161-376 |
15. | Hoogstraal, H. (1985) Adv. Parasitol. 24, 135-238[Medline] [Order article via Infotrieve] |
16. | Fujisaki, K. (1978) Natl. Inst. Anim. Health Quart. (Yatabe) 18, 27-38 |
17. | Fujisaki, K., Kawazu, S., and Kamio, T. (1994) Parasitol. Today 10, 31-33[Medline] [Order article via Infotrieve] |
18. | Rand, K. N., Moore, T., Srikantha, A., Spring, K., Tellam, R., Willadsen, P., and Cobon, G. S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9657-9661[Abstract] |
19. |
Riding, G. A.,
Jarmey, J.,
McKenna, R. V.,
Pearson, R.,
Cobon, G. S.,
and Willadsen, P. A.
(1994)
J. Immunol.
153,
5158-5166 |
20. | Zaim, M., and Guillet, P. (2002) Trends Parasitol. 18, 161-163[CrossRef][Medline] [Order article via Infotrieve] |
21. | Willadsen, P., and Kemp, D. H. (1988) Parasitol. Today 4, 196-198[CrossRef][Medline] [Order article via Infotrieve] |
22. | You, M., Xuan, X., Tsuji, N., Kamio, T., Igarashi, I., Nagasawa, H., Mikami, T., and Fujisaki, K. (2001) Insect Biochem. Mol. Biol. 32, 67-73[CrossRef][Medline] [Order article via Infotrieve] |
23. | Tsuji, N., Kamio, T., Isobe, T., and Fujisaki, K. (2001) Insect Mol. Biol. 10, 121-129[Medline] [Order article via Infotrieve] |
24. | Gopalakrishnan, B., Muthukrishnan, S., and Kramer, K. J. (1995) Insect Biochem. Mol. Biol. 25, 255-265[CrossRef] |
25. | Kramer, K. J., Corpuz, L. M., Choi, H., and Muthukrishnan, S. (1993) Insect Biochem. Mol. Biol. 23, 691-701[CrossRef][Medline] [Order article via Infotrieve] |
26. | Kim, M. G., Shin, S. W., Bae, K. S., Kim, S. C., and Park, H. Y. (1998) Insect Biochem. Mol. Biol. 28, 163-171[CrossRef][Medline] [Order article via Infotrieve] |
27. | Chomczynski, P., and Sacchi, N. (1987) Microbiol. Immunol. 162, 156-159 |
28. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
29. | O'Farrell, P. Z., Goodman, H. M., and O'Farrell, P. H. (1977) Cell 12, 1133-1141[Medline] [Order article via Infotrieve] |
30. | Tsuji, N., Morales, T. H., Ozols, V. V., Carmody, A. B., and Chandrashekar, R. (1998) Mol. Biochem. Parasitol. 97, 69-79[CrossRef][Medline] [Order article via Infotrieve] |
31. | McCreath, K. J., and Goody, G. W. (1992) J. Microbiol. Methods 14, 229-237[CrossRef] |
32. | Carmody, W. R. (1961) J. Chem. Educ. 38, 550-559 |
33. |
Chandrashekar, R.,
Tsuji, N.,
Morales, T.,
Ozols, V.,
and Mehta, K.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
531-536 |
34. | Rosenfeld, J., Capdevielle, J., Claude, J., Guillemot, C., and Ferrara, P. (1992) Anal. Biochem. 203, 173-179[Medline] [Order article via Infotrieve] |
35. | Majima, E., Koike, H., Hong, Y-M., Shinohara, Y., and Terada, H. (1993) J. Cell. Biochem. 268, 22181-22187 |
36. |
Furuse, M.,
Fujita, K.,
Hiiragi, T.,
Fujimoto, K.,
and Tsukita, S.
(1998)
J. Cell Biol.
141,
1539-1550 |
37. | Sinnott, M. L. (1990) Chem. Rev. 90, 1171-1202 |
38. | Shinoda, T., Kobayashi, J., Matsui, M., and Chinzei, Y. (2001) Insect Biochem. Mol. Biol. 25, 521-532 |
39. | Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G. (1997) Protein Eng. 10, 1-6[Abstract] |
40. | Tarentino, A. L., and Plummer, T. H. (1994) Methods Enzymol. 230, 44-57[Medline] [Order article via Infotrieve] |
41. |
Watanabe, T.,
Kobori, K.,
Miyashita, K.,
Fujii, T.,
Sakai, S.,
Uchida, M.,
and Tanaka, H.
(1993)
J. Biol. Chem.
268,
18567-18572 |
42. |
Owhashi, M.,
Arita, H.,
and Hayai, N.
(2000)
J. Biol. Chem.
275,
1279-1286 |
43. |
Strieter, R. M.,
Polverini, P. J.,
Kunkel, S. L.,
Arenberg, D. A.,
Burdick, M. D.,
Kasper, J.,
Dzuiba, J.,
Damme, J. V.,
Walz, A.,
Marriott, D.,
Chan, S. Y.,
Roczniak, S.,
and Shanafelt, A. B.
(1995)
J. Biol. Chem.
270,
27348-27357 |
44. | Zhu, X., Zhang, H., Fukamizo, T., Muthukrishnan, S., and Kramer, K. J. (2001) Insect Biochem. Mol. Biol. 31, 1221-1230[CrossRef][Medline] [Order article via Infotrieve] |
45. | Fukamizo, T., and Kramer, K. J. (1987) Insect Biochem. 17, 547-550[CrossRef] |
46. | Huber, M., Cabib, E., and Miller, L. H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2807-2810[Abstract] |
47. | Shahabuddin, M., Toyoshima, T., Aikawa, M., and Kasow, D. C. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4266-4270[Abstract] |
48. |
Shen, Z.,
and Jacobs-Lorena, M.
(1997)
J. Biol. Chem.
272,
28895-28900 |
49. | Rudzinska, M. A., Spielman, A., Lewengrub, S., Piesman, J., and Karakashian, S. (1982) Cell Tissue Res. 221, 471-481[Medline] [Order article via Infotrieve] |
50. | Maeda, S., Volrath, S. L., Hanzlik, T. N., Harper, S. A., Majima, K., Maddox, D. W., Hammock, B. D., and Fowler, E. (1991) Virology 184, 777-780[Medline] [Order article via Infotrieve] |
51. | Tomalski, M. D., and Miller, L. K. (1991) Nature 352, 82-85[CrossRef][Medline] [Order article via Infotrieve] |
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