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Differential activation of the lectin and antimicrobial peptide genes in Sarcophaga peregrina (the flesh fly).

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Archives of Insect Biochemistry and Physiology 69:189–198 (2008)
Differential Activation of the Lectin and
Antimicrobial Peptide Genes in Sarcophaga peregrina
(the Flesh Fly)
Takahiro Tanji,1,2,3 Hirohisa Shiraishi,1,3 Shunji Natori,1,2 and Ayako Ohashi-Kobayashi1,3,
Sarcophaga lectin is an immune defense protein which is transcriptionally induced upon immune challenge in the flesh fly,
Sarcophaga peregrina. So far, we have revealed that the Sarcophaga lectin gene has multiple NF-kB -binding motifs in its
promoter. Here we showed that the nuclear extracts from Sarcophaga-derived culture cells, NIH-Sape-4, and larval fat bodies
have binding activity to the multiple kB motifs in the lectin gene promoter, some of which were responsive to immune stimuli.
We also compared the expression profiles of the lectin gene with those of the antibacterial peptide genes from the point of view
of inducers, expression tissues and local induction in digestive tracts. In each case, the lectin gene was activated in different
manners from other inducible defense genes. These results indicate the complex regulation of the lectin gene, possibly by NF-kB
-related transcription factors. Arch. Insect Biochem. Physiol. 69:189–198, 2008.
& 2008 Wiley-Liss, Inc.
KEYWORDS: Sarcophaga lectin; insect immunity; NF-kB
INTRODUCTION
Insects synthesize a variety of defense proteins
to protect themselves from microbial infection
(Bulet et al., 2004; Christophides et al., 2004;
Ferrandon et al., 2007; Kanost et al., 2004;
Lemaitre and Hoffmann, 2007; Tanji and Ip,
2005). These proteins include antibacterial ones,
antifungal ones, and lectins. Macromolecules derived from microbial cell walls, such as peptidoglycans and b-1,3-glucans, trigger activation of
the genes for these insect defense proteins.
In Drosophila melanogaster, it is well known that
the two distinct signaling pathways, the Toll and
immune deficiency (IMD) pathways, are activated
by different microbial components respectively to
induce the expression of antimicrobial peptide genes
systemically; peptidoglycans from Gram-positive
bacteria and b-1,3-glucans from fungi activate the
Toll pathway, while peptidoglycans from Gram-negative bacteria activate the IMD pathway. The signal
from each pathway recruits adequate NF-kB -related
transcription factors (Dorsal, DIF and Relish), which
bind to NF-kB -binding motifs in the promoters of
antimicrobial peptide genes with different binding
preferences depending on the sequences (Senger
et al., 2004). The preferences confer specificity to the
innate immunity to some extent. For example, the
infection of fungi, which activates Dorsal/DIF
through the Toll pathway, leads to the activation of
the antifungal peptide Drosomycin gene via the Dorsal/DIF-binding site in its promoter. On the other
hand, the infection of Gram-negative bacteria, which
activates Relish through the IMD pathway, leads to
the activation of the anti-Gram-negative bacterial
peptide Diptericin gene via the Relish-binding site in
1
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
2
The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan
3
Faculty of Pharmaceutical Sciences, Iwate Medical University, Yahaba, Iwate 028-3694, Japan
*Correspondence to: A. Ohashi-Kobayashi, Faculty of Pharmaceutical Sciences, Iwate Medical University, Yahaba, Iwate 028-3694, Japan. E-mail: aohashi@iwate-med.ac.jp
Received 15 March 2008; Accepted 10 July 2008
& 2008 Wiley-Liss, Inc.
DOI: 10.1002/arch.20280
Published online in Wiley InterScience (www.interscience.wiley.com)
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its promoter. Recently, several NF-kB-related transcription factors have also been cloned from other
insect species including mosquitoes, beetle and silkworm, and their functions in immunity have been
characterized (Barillas-Mury et al., 1996; Sagisaka
et al., 2004; Shin et al., 2002; Shin et al., 2005;
Tanaka et al., 2007; Tanaka et al., 2005). These reports demonstrated that the selective activation of
immune defense by multiple NF-kB -related transcription factors is common among insects.
Not only the systemic but also the local
immune response is an important factor for the
fight against the pathogen at the site of infection.
There, the activation of the antimicrobial peptide
genes is also regulated in a different manner in
Drosophila (Tzou et al., 2000).
We have been studying the defense proteins of
Sarcophaga peregrina (flesh fly). In addition to
many antimicrobial peptides, such as Sarcotoxin I
(Sarcophaga homologue of Cecropin), Sarcotoxin II
(that of Attacin), and Sapecin (that of Defensin)
(Ando et al., 1987; Matsuyama and Natori, 1988;
Okada and Natori, 1983), a lectin was found to be
produced upon immune challenge (Komano et al.,
1980). They are induced at the transcriptional level
like many antimicrobial peptides in Drosophila
(Ando and Natori, 1988; Matsumoto et al., 1986;
Matsuyama and Natori, 1988; Takahashi et al.,
1985). However, the profiles of the induction were
yet to be investigated in details.
