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*
M, F, L Science Center, Tensei-suisan Co., Karatsu, Saga, Japan;
Institute for Virus Research, Kyoto University, Shogoin, Sakyo-ku, Kyoto, Japan;
Department of Medical Zoology, Kyoto Prefectural University of Medicine, Kawaramachi-hirokoji, Kyoto, Japan; and
§
Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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During screening of biological response modifiers, particularly those regulating cell growth, from marine fishes and plants, we observed that several mammalian tumor cells exposed to extracts from visceral organs of Chub mackerel underwent morphological changes resembling those of apoptosis. This activity was heat, pH, and protease sensitive, and Fas and TNF receptor independent (our unpublished observations).
In the current study, we report the purification, cDNA cloning, and characterization of this protein factor designated as apoptosis-inducing protein (AIP).3 AIP was found to be induced in fish by infection with larval nematode, and possesses the basic dinucleotide-binding motif and COOH-terminal endoplasmic reticulum (ER) retention signal, indicating that it is a novel structural and functional reticuloplasmin. Evidence is presented that AIP is secreted from the ER in vivo and in vitro as a functional molecule with apoptosis-inducing activity, and therefore has potential importance in host defense system against invading parasites.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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All packed columns used for purification were from Pharmacia
(Piscataway, NJ). All fractions were assayed for apoptosis-inducing
activity, and all procedures were done at 4°C. Lyophilized visceral
extracts from Anisakis simplex-infected C.
mackerel were dissolved in 100 mM Tris-HCl (pH 7.5) and
centrifuged at 27,000 x g for 30 min. The supernatant
was subjected to ammonium sulfate fractionation, and the precipitate
obtained at 55–95% saturation with ammonium sulfate was dissolved in
20 mM Tris-HCl (pH 7.5), and applied to a HiLoad 16/60 Superdex 200 pg
gel-filtration column in the same buffer. Fractions that contained
apoptosis-inducing activity (active fractions) were pooled, applied to
a Con A-Sepharose column, eluted with 0.5 M
methyl-
-D-mannopyranoside, and concentrated by
ultrafiltration with a 50-kDa molecular mass cutoff membrane.
Concentrated material was applied to a HiLoad Superdex 200 column
equilibrated with buffer A (20 mM bis-Tris, pH 6.4, 100 mM NaCl) and
eluted with the same buffer. Active fractions were applied to a Mono Q
HR 5/5 column equilibrated with buffer A and eluted with a linear
gradient up to 1 M NaCl. Active fractions were concentrated, applied to
a Superdex 200 HR 10/30 gelfiltration column equilibrated with PBS, and
eluted with the same buffer. Active fractions were sterilized and
stored in small portions at -80°C. The concentration of protein was
determined by the bicinchoninic acid assay (Pierce, Rockford, IL) with
BSA as a standard.
cDNA cloning
To determine the N-terminal sequence, two protein bands of 62 and 64 kDa were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and submitted for sequence analysis. To determine the internal sequence, the Coomassie blue-stained protein band was excised from the SDS-PAGE gel. After in-gel digestion with V8 protease, the sequences of four peptides were determined. The following peptide sequences and residue numbers are based on the derived amino acid sequence of the AIP gene (residues 1–524): EHLADXLEDKDYDTLLQTLD (residues 31–50, N-terminal sequences of two polypeptides), FVMTDDNTFY (residues 138–147), MIYDQADV (residues 246–253), AFLSVLDVP (residues 272–280), and SLLFLGASDE (residues 408–417). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. X, Indicates unidentified amino acid. Two degenerate primers corresponding to the peptide sequences EHLADCLEDKDYDTLLQTLD and MIYDQADV were designed: GA(A/G)GA(C/T)AA(A/G)GA(C/T)TA(C/T)GA(C/T)AC and TC(A/C/G/T)GC(C/T)TG(A/G)TC(A/G)TA(A/G)TACAT, and used as primers for RT-PCR. Total RNA was prepared from the dissected capsule of larva-infected C. mackerel by homogenization in TRIZOL (Life Technologies, Grand Island, NY) and used to synthesize the first-strand cDNA with Superscript reverse transcriptase (Life Technologies). Poly(A) RNA was isolated from total RNA by using oligo(dT) mRNA purification kit (Pharmacia) and used to construct the capsule cDNA library (SuperScript plasmid system for cDNA synthesis and plasmid cloning; Life Technologies). RT-PCR conditions used were 94°C for 1 min, 56°C for 1 min, and 72°C for 2 min (35 cycles). A 644-bp PCR product was obtained and subsequently sequenced after subcloning into the pCR vector using the TA cloning kit (Invitrogen, San Diego, CA). A capsule cDNA library was screened with this 645-bp PCR product, and the largest insert was sequenced on both strands.
