Causative Nematode of Human Anisakiasis , a Anisakis Simplex

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Purification and Cloning of an Apoptosis-Inducing Protein Derived from Fish Infected with Anisakis simplex, a Causative Nematode of Human Anisakiasis This information is current as of September 23, 2021. Sang-Kee Jung, Angela Mai, Mitsunori Iwamoto, Naoki Arizono, Daisaburo Fujimoto, Kazuhiro Sakamaki and Shin Yonehara J Immunol 2000; 165:1491-1497; ; doi: 10.4049/jimmunol.165.3.1491 Downloaded from http://www.jimmunol.org/content/165/3/1491

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Puriﬁcation and Cloning of an Apoptosis-Inducing Protein Derived from Fish Infected with Anisakis simplex, a Causative Nematode of Human Anisakiasis1

Sang-Kee Jung2*† Angela Mai,*† Mitsunori Iwamoto,* Naoki Arizono,‡ Daisaburo Fujimoto,§ Kazuhiro Sakamaki,† and Shin Yonehara2†

While investigating the effect of marine products on cell growth, we found that visceral extracts of Chub mackerel, an ocean fish, had a powerful and dose-dependent apoptosis-inducing effect on a variety of mammalian tumor cells. This activity was strikingly dependent on infection of the C. mackerel with the larval nematode, Anisakis simplex. After purification of the protein responsible for the apoptosis-inducing activity, we cloned the corresponding gene and found it to be a flavoprotein. This protein, termed

apoptosis-inducing protein (AIP), was also found to possess an endoplasmic reticulum retention signal (C-terminal KDEL se- Downloaded from quence) and H2O2-producing activity, indicating that we had isolated a novel reticuloplasimin with potent apoptosis-inducing activity. AIP was induced in ﬁsh only after infection with larval nematode and was localized to capsules that formed around larvae to prevent their migration to host tissues. Our results suggest that AIP may function to impede nematode infection. The Journal of Immunology, 2000, 165: 1491–1497.