Inducible lectins have also been found from
other insects such as hornworm (Yu et al., 1999;
Yu and Kanost, 2000), which supports the idea
that the existence of an inducible lectin is not
specific to flesh fly but general in insects. Although
the activation of the antimicrobial peptide genes
has been well characterized in Drosophila, the
activation of the lectin genes has been poorly
analyzed, since no lectin genes are known to be
induced intensively upon infection in this model
animal. Therefore, to characterize the expression
profiles of the Sarcophaga lectin gene will provide
us a novel insight into the expression manner of an
unexplored set of inducible lectins involved in
immune defense. In this work, we compared the
expression of the Sarcophaga lectin gene with the
antimicrobial peptide genes and showed that it is
regulated in different manners in many aspects.
MATERIALS AND METHODS
Cells and Animals
The Sarcophaga embryonic cell line, NIH-Sape4, was routinely cultured in M-M medium at 251C
as described in (Komano et al., 1987).
Flesh flies, Sarcophaga peregrina, were kept at
271C with dry milk, sugar cubes and fresh water.
Larvae were reared on pork liver, and when they
crawled upward at the third instar, they were collected, washed and kept in a plastic container with
a small amount of water. Larvae pupated 16 h after
their transfer to dry conditions (Ohtaki, 1966).
Electrophoretic Mobility Shift Assay
A nuclear extract from NIH-Sape-4 cells was
prepared as described in (Kobayashi et al., 1993).
For the nuclear extract from fat body of the third
instar larvae, larvae ware pricked with a thin needle
and dissected 6 h later. Fat bodies from 30 larvae
were rinsed in Grace’s Insect Cell Culture Medium
(Invitrogen), kept in five volumes of hypotonic
buffer for 10 min on ice and centrifuged at
1,000 g for 10 min. The pellet was resuspended
in two volumes of hypotonic buffer and homogenized with a Teflon homogenizer. Then the
homogenate was centrifuged at 1,000 g for
10 min. The pellet was centrifuged again at
25,000 g for 20 min after the resuspension in
500 ml of hypotonic buffer and nuclei was collected
as pellet. The nuclear extract was prepared as that
from NIH-Sape-4 cells.
The nuclear extract (10 mg protein) was subjected to electrophoretic mobility shift assay as
described in (Kobayashi et al., 1993).
The sequences of synthetic oligodeoxyribonucleotides used as probes were listed in
Figure 1 with a few flanking bases. The mutated
NF-kB -binding motif (50 -CATATTAATACCCTG-30 )
was used as a nonspecific competitor.
Archives of Insect Biochemistry and Physiology December 2008
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Activation of the Sarcophaga lectin gene
191
Fig. 1. Electrophoretic mobility shift assays demonstrating specific binding of nuclear protein to the NF-kB-binding
motifs in the promoter of the Sarcophaga lectin gene. (A) kB motifs to which the binding activity was detected was shown
along with the consensus sequence (Hultmark, 1993). (B) Nuclear extract was prepared from NIH-Sape-4 cells. The
binding activity to two probes was shown. Sixty-fold specific (S) and nonspecific (N) cold probes were also applied to
examine the specificity of the binding activity. Bound and free probes were indicated by arrowheads. Nonspecific binding
signal was indicated by an asterisk. (C) Nuclear extract was prepared from the fat body of the third instar larvae of
Sarcophaga peregrina 6 h after body injury.
Infection of the Third Instar Larvae
As foreign substances, Escherichia coli (K-12
594), Staphylococcus aureus (Cowan I strain), spores
of Beauveria bassiana (IFO No. 31676) and NIHSape-4 cells were used for the injection. They were
rinsed with insect saline (130 mM NaCl, 5 mM KCl
and 1 mM CaCl2) and resuspended at the final
concentration of 20%, 2% and 0.2% (v/v) before
injection. The suspension (5 ml) was subjected into
the body cavity with an injection needle.
For feeding experiments, lipopolysaccharide
from Escherichia coli O55: B5 (Sigma-Aldrich),
peptidoglycan from Micrococcus luteus (Sigma-Aldrich), and zymosan from Saccharomyces cerevisiae
(Sigma-Aldrich) were also used at the final concentration of 0.125 mg/ml. Larvae were soaked
in distilled water containing microorganisms or
microbial components for 6 h. Feeding time period
Archives of Insect Biochemistry and Physiology December 2008
was determined by confirming that India ink
reached the midgut after soaking in the ink.