Monoclonal Ab
Splenocytes from BALB/c female mouse immunized with purified AIP
were fused with mouse myeloma FOX-NY, and the resultant hybridomas were
screened by ELISA and immunoblotting of the partially purified AIP.
Positive supernatants were then tested for specificity by determining
whether they contained Abs capable of immunodepleting
apoptosis-inducing activity in visceral extracts of C.
mackerel. Three monoclonal clones that produce Abs termed
I38A, I32D, and I310H were subsequently obtained. These Abs recognized
only 62- and 64-kDa polypeptides in visceral extracts of infected fish,
and visceral extracts immunodepleted with these mAbs completely lost
apoptosis-inducing activity. The isotypes of these were all IgG1
.
Apoptosis assays
For cytotoxicity assay, human promyelocytic leukemia cells HL-60 were seeded at 30,000 cells/well in a 96-well flat-bottom microtiter plate and cultured at 37°C with various amounts of AIP for 12 h or indicated times. Cell viability was measured by MTS assay (Promega, Madison, WI). The absorbance of MTS formazan was measured at an OD of 490 nm using an automated microplate reader. The percentage of viable cells was calculated as follows: ((experimental OD value - spontaneous OD value)/(maximum OD value - spontaneous OD value)) x 100. The spontaneous OD value was determined by adding SDS to a final concentration of 1%, whereas the maximum OD value was determined by incubating the cells with medium alone.
For DNA fragmentation assay, cells were centrifuged for 4 min, 400 x g at the end of each incubation period. The pellet was resuspended in lysis buffer (10 mM Tris, pH 8, 400 mM NaCl, 2 mM EDTA, 0.25% SDS, 0.2 mg/ml proteinase K) and digested for 2 h at 56°C. After digestion was complete, 1/4 vol of saturated NaCl was added and centrifuged at 12,000 rpm for 10 min. The supernatant containing the DNA was treated with 0.2 mg/ml RNase A for 2 h at 37°C, and DNA was precipitated with ethanol. Electrophoresis was carried out on 2% agarose gel containing 0.5 µg/ml ethidium bromide.
For DNA content analysis, the cells were harvested at various times after treatment with AIP, fixed with 70% ethanol, and then incubated with PBS containing 50 µg/ml RNase A at 37°C for 30 min. The DNA content of the cells was analyzed with a flow cytometry after staining with 10 µg/ml propidium iodide.
For microscopic analysis, cells were fixed with 1% glutaraldehyde, stained with 10 µg/ml Hoechst 33258. The specimens were analyzed under phase contrast and fluorescent light using a Zeiss Axiovert microscope.
Calcium perturbation
A23187 were administered to NIH3T3 cells in fresh serum-free medium with insulin, transferrin, and selenium. At the end of the incubation, samples of the culture medium were concentrated by ultrafiltration with a 50-kDa molecular mass cutoff membrane and retained for apoptosis assay and immunoblot analysis. Cells were washed with PBS, disrupted by three cycles of freezing-thawing, and centrifuged at 15,000 rpm. The supernatant was used for immunoblot analysis
Hydrogen peroxide measurement
The release of H2O2 under AIP catalysis was evidenced by peroxidase/o-phenylenediamine dihydrochloride (OPD) method. In the presence of H2O2 and peroxidase, OPD is oxidized and forms a completely soluble end product with maximum absorbance at 490 nm after the reaction is stopped with H2SO4. AIP purified from larva-infected fish or expressed in COS-7 cells transfected with the AIP gene was incubated with 5 mM substrate, 10 U/ml peroxidase, and 500 µg/ml OPD for 2 h at 37°C. The reaction was stopped with the equal volume of 2 N H2SO4, and the amount of H2O2 production was determined by comparison with a standard curve that had been generated using fresh dilutions of stock H2O2.