lterations in cell survival and growth contribute to the with apoptosis-inducing activity, and therefore has potential im- http://www.jimmunol.org/ pathogenesis of a number of human diseases, including portance in host defense system against invading parasites. A cancer, infection, autoimmune diseases, and neurode- generative disorders (1). Apoptosis can be triggered by a variety of Materials and Methods extrinsic and intrinsic signals. Purification of AIP During screening of biological response modifiers, particularly All packed columns used for purification were from Pharmacia (Piscat- those regulating cell growth, from marine fishes and plants, we away, NJ). All fractions were assayed for apoptosis-inducing activity, and observed that several mammalian tumor cells exposed to extracts all procedures were done at 4°C. Lyophilized visceral extracts from Ani- from visceral organs of Chub mackerel underwent morphological sakis simplex-infected C. mackerel were dissolved in 100 mM Tris-HCl changes resembling those of apoptosis. This activity was heat, pH, (pH 7.5) and centrifuged at 27,000 ϫ g for 30 min. The supernatant was by guest on September 23, 2021 and protease sensitive, and Fas and TNF receptor independent (our subjected to ammonium sulfate fractionation, and the precipitate obtained at 55–95% saturation with ammonium sulfate was dissolved in 20 mM unpublished observations). Tris-HCl (pH 7.5), and applied to a HiLoad 16/60 Superdex 200 pg gel- In the current study, we report the purification, cDNA cloning, filtration column in the same buffer. Fractions that contained apoptosis- and characterization of this protein factor designated as apoptosis- inducing activity (active fractions) were pooled, applied to a Con A-Sepha- ␣ inducing protein (AIP).3 AIP was found to be induced in fish by rose column, eluted with 0.5 M methyl- -D-mannopyranoside, and concentrated by ultrafiltration with a 50-kDa molecular mass cutoff mem- infection with larval nematode, and possesses the basic dinucle- brane. Concentrated material was applied to a HiLoad Superdex 200 col- otide-binding motif and COOH-terminal endoplasmic reticulum umn equilibrated with buffer A (20 mM bis-Tris, pH 6.4, 100 mM NaCl) (ER) retention signal, indicating that it is a novel structural and and eluted with the same buffer. Active fractions were applied to a Mono functional reticuloplasmin. Evidence is presented that AIP is se- Q HR 5/5 column equilibrated with buffer A and eluted with a linear gra- creted from the ER in vivo and in vitro as a functional molecule dient 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 Ϫ80°C. The concentration of protein was determined by *M, F, L Science Center, Tensei-suisan Co., Karatsu, Saga, Japan; †Institute for Virus the bicinchoninic acid assay (Pierce, Rockford, IL) with BSA as a standard. Research, Kyoto University, Shogoin, Sakyo-ku, Kyoto, Japan; ‡Department of Med- ical Zoology, Kyoto Prefectural University of Medicine, Kawaramachi-hirokoji, cDNA cloning Kyoto, Japan; and §Department of Applied Biological Science, Faculty of Agricul- ture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan To determine the N-terminal sequence, two protein bands of 62 and 64 kDa were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride Received for publication January 18, 2000. Accepted for publication May 19, 2000. membrane, and submitted for sequence analysis. To determine the internal The costs of publication of this article were defrayed in part by the payment of page sequence, the Coomassie blue-stained protein band was excised from the charges. This article must therefore be hereby marked advertisement in accordance SDS-PAGE gel. After in-gel digestion with V8 protease, the sequences of with 18 U.S.C. Section 1734 solely to indicate this fact. four peptides were determined. The following peptide sequences and res- 1 Sequence data have been submitted to the European Molecular Biology Laboratory idue numbers are based on the derived amino acid sequence of the AIP (EMBL) databases under accession number AJ400871. gene (residues 1–524): EHLADXLEDKDYDTLLQTLD (residues 31–50, 2 Address correspondence and reprint requests to Dr. Sang-Kee Jung, M, F, L Science N-terminal sequences of two polypeptides), FVMTDDNTFY (residues Center, Tensei-suisan Co., 1-25 Nakase-dori, Karatsu, Saga 847-0193, Japan; or Dr. 138–147), MIYDQADV (residues 246–253), AFLSVLDVP (residues Shin Yonehara, Institute for Virus Research, Kyoto University, Shogoin, Sakyo-ku, 272–280), and SLLFLGASDE (residues 408–417). Single-letter abbrevi- Kyoto 606-8507, Japan. E-mail address: [email protected] or syonehar@ ations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; virus.kyoto-u.ac.jp E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, 3 Abbreviations used in this paper: AIP, apoptosis-inducing protein; BiP, Ig heavy Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. X, chain binding protein; ER, endoplasmic reticulum; Fig 1, IL-4-induced mouse B cell Indicates unidentified amino acid. Two degenerate primers corresponding gene; LAO, L-amino acid oxidase; OPD, o-phenylenediamine dihydrochloride. to the peptide sequences EHLADCLEDKDYDTLLQTLD and MIY

DQADV were designed: GA(A/G)GA(C/T)AA(A/G)GA(C/T)TA(C/ with the equal volume of2NH2SO4, and the amount of H2O2 production T)GA(C/T)AC and TC(A/C/G/T)GC(C/T)TG(A/G)TC(A/G)TA(A/G)TA was determined by comparison with a standard curve that had been gen-