Northern Blot Analysis
Total RNA was prepared from dissected tissues
and 20 mg of RNA was subjected to Northern blot
hybridization as described in (Kobayashi et al.,
1993).
RESULTS
The Sarcophaga Lectin Gene Has Multiple NF-kBBinding Motifs in the Promoter
Many antimicrobial peptide genes in Drosophila
have multiple NF-kB-binding motifs in their
promoters (Engström, 1997; Hultmark, 1993).
Recently, it has been shown that, not only multiple
kB motifs are necessary, but cooperative regulation
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by these motifs are required for the maximum and
adequate induction (Tanji et al., 2007).
The Sarcophaga lectin gene is produced upon
injury of body cavity, mainly in fat body (Komano
et al., 1983). Before, one kB motif was found at
about 0.3 kb 5’-upstream of the lectin gene. This
motif is required for the activation of the lectin
gene promoter at least in NIH-Sape-4 cells, which
are an embryonic cell line of Sarcophaga and
known to express the lectin gene under the normal
culture condition (Kobayashi et al., 1993; Shiraishi
et al., 2000). Later, we sequenced up to 3.1 kb of
the 5’-upstream sequence of the lectin gene (the
sequence deposited in the DDBJ/EMBL/GenBank
nucleotide sequence databases with the accession
number AB054644), and found many putative kB
motifs in the distal region of its promoter (Tanji
et al., 2002).
As a first step, we performed electrophoretic
mobility shift assay using nuclear extract from
NIH-Sape-4 to find out whether it has the binding
activity to these putative kB motifs; the binding
activity to 11 out of 13 putative kB motifs was
detected (Fig. 1A,B). Then we moved on to nuclear
extract from the fat body of the third instar larvae
and it also showed binding activity to those 11 kB
motifs (Fig. 1C). Among them, the binding activity
to two sites, 1.5 kb and 0.6 kb 5’-upstream of the
lectin gene respectively, increased modestly when
the extract from injured larvae was subjected,
suggesting that these kB motifs participate in the
activation of the lectin gene upon injury and
infection.
Differential Induction of the Immune Defense Genes
Expression by Various Pathogens and Tumorous Cells
In Drosophila, it has been known that the infection of Gram-negative bacteria activates Relish
via the IMD pathway, and the infection of Grampositive bacteria and fungi activates other NF-kB related transcription factors Dorsal and DIF via the
Toll pathway. Hence, different kinds of pathogens
induce the antimicrobial peptide genes in different
manners (Lemaitre et al., 1997).
To investigate the expression profile of the
Sarcophaga lectin gene, we injected various kinds of
pathogens into the hemocoel of the third instar
larvae, and investigated the induction of the Sarcophaga lectin gene 20 hours after injection by
Northern blot. It has been reported that the induction by injury itself and/or saline injection
almost disappears by then; the gene expression
induced by foreign substances still persists
(Takahashi et al., 1986). We subjected Escherichia
coli as Gram-negative bacteria, Staphylococcus aureus
as Gram-positive bacteria, and Beauveria bassiana as
fungi. In addition to them, we also examined the
effect of NIH-Sape-4 cells as tumorous cells from
the identical species; tumor cells would be harmful
as well as pathogenic microorganisms, and they
should be eliminated when they are inside the
body. Then the expression of the lectin gene was
compared with that of sarcotoxin I and sarcotoxin
II genes. Both sarcotoxin I and II genes have been
shown to be tandemly aligned in the genome
(Kanai and Natori, 1989, 1990), and the sequences
for sarcotoxin IA and sarcotoxin II unit 3 were used
for the probes as representatives of each gene
cluster. These probes can cross-react with the other
transcripts expressed from the respective clusters.
Though body injury and injection of saline itself induced the lectin gene expression, all kinds of
the examined stimulants increased the expression
level (Fig. 2). Especially, E. coli and B. bassiana
increased the expression in a dose-dependent
manner. When compared with the antimicrobial
peptide genes, the induction of sarcotoxin II genes
expression was relatively indiscriminate with low
induction efficiency. On the contrary, the induction of the sarcotoxin I genes expression was rather
specific with high induction efficiency; i.e., it was
not induced by the infection of fungi B. bassiana
well except the relatively weak induction at the
lowest concentration. Furthermore, the concentration of foreign substances that saturates the induction was different among those immune
defense genes. Sarcotoxin I genes were regulated
in a dose-dependent manner when induced by
S. aureus and NIH-Sape-4 cells. These results
indicate that the induction of the Sarcophaga lectin
Archives of Insect Biochemistry and Physiology December 2008
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Fig. 2. Specificity of the induction of the immune defense genes expression in the fat body of the third instar
larvae by injection of foreign pathogens and tumorous
cells. Foreign pathogens and NIH-Sape-4 cells suspension
in insect saline (0.2%, 2% and 20%) was injected into the
third instar larvae, and the fat bodies were dissected 20 h
after stimulation. The total RNA was subjected to Northern
blot.
gene expression is similar to that of sarcotoxin II
genes expression, and sarcotoxin I genes are regulated in a different manner.