Plasmid construction and transfections
Full-length AIP cDNA was introduced into a mammalian expression plasmid pEF-BOS (2) (pEF-AIP). To exchange signal sequence of AIP for mammalian signal sequence, the region coding aa 31–524 and stop codon was introduced into a SfiI site of pSecTag2 vector (Invitrogen) (pSec-AIP). pEF-AIP and pSec-AIP were transfected into Cos-7 cells using a Bio-Rad (Richmond, CA) Gene Pulser apparatus and into NIH3T3 cells by the Lipofectamine-Plus method (Life Technologies), respectively.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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We have attempted to search for the source of factors involved in
controlling cell growth by screening water-soluble materials of marine
fishes and plants, then found that visceral extracts of C.
mackerel have a powerful and dose-dependent cytotoxic effect on a
variety of mammalian tumor cells. The activity in visceral extracts of
C. mackerel was likely to be mediated by protein components,
as suggested by its molecular size (>50 kDa) and its sensitivity to
trypsin and heat (data not shown; see Material and Methods).
Several chromatographic steps were used to purify this factor, as
detailed in Materials and Methods. This procedure provided
an 8000-fold increase in sp. act. After the final gel-filtration step,
SDS-PAGE followed by silver staining revealed two protein bands of 62
and 64 kDa (Fig. 1
A). The two
polypeptides had the same N-terminal sequence (see the cDNA cloning in
Materials and Methods), suggesting that it is truncated or
modified form of one another. Cytotoxic activity was eluted from
gel-filtration column at about 135 kDa (data not shown), indicating
that both proteins existed as dimer in solution.
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To verify that the polypeptide was indeed apoptosis-inducing
factor, three mAbs were generated against purified apoptosis-inducing
factor and used to immunodeplete apoptosis-inducing activity in
C. mackerel extracts. These Abs recognized only 62- and
64-kDa protein bands on immunoblot of C. mackerel visceral
extract and purified apoptosis-inducing sample (Fig. 1
B).
Immunodepletion of C. mackerel viscera extracts
with these mAbs completely removed apoptosis-inducing activity (see mAb
in Materials and Methods). These results showed that the 62-
and 64-kDa proteins were responsible for the apoptosis-inducing
activity. We designated these proteins AIP. AIP was found to possess
strong apoptosis activity. Cytolytic activity was examined using the
human leukemia cell HL-60. The median cytolytic dose was observed at 5
ng/ml AIP (Fig. 1C), and cells were completely killed by
incubation for 24 h in the presence of 20 ng/ml AIP (Fig. 1D). Oligonucleosome-length DNA fragments, which are
characteristic of apoptosis, were observed within 2 h in HL-60
cells treated with 20 ng/ml AIP (Fig. 2A). Nuclear staining (Fig. 2B) and flow cytometry analysis (Fig. 2C) also
confirmed other typical apoptotic features.
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Next, we examined tissue expression of AIP to select an organ
suitable for the isolation of the gene encoding AIP. Unexpectedly,
neither AIP nor apoptosis-inducing activity was detected in any of the
tissues examined (data not shown), suggesting that the expression of
AIP is conditionally regulated in fish. We found that the induction of
AIP depends on the infection by the larval nematode, A.
simplex. Apoptosis-inducing activity and AIP could be detected in
visceral extracts from infected fish (five samples), but not in those
from uninfected fish (five samples) (Fig. 3A–D). Furthermore, AIP was
detected in extracts from tissues infected with larvae (data not
shown), particularly in capsules that surrounded the larvae on the
surface of the tissue (see below).
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The N terminus and four V8 protease-digested peptides of the
purified AIP were sequenced. On the basis of the sequences (see cDNA
cloning in Materials and Methods), degenerate PCR primers
were designed, and RT-PCR was used to amplify a single 645-bp product
(residues 38–252) (Fig. 4A)
from the total RNA of capsules containing larva, in which AIP
transcripts were mainly detected. This PCR product was then used to
isolate the full-length 2025-bp cDNA from a cDNA library constructed
from the mRNA of capsules containing larva. The cDNA contained an open
reading frame for a protein of 524 aa with a predicted
Mr of 55,000 (Fig. 4A). The
N-terminal and four V8 protease-digested peptide sequences were found
within the open reading frame. SignalP and PSORT analyses indicated the
presence of a signal peptide sequence, but not one that would be
cleavable in mammals. However, N terminus of purified, mature AIP
started at position 31 amino acid (Glu), indicating that the signal
peptide is cleaved out in fish. Five potential
N-glycosylation sites were observed in the amino acid
sequence of AIP, which may explain the difference between the
Mr determined by SDS-PAGE and that
predicted from the amino acid sequence.