CAT, and used as primers for RT-PCR. Total RNA was prepared from the erated using fresh dilutions of stock H2O2. dissected capsule of larva-infected C. mackerel by homogenization in TRIZOL (Life Technologies, Grand Island, NY) and used to synthesize the Plasmid construction and transfections first-strand cDNA with Superscript reverse transcriptase (Life Technolo- gies). Poly(A) RNA was isolated from total RNA by using oligo(dT) Full-length AIP cDNA was introduced into a mammalian expression plas- mRNA purification kit (Pharmacia) and used to construct the capsule mid pEF-BOS (2) (pEF-AIP). To exchange signal sequence of AIP for cDNA library (SuperScript plasmid system for cDNA synthesis and plas- mammalian signal sequence, the region coding aa 31–524 and stop codon mid cloning; Life Technologies). RT-PCR conditions used were 94°C for was introduced into a SfiI site of pSecTag2 vector (Invitrogen) (pSec-AIP). 1 min, 56°C for 1 min, and 72°C for 2 min (35 cycles). A 644-bp PCR pEF-AIP and pSec-AIP were transfected into Cos-7 cells using a Bio-Rad product was obtained and subsequently sequenced after subcloning into the (Richmond, CA) Gene Pulser apparatus and into NIH3T3 cells by the pCR vector using the TA cloning kit (Invitrogen, San Diego, CA). A cap- Lipofectamine-Plus method (Life Technologies), respectively. sule cDNA library was screened with this 645-bp PCR product, and the largest insert was sequenced on both strands. Results and Discussion AIP purification Monoclonal Ab We have attempted to search for the source of factors involved in Splenocytes from BALB/c female mouse immunized with purified AIP were fused with mouse myeloma FOX-NY, and the resultant hybridomas controlling cell growth by screening water-soluble materials of were screened by ELISA and immunoblotting of the partially purified AIP. marine fishes and plants, then found that visceral extracts of C. Positive supernatants were then tested for specificity by determining mackerel have a powerful and dose-dependent cytotoxic effect on whether they contained Abs capable of immunodepleting apoptosis-induc- a variety of mammalian tumor cells. The activity in visceral ex- Downloaded from ing activity in visceral extracts of C. mackerel. Three monoclonal clones tracts of C. mackerel was likely to be mediated by protein com- that produce Abs termed I38A, I32D, and I310H were subsequently ob- Ͼ tained. These Abs recognized only 62- and 64-kDa polypeptides in visceral ponents, as suggested by its molecular size ( 50 kDa) and its extracts of infected fish, and visceral extracts immunodepleted with these sensitivity to trypsin and heat (data not shown; see Material and mAbs completely lost apoptosis-inducing activity. The isotypes of these Methods). Several chromatographic steps were used to purify this were all IgG1 ␬. factor, as detailed in Materials and Methods. This procedure pro- vided an 8000-fold increase in sp. act. After the final gel-filtration Apoptosis assays http://www.jimmunol.org/ step, SDS-PAGE followed by silver staining revealed two protein For cytotoxicity assay, human promyelocytic leukemia cells HL-60 were bands of 62 and 64 kDa (Fig. 1A). The two polypeptides had the 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. same N-terminal sequence (see the cDNA cloning in Materials Cell viability was measured by MTS assay (Promega, Madison, WI). The and Methods), suggesting that it is truncated or modified form of absorbance of MTS formazan was measured at an OD of 490 nm using an one another. Cytotoxic activity was eluted from gel-filtration col- automated microplate reader. The percentage of viable cells was calculated umn at about 135 kDa (data not shown), indicating that both pro- as follows: ((experimental OD value Ϫ spontaneous OD value)/(maximum OD value Ϫ spontaneous OD value)) ϫ 100. The spontaneous OD value teins existed as dimer in solution. was determined by adding SDS to a final concentration of 1%, whereas the maximum OD value was determined by incubating the cells with medium AIP possesses strong apoptosis-inducing activity by guest on September 23, 2021 alone. To verify that the polypeptide was indeed apoptosis-inducing fac- For DNA fragmentation assay, cells were centrifuged for 4 min, 400 ϫ g at the end of each incubation period. The pellet was resuspended in lysis tor, three mAbs were generated against purified apoptosis-inducing buffer (10 mM Tris, pH 8, 400 mM NaCl, 2 mM EDTA, 0.25% SDS, 0.2 factor and used to immunodeplete apoptosis-inducing activity in C. mg/ml proteinase K) and digested for2hat56°C. After digestion was mackerel extracts. These Abs recognized only 62- and 64-kDa complete, 1/4 vol of saturated NaCl was added and centrifuged at 12,000 protein bands on immunoblot of C. mackerel visceral extract and rpm for 10 min. The supernatant containing the DNA was treated with 0.2 purified apoptosis-inducing sample (Fig. 1B). Immunodepletion of mg/ml RNase A for2hat37°C, and DNA was precipitated with ethanol. Electrophoresis was carried out on 2% agarose gel containing 0.5 ␮g/ml C. mackerel viscera extracts with these mAbs completely removed ethidium bromide. apoptosis-inducing activity (see mAb in Materials and Methods). For DNA content analysis, the cells were harvested at various times These results showed that the 62- and 64-kDa proteins were re- after treatment with AIP, fixed with 70% ethanol, and then incubated with sponsible for the apoptosis-inducing activity. We designated these PBS containing 50 ␮g/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 proteins AIP. AIP was found to possess strong apoptosis activity. ␮g/ml propidium iodide. Cytolytic activity was examined using the human leukemia cell For microscopic analysis, cells were fixed with 1% glutaraldehyde, HL-60. The median cytolytic dose was observed at 5 ng/ml AIP stained with 10 ␮g/ml Hoechst 33258. The specimens were analyzed under (Fig. 1C), and cells were completely killed by incubation for 24 h phase contrast and fluorescent light using a Zeiss Axiovert microscope. in the presence of 20 ng/ml AIP (Fig. 1D). Oligonucleosome- Calcium perturbation length DNA fragments, which are characteristic of apoptosis, were observed within2hinHL-60 cells treated with 20 ng/ml AIP (Fig. A23187 were administered to NIH3T3 cells in fresh serum-free medium with insulin, transferrin, and selenium. At the end of the incubation, sam- 2A). Nuclear staining (Fig. 2B) and flow cytometry analysis (Fig. ples of the culture medium were concentrated by ultrafiltration with a 50- 2C) also confirmed other typical apoptotic features. kDa molecular mass cutoff membrane and retained for apoptosis assay and immunoblot analysis. Cells were washed with PBS, disrupted by three Induction of AIP strikingly depends on infection of the C. cycles of freezing-thawing, and centrifuged at 15,000 rpm. The supernatant mackerel with the larval nematode was used for immunoblot analysis Next, we examined tissue expression of AIP to select an organ Hydrogen peroxide measurement suitable for the isolation of the gene encoding AIP. Unexpectedly,