Differential Activation of Immune Defense Genes at
Various Tissues
Though a fat body and hemocytes are tissues
responsible for systemic immune response including the production of antimicrobial peptides,
it has been known in Drosophila that the epithelial
tissues also participate in local immune response
(Tzou et al., 2000). We analyzed the expression of
the Sarcophaga lectin gene at various tissues 6 h
after body injury by Northern blot, and compared
it with the expression of the antimicrobial peptide
genes.
As reported before, the induction of the Sarcophaga lectin and sarcotoxin II genes expression was
detected in fat body, while that of the sapecin gene
expression was detected in hemocytes. But some
other tissues also expressed these genes after injury
(Fig. 3). The expression of the Sarcophaga lectin
gene was also induced strongly in epidermis and/
or muscle, and modestly in digestive tracts and
tracheae. The expression of sarcotoxin II genes was
Archives of Insect Biochemistry and Physiology December 2008
Activation of the Sarcophaga lectin gene
193
induced in tracheae and hemocytes at the intermediate level, and modestly in digestive tracts and
epidermis and/or muscle. The expression of sapecin gene was induced in cuticle and/or muscle,
tracheae and fat body. These results indicate that
these defense genes are induced in many tissues to
participate in local immunity, but with the different distribution from one another. It should also
be noted that the lectin gene expression was not
detectable in hemocytes like sarcotoxin II and
sapecin, suggesting that the relatively extended
induction was not quite ubiquitous, but was
under restricted regulation. Furthermore, the constitutive expression of the Sarcophaga lectin and
sarcotoxin II genes was detected at the cephalic
portion though the exact expression tissue was not
identifiable.
Feeding on Microorganisms and Microbial Components
Induces the Expression of the Immune Defense Genes
For insects, natural infection from digestive tract
is the most likely way to get an infection in the
wild, and induction of immune defense genes in
the tract by bacterial diet has been reported in
other insect species (Freitak et al., 2007; Vodovar
et al., 2005). From the tissue expression profiles,
immune defense genes including the Sarcophaga
lectin gene were turned out to be induced upon
body injury not only in the fat body and/or hemocytes but also in epithelial tissues like digestive
tracts. Therefore, these genes were expected to
participate in local immunity in the digestive tract.
To mimic the natural infection, we fed the third
instar larvae on microorganisms or microbial
compounds, and examined the expression of the
Sarcophaga lectin gene in the midgut along with the
antimicrobial peptide genes by Northern blot. We
subjected Escherichia coli (Gram-negative bacteria),
Staphylococcus aureus (Gram-positive bacteria) and
Beauveria bassiana (fungi) as microorganisms. As
microbial compounds, we examined lipopolysaccharide (Gram-negative compound), peptidoglycan from Gram-positive bacteria and
zymosan (fungal compound).
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Takahiro Tanji et al.
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Fig. 3. Expression tissues of the immune defense genes in the third instar larvae. Larvae were injured 6 h before
dissecting, and the total RNA from the dissected tissues was subjected to Northern blot. Lane 1, cephalic portion; lane 2,
digestive tracts; lane 3, epidermis and muscle; lane 4, tracheae; lane 5, fat body; lane 6, hemocytes.
Fig. 4. Induction of the immune defense genes expression in the third instar larvae by feeding on microorganisms and microbial components. Larvae were soaked in the
suspension for 6 h, and the total RNA from the midgut was
subjected to Northern blot. Lane 1, mock; lane 2, E. coli;
lane 3, S. aureus; lane 4, B. bassiana; lane 5, lipopolysaccharide; lane 6, peptidoglycan; lane 7, zymosan.
Though the expression of sarcotoxin I and
sarcotoxin II genes were not detectable under any
conditions (data not shown), the expression of the
Sarcophaga lectin and sapecin gene was detected
(Fig. 4). Essentially, the expression of both genes
was induced by all kinds of inducers examined, but
the preferences were different. The expression of
the Sarcophaga lectin gene was strongly induced by
E. coli but not by B. bassiana. On the other hand,
the sapecin gene expression was induced by
B. bassiana as strongly as by E. coli. As for microbial
compounds, the induction of the sapecin gene
expression was stronger than that of the Sarcophaga
lectin gene, especially by peptidoglycan from
Gram-positive bacteria. These results indicate that
the local induction of the Sarcophaga lectin gene in
midgut is regulated in a different manner from that
of the antimicrobial peptide genes.