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ß dinucleotide-binding fold commonly
found in flavin adenine dinucleotide- and NADPH-binding proteins
(3, 4). Indeed, purified AIP showed an absorption spectrum
resembling that of flavoprotein (data not shown). This observation
strongly suggests that binding of flavin might be required for AIP
function. Interestingly, the carboxyl-terminal region of AIP was found
to possess a KDEL sequence, which has been postulated to be both
necessary and sufficient for proteins to be retained in the lumen of
ER. Indeed, AIP was predominantly detected in the ER of murine
fibroblast cells transfected with the AIP gene (data not shown).
The amino acid sequence of AIP displays 41% overall identity to those
of two flavoproteins, the predicted IL-4-induced mouse B cell gene
(Fig 1) protein (5) of unknown function and
snake venom L-amino acid oxidase (LAO) (6, 7) (Fig. 4B). We therefore investigated whether AIP
could catalyze H2O2
production. AIP purified from larva-infected fish or expressed in COS-7
cells transfected with the AIP gene oxidized
L-amino acids, especially
L-lysine (Fig. 4C). Then, we examined
the effect of scavengers of
H2O2 on the apoptosis
induced by AIP. Cotreatment of HL-60 cells with AIP and catalase
decreased AIP-induced apoptosis by 85% (Fig. 4D). These
results indicated that AIP is the first member of LAO family to possess
an ER retention signal, and that AIP-induced apoptosis is mainly
mediated by H2O2.
Intestinal infection with nematode parasites causes polarization of the
immune responses to the Th2 type in animals (8). Th2
cytokines, especially IL-4, play a central role in host defense against
nematode infection (9, 10, 11). In fish, it is also possible
that the infection by gastrointestinal nematode A. simplex
induces a Th2-like immune response. If so, it could be speculated that
AIP induction is associated with elevated levels of Th2-like cytokines
in fish after nematode infection and that this induction occurs in a
similar manner to that of Fig 1 in response to IL-4 in
mammals. However, the predicted Fig 1 protein does not
possess the ER retention signal (5), and Fig 1
protein expressed in COS-7 did not show apoptosis-inducing activity in
vitro even at concentration 100 times higher than the minimal
concentration of AIP required to induce apoptosis, although it does
possess H2O2-producing
activity (data not shown). Thus, Fig 1 protein appears
functionally unrelated to AIP. While the physiological role of snake
venom LAO is unclear, its antibacterial and apoptosis-inducing
activities have been demonstrated (6, 7). LAO is also not
an ER rumen protein and does not induce apoptosis in HL-60 cells at
concentrations lower than 2.5 µg/ml even by 24-h treatment
(6), while treatment with 20 ng/ml of AIP induced
apoptosis in HL-60 cells within 2 h (Fig. 2). In these respects,
snake venom LAO appears to be more similar to Fig 1 protein
than to AIP.
AIP predominantly localizes in the inner cavity of capsule surrounding the larvae in vivo and is efficiently secreted into the medium as a functional protein with apoptosis-inducing activity in vitro through calcium perturbation
Larval nematodes are found in the abdominal cavity on the surface
of visceral organs or in the peritoneal cavity, as well as in the
intestine of infected C. mackerel. The host forms a capsule
around the larvae to prevent their migration from the abdominal region
into various viscera. Apoptosis-inducing activity or AIP was 400 times
higher in the capsule than in whole viscera extracts from fish infected
with larvae (Fig. 5, A and
B), and was not detected in free larvae (data not shown) or
larvae within the capsule (Fig. 5, A and B). As
revealed by Southern blot analysis, AIP is a fish-derived protein (Fig. 5C). Therefore, AIP is produced by larva-infected fish and
appears to be concentrated in the capsule surrounding the larvae.
Interestingly, cytolytic activity was not found in viscera extracts
from sardines, even when infected with larvae (data not shown). In
sardines, localization of Anisakis larvae is restricted to
the muscular and glandular parts of the esophagus and intestine or
intestinal wall, and larvae have never been found on visceral organs or
in the abdominal cavity of these fish, suggesting that the early third
stage larvae have not the ability to penetrate through the digestive
tract of fishes. These results implied that AIP induction is associated
with the capacity of host fish to encapsulate larvae after they have
penetrated the abdominal cavity.