DISCUSSION
From electrophoretic mobility shift assay, it was
suggested that the Sarcophaga lectin gene expression was regulated by multiple NF-kB -binding
motifs in its promoter. The Drosophila genome
encodes three NF-kB -related transcription factors,
Dorsal, DIF, and Relish. As for Sarcophaga, its
genome probably encodes at least three NF-kB-related transcription factors. The partial cDNA for
the Dorsal homologue was cloned from NIH-Sape4 cDNA library, and the Sarcophaga fat body and
Archives of Insect Biochemistry and Physiology December 2008
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NIH-Sape-4 cells have a protein that cross-reacts
with an anti-Drosophila DIF antibody with the
equivalent molecular weight (unpublished data).
Whereas, the existence of the Relish homologue is
still in question. Besides, Sarcophaga has another
transcription factor, SRAM (Sarcophaga-derived
Rel/Ankyrin Molecule), of which no homologues
have been identified in any other species (Shiraishi
et al., 2000). SRAM is a sole factor detected to bind
to the most proximal kB motif of the lectin gene
specifically (Kobayashi et al., 1993). Some of these
three factors may cooperatively regulate the expression of the lectin gene utilizing multiple kB
motifs.
In Drosophila, the binding specificity of three
NF-kB -related transcription factors was extensively
analyzed (Senger et al., 2004). Though the DIF
SELEX assay showed ambiguous sequences
following
the
first
three
G’s,
Dorsal
(GGGWWWHCBN) and Relish (GGGAHNYMYN)
have more restrictive specificity. The sequence of
the kB motif at 1.5 kb 5’-upstream of the lectin
gene is close to the Dorsal binding consensus. The
other one at 0.6 kb 5’-upstream of the gene is close
to both Dorsal and Relish binding consensuses.
We also found out that the recombinant SRAM can
bind to these two kB motifs (unpublished data).
Although the conservation of binding consensuses
between Drosophila and Sarcophaga is not clear, it is
likely considering the similarity of amino acid
sequences of Dorsal from these two species. Our
speculation is that multiple NF-kB -related transcription factors contribute to the expression of the
lectin gene.
In the fat body, the Sarcophaga lectin gene expression was induced by many pathogens and tumorous cells. Because specificity of the induction is
different among immune defense genes, it is reasonable to think that more than one signaling
pathway participates in the response, like the Toll
and IMD pathways in Drosophila. More restricted
expression of sarcotoxin I genes than that of the
Sarcophaga lectin and sarcotoxin II genes suggests
that the former gene is activated by one pathway
and the latter genes are activated by either another
pathway or multiple pathways.
Archives of Insect Biochemistry and Physiology December 2008
Activation of the Sarcophaga lectin gene
195
In Drosophila, the gene encoding Cecropin, the
homologue of sarcotoxin I, is regulated by both the
Toll and IMD pathways (Corbo and Levine, 1996;
Lemaitre et al., 1996). On the other hand, the gene
encoding Attacin, the homologue of sarcotoxin II,
is preferably regulated by the IMD pathway
(Georgel et al., 2001). Cecropin is both antibacterial and antifungal (Ekengren and Hultmark,
1999; Samakovlis et al., 1990) and Attacin
homologue in other insect species are specific to
Gram-negative bacteria (Ando et al., 1987; Hultmark et al., 1983). Combined with this knowledge,
Drosophila selectively produces antimicrobial peptides which attack infected pathogens.
The expression pattern of sarcotoxin I and
sarcotoxin II genes doesn’t seem to correspond to
each antimicrobial spectrum. However, this paradox may be explainable considering the environment where flesh flies live in the wild; it is
probably enriched with Gram-negative bacteria,
and once they are injured or infected, the defense
against Gram-negative bacteria by anti-Gramnegative bacterial peptides like sarcotoxin II is
required. Possibly, Sarcophaga lectin is also among
those immune defense proteins; or the lectin is
induced with wide spectrum and acts against a
variety of pathogens.
Interestingly, the injection of NIH-Sape-4 also
increased the expression of these immune defense
genes. Cecropin is known to have antitumor activity both in vitro and in vivo (Moore et al., 1994;
Winder et al., 1998). Sarcophaga lectin activates
murine macrophages, resulting in production of
tumor necrosis factor-a and lysis of tumor cells
(Itoh et al., 1984, 1985; Yamazaki et al., 1983).
These immune defense proteins may function in
the elimination of tumor cells in the fly.
Contrary to the similarity in the induction by
foreign substances in fat body, the expression
pattern in each tissue is not similar between the
Sarcophaga lectin and sarcotoxin II genes. Although
they were both mainly induced in fat body, the
sarcotoxin II genes but not the Sarcophaga lectin
gene were induced in hemocytes, another main
immune defense tissues. Furthermore, the expression of the Sarcophaga lectin gene is abundant in
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Takahiro Tanji et al.