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, A and B), suggesting that
AIP is secreted from the ER. Proteins that contain the C-terminal ER
retention signal (KDEL) are found in the ER lumen and are called
reticuloplasmins. The major reticuloplasmins have been proposed to
function as molecular chaperones during protein assembly and
degradation and for calcium storage (12, 13, 14). These
proteins are transferred into the ER with the help of their signal
peptide and are retained there by continuous retrieval from a post-ER
compartment, which relies on a pH-dependent interaction of the
retention signal with a specific receptor (15, 16). Some
proteins with the KDEL retention signal were reported to be exported
from the ER to plasma membranes or extracellular medium under normal
conditions or by an activation-dependent mechanism
(17, 18, 19). To investigate whether AIP can be secreted as a
functional molecule, we constructed pSecAIP, which contained the murine
Ig -chain leader sequence in place of the signal sequence of AIP, to
fully function for translocation into ER of mammalian cells. NIH3T3
cells were cotransfected with the pSec-AIP and Escherichia coli
lacZ gene, and stained for ß-galactosidase. Transfected cells
were identified by their blue color, and apoptotic cells were
determined microscopically. The typical morphological properties of
apoptotic cells were not observed even more than 48 h after
transfection (Fig. 5D), while 12-h AIP treatment (20 ng/ml)
of control NIH3T3 cells was found to induce membrane blebbing, cell
rounding, and cell detachment from the plates, which are characteristic
features of apoptosis (data not shown). These observations indicated
that overexpression of AIP within cells does not result in apoptosis.
However, AIP was efficiently secreted into the medium as a functional
protein with apoptosis-inducing activity when the transfected murine
fibroblast cells were treated with the calcium ionophore A23187 (Fig. 5, E and F). We also found a progressive increase
in the amount of endogenous Ig heavy chain binding protein (BiP, also
known as Grp78) in the medium of the same ionophore-treated cells (Fig. 5F). Such treatments with calcium ionophores have been shown
to cause secretion of reticuloplasmins, including BiP, endoplasmin,
protein disulfide isomerase, and calreticulin, suggesting a role for
calcium ions in the retention system (17). Taken together,
these results suggest that AIP may be secreted by a similar mechanism
into the capsule cavity from, as yet, unidentified capsular cells, and
that the secreted AIP confers resistance to larval nematodes. The ingestion of larval nematode, A. simplex, was reported to cause a parasitic disease known as anisakiasis in humans (20, 21). Several species of larval nematodes including Anisakis larvae undergo extensive migration within their intermediate hosts, and the migrated visceral larvae cause a severe inflammatory disease characterized by hepatomegaly, eosinophilia, and hypergammaglobulinemia (22, 23, 24). Encapsulation of the larvae by the host certainly prevents their migration and growth, and may function as a major defense system against larval infection. We purified infection-specific AIP and cloned the corresponding gene. AIP induction in fish is the result of an interaction between parasite and host, and is mainly restricted into the capsules surrounding larval nematode. Recently, it has been reported that catalase is needed to extend lifespan in Caenorhabditis elegans by protecting nematodes from oxidative damages (25). The formation of capsules by the host may serve to confine larvae to an area in which AIP expression can strongly suppress their vitality and ability to invade host tissues. In addition, because AIP may play a crucial role in determining resistance to nematode infection, we hope that this study will lead to a better understanding of host defense system against larval nematodes and provide a conceptual basis for the development of novel and more efficient means for combating this type of infection.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sang-Kee Jung, M, F, L Science Center, Tensei-suisan Co., 1-25 Nakase-dori, Karatsu, Saga 847-0193, Japan; or Dr. Shin Yonehara, Institute for Virus Research, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
3 Abbreviations used in this paper: AIP, apoptosis-inducing protein; BiP, Ig heavy chain binding protein; ER, endoplasmic reticulum; Fig 1, IL-4-induced mouse B cell gene; LAO, L-amino acid oxidase; OPD, o-phenylenediamine dihydrochloride.
Received for publication January 18, 2000. Accepted for publication May 19, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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ß-fold in proteins, using an amino acid sequence fingerprint. J. Mol. Biol. 187:101.[Medline]
, an interleukin 4-induced mouse B cell gene isolated by cDNA representational difference analysis. Proc. Natl. Acad. Sci. USA. 94:2507. protein. Biochem. Biophys. Res. Commun. 248:476.[Medline]
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