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epidermis or muscle after body injury while that of
sarcotoxin II genes is more abundant in tracheae.
Sarcotoxin II may play a role in local immune response in tracheae. One interesting possibility is
that Sarcophaga lectin is excreted through cuticle
upon injury and infection, and acts on pathogens
surrounding the animals. The biological meaning
of this differential expression is yet to be elucidated.
The Northern blot analysis using Sarcophaga
tissues also showed that the cephalic portion expressed the Sarcophaga lectin and sarcotoxin II
genes constitutively. Though many tissues such as
imaginal discs and brain are included in this portion, we consider salivary glands the most likely
candidate of the expression tissue, for we detected
the activation of the lectin gene promoter in Drosophila salivary glands by reporter assay (Tanji
et al., 2002). Salivary glands are also important
tissues for the first line immune defense against
pathogens invading through mouth with foods by
secreting antimicrobial substances. For example,
the constitutive activation of the antifungal peptide
Drosomycin gene promoter has been reported in
both larval and adult stages of Drosophila
salivary glands (Ferrandon et al., 1998; Tzou et al.,
2000). Furthermore, the secretion of antimicrobial peptides, such as histatins and b-defensins, in
vertebrate saliva has been reported (MacKay
et al., 1984; Mathews et al., 1999; Pollock et al.,
1984).
Finally, we showed that the expression of the
Sarcophaga lectin gene was also induced in larval
midgut by feeding on microorganisms and microbial components as well as sapecin gene. Previously, we discussed the possibility of the
participation of the lectin in digestive tracts (Tanji
et al., 2002). We assumed that the constitutive
activation of the lectin gene promoter in the
transgenic Drosophila digestive tracts was caused by
food components. Drosophila standard food contains yeast. The induction of the lectin gene by
zymosan from S. cerevisiae supports the idea that
the yeast activated the promoter. In summary, the
expression of the Sarcophaga lectin gene is induced
by various stimulants in many tissues, and the
induction is rather indiscriminate than that of
some antimicrobial peptide genes. Possibly,
Sarcophaga lectin functions in many occasions
including both systemic and local immune
defenses.
LITERATURE CITED
Ando K, Natori S. 1988. Molecular cloning, sequencing, and
characterization of cDNA for sarcotoxin IIA, an inducible
antibacterial protein of Sarcophaga peregrina (flesh fly).
Biochemistry 27:1715–1721.
Ando K, Okada M, Natori S. 1987. Purification of sarcotoxin
II, antibacterial proteins of Sarcophaga peregrina (flesh fly)
larvae. Biochemistry 26:226–230.
Barillas-Mury C, Charlesworth A, Gross I, Richman A, Hoffmann JA, Kafatos FC. 1996. Immune factor Gambif1, a new
rel family member from the human malaria vector, Anopheles gambiae. EMBO J 15:4691–4701.
Bulet P, Stocklin R, Menin L. 2004. Anti-microbial peptides:
from invertebrates to vertebrates. Immunol Rev 198:
169–184.
Christophides GK, Vlachou D, Kafatos FC. 2004. Comparative
and functional genomics of the innate immune system in
the malaria vector Anopheles gambiae. Immunol Rev
198:127–148.
Corbo JC, Levine M. 1996. Characterization of an immunodeficiency mutant in Drosophila. Mech Dev 55:
211–220.
Ekengren S, Hultmark D. 1999. Drosophila cecropin as an
antifungal agent. Insect Biochem Mol Biol 29:965–972.
Engström Y. 1997. Insect immune gene regulation. In: Brey PT
and Hultmark D, editors. Molecular Mechanisms of Immune Responses in Insects. London: Chapman & Hall.
p 211–244.
Ferrandon D, Imler JL, Hetru C, Hoffmann JA. 2007. The
Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections. Nat Rev Immunol 7:862–874.
Ferrandon D, Jung AC, Criqui M, Lemaitre B, UttenweilerJoseph S, Michaut L, Reichhart J, Hoffmann JA. 1998. A
Archives of Insect Biochemistry and Physiology December 2008
_______________________________________
Activation of the Sarcophaga lectin gene
197
drosomycin-GFP reporter transgene reveals a local immune
response in Drosophila that is not dependent on the Toll
pathway. EMBO J 17:1217–1227.
(fleshfly) lectin and detection of sarcotoxins in the culture
medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina. Biochem J 248:217–222.
Freitak D, Wheat CW, Heckel DG, Vogel H. 2007. Immune
system responses and fitness costs associated with consumption of bacteria in larvae of Trichoplusia ni. BMC Biol
5:56.
Komano H, Mizuno D, Natori S. 1980. Purification of lectin
induced in the hemolymph of Sarcophaga peregrina larvae
on injury. J Biol Chem 255:2919–2924.
Georgel P, Naitza S, Kappler C, Ferrandon D, Zachary D,
Swimmer C, Kopczynski C, Duyk G, Reichhart JM, Hoffmann JA. 2001. Drosophila immune deficiency (IMD) is a
death domain protein that activates antibacterial defense
and can promote apoptosis. Dev Cell 1:503–514.
Hultmark D. 1993. Immune reactions in Drosophila and
other insects: a model for innate immunity. Trends Genet
9:178–183.
Hultmark D, Engström A, Anderson K, Steiner H, Bennich H,
Boman HG. 1983. Insect immunity. Attacins, a family of
antibacterial proteins from Hyalophora cecropia. EMBO J
2:571–576.
Itoh A, Iizuka K, Natori S. 1984. Induction of TNF-like factor
by murine macrophage-like cell line J774.1 on treatment
with Sarcophaga lectin. FEBS Lett 175:59–62.
Itoh A, Iizuka K, Natori S. 1985. Antitumor effect of Sarcophaga lectin on murine transplanted tumors. Jpn J Cancer
Res 76:1027–1033.
Kanai A, Natori S. 1989. Cloning of gene cluster for sarcotoxin
I, antibacterial proteins of Sarcophaga peregrina. FEBS Lett
258:199–202.
Kanai A, Natori S. 1990. Analysis of a gene cluster for sarcotoxin II, a group of antibacterial proteins of Sarcophaga
peregrina. Mol Cell Biol 10:6114–6122.
Kanost MR, Jiang H, Yu XQ. 2004. Innate immune responses
of a lepidopteran insect, Manduca sexta. Immunol Rev
198:97–105.
Kobayashi A, Matsui M, Kubo T, Natori S. 1993. Purification
and characterization of a 59-kilodalton protein that specifically binds to NF-kB-binding motifs of the defense protein
genes of Sarcophaga peregrina (the flesh fly). Mol Cell Biol
13:4049–4056.
Komano H, Kasama E, Nagasawa Y, Nakanishi Y, Matsuyama
K, Ando K, Natori S. 1987. Purification of Sarcophaga
Archives of Insect Biochemistry and Physiology December 2008
Komano H, Nozawa R, Mizuno D, Natori S. 1983. Measurement of Sarcophaga peregrina lectin under various physiological conditions by radioimmunoassay. J Biol Chem
258:2143–2147.
Lemaitre B, Hoffmann J. 2007. The host defense of Drosophila
melanogaster. Annu Rev Immunol 25:697–743.
Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA.
1996. The dorsoventral regulatory gene cassette spätzle/
Toll/cactus controls the potent antifungal response in
Drosophila adults. Cell 86:973–983.
Lemaitre B, Reichhart JM, Hoffmann JA. 1997. Drosophila host
defense:
differential
induction
of
antimicrobial
peptide genes after infection by various classes of
microorganisms. Proc Natl Acad Sci USA 94:14614–
14619.
MacKay BJ, Denepitiya L, Iacono VJ, Krost SB, Pollock JJ. 1984.
Growth-inhibitory and bactericidal effects of human parotid salivary histidine-rich polypeptides on Streptococcus
mutans. Infect Immun 44:695–701.
Mathews M, Jia HP, Guthmiller JM, Losh G, Graham S,
Johnson GK, Tack BF, McCray Jr PB. 1999. Production of b-defensin antimicrobial peptides by the
oral mucosa and salivary glands. Infect Immun 67:
2740–2745.
Matsumoto N, Okada M, Takahashi H, Ming QX, Nakajima Y,
Nakanishi Y, Komano H, Natori S. 1986. Molecular cloning
of a cDNA and assignment of the C-terminal of sarcotoxin
IA, a potent antibacterial protein of Sarcophaga peregrina.
Biochem J 239:717–722.
Matsuyama K, Natori S. 1988. Molecular cloning of cDNA for
sapecin and unique expression of the sapecin gene during
the development of Sarcophaga peregrina. J Biol Chem
263:17117–17121.
Moore AJ, Devine DA, Bibby MC. 1994. Preliminary experimental anticancer activity of cecropins. Pept Res
7:265–269.
198
Takahiro Tanji et al.
_______________________________________________________
Ohtaki T. 1966. On the delayed pupation of the flesh fly,
Sarcophaga peregrina Robineau-Desvoidy. Jap J Med Sci Biol
19:97–104.
Okada M, Natori S. 1983. Purification and characterization of
an antibacterial protein from haemolymph of Sarcophaga
peregrina (flesh-fly) larvae. Biochem J 211:727–734.
Pollock JJ, Denepitiya L, MacKay BJ, Iacono VJ. 1984. Fungistatic and fungicidal activity of human parotid salivary
histidine-rich polypeptides on Candida albicans. Infect Immun 44:702–707.
Sagisaka A, Tanaka H, Furukawa S, Yamakawa M. 2004.
Characterization of a homologue of the Rel/NF-kB transcription factor from a beetle, Allomyrina dichotoma. Biochim Biophys Acta 1678:85–93.
Tanaka H, Matsuki H, Furukawa S, Sagisaka A, Kotani E, Mori
H, Yamakawa M. 2007. Identification and functional analysis of relish homologs in the silkworm, Bombyx mori.
Biochim Biophys Acta 1769:559–568.
Tanaka H, Yamamoto M, Moriyama Y, Yamao M, Furukawa S,
Sagisaka A, Nakazawa H, Mori H, Yamakawa M. 2005. A
novel Rel protein and shortened isoform that differentially
regulate antibacterial peptide genes in the silkworm Bombyx
mori. Biochim Biophys Acta 1730:10–21.
Tanji T, Hu X, Weber AN, Ip YT. 2007. Toll and IMD pathways
synergistically activate an innate immune response in
Drosophila melanogaster. Mol Cell Biol 27:4578–4588.
Tanji T, Ip YT. 2005. Regulators of the Toll and Imd pathways
in the Drosophila innate immune response. Trends Immunol 26:193–198.
Samakovlis C, Kimbrell DA, Kylsten P, Engström A, Hultmark
D. 1990. The immune response in Drosophila: pattern of
cecropin expression and biological activity. EMBO J
9:2969–2976.
Tanji T, Kobayashi A, Natori S. 2002. Activation of the Sarcophaga lectin gene promoter in transgenic Drosophila. Arch
Insect Biochem Physiol 50:131–138.
Senger K, Armstrong GW, Rowell WJ, Kwan JM, Markstein M,
Levine M. 2004. Immunity regulatory DNAs share
common organizational features in Drosophila. Mol Cell
13:19–32.
Tzou P, Ohresser S, Ferrandon D, Capovilla M, Reichhart JM,
Lemaitre B, Hoffmann JA, Imler JL. 2000. Tissue-specific
inducible expression of antimicrobial peptide genes in
Drosophila surface epithelia. Immunity 13:737–748.
Shin SW, Kokoza V, Ahmed A, Raikhel AS. 2002. Characterization of three alternatively spliced isoforms of the Rel/NFkB transcription factor Relish from the mosquito Aedes aegypti. Proc Natl Acad Sci USA 99:9978–9983.
Vodovar N, Vinals M, Liehl P, Basset A, Degrouard J, Spellman
P, Boccard F, Lemaitre B. 2005. Drosophila host defense after
oral infection by an entomopathogenic Pseudomonas
species. Proc Natl Acad Sci U S A 102:11414–11419.
Shin SW, Kokoza V, Bian G, Cheon HM, Kim YJ, Raikhel AS.
2005. REL1, a homologue of Drosophila dorsal, regulates
toll antifungal immune pathway in the female mosquito
Aedes aegypti. J Biol Chem 280:16499–16507.
Winder D, Gunzburg WH, Erfle V, Salmons B. 1998. Expression of antimicrobial peptides has an antitumour effect
in human cells. Biochem Biophys Res Commun 242:
608–612.
Shiraishi H, Kobayashi A, Sakamoto Y, Nonaka T, Mitsui Y,
Aozasa N, Kubo T, Natori S. 2000. Molecular cloning and
characterization of SRAM, a novel insect rel/ankyrin-family
protein present in nuclei. J Biochem 127:1127–1134.
Yamazaki M, Ikenami M, Komano H, Tsunawaki S, Kamiya H,
Natori S, Mizuno D. 1983. Polymorphonuclear leukocytemediated cytolysis induced by animal lectin. Gan 74:
576–583.
Takahashi H, Hiroto H, Natori S. 1986. Expression of the
lectin gene in Sarcophaga peregrina during normal development and under conditions where the defence mechanism is activated. J Insect Physiol 32:771–779.
Yu XQ, Gan H, Kanost MR. 1999. Immulectin, an inducible Ctype lectin from an insect, Manduca sexta, stimulates activation of plasma prophenol oxidase. Insect Biochem Mol
Biol 29:585–597.
Takahashi H, Komano H, Kawaguchi N, Kitamura N, Nakanishi S, Natori S. 1985. Cloning and sequencing of cDNA
of Sarcophaga peregrina humoral lectin induced on injury of
the body wall. J Biol Chem 260:12228–12233.
Yu XQ, Kanost MR. 2000. Immulectin-2, a lipopolysaccharidespecific lectin from an insect, Manduca sexta, is induced in
response to gram-negative bacteria. J Biol Chem 275:
37373–37381.
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