The release of H2O2 under AIP catalysis was evidenced by peroxidase/o- neither AIP nor apoptosis-inducing activity was detected in any of phenylenediamine dihydrochloride (OPD) method. In the presence of H2O2 the tissues examined (data not shown), suggesting that the expres- and peroxidase, OPD is oxidized and forms a completely soluble end prod- sion of AIP is conditionally regulated in fish. We found that the uct with maximum absorbance at 490 nm after the reaction is stopped with induction of AIP depends on the infection by the larval nematode, 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 A. simplex. Apoptosis-inducing activity and AIP could be detected peroxidase, and 500 ␮g/ml OPD for2hat37°C. The reaction was stopped in visceral extracts from infected fish (five samples), but not in The Journal of Immunology 1493 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 1. Analyses of the apoptosis-inducing factors by SDS-PAGE, Western blot analysis, and an apoptosis assay using HL-60 cells. A partially purified fraction, prepared from the lyophilized visceral extracts of larva-infected C. mackerel, was subjected to sequential chromatography on FIGURE 2. Induction of apoptosis by AIP. HL-60 cells were incubated Con A-Sepharose (lane 1), Mono Q (lane 2), and Superdex 200 HR 10/30 with 20 ng/ml of AIP for the indicated times (h). A, Total cellular DNA was gel-filtration column (lane 3). Active fractions were resolved by SDS- prepared from cells and electrophoresed on a 2% agarose gel. M, 123-bp PAGE, and subjected to either silver staining (A) or immunoblot analysis DNA ladder markers. B, Cells were fixed with 1% glutaraldehyde, stained (B) using anti-AIP mAbs, which were proficient for immunodepleting ap- with Hoechst 33258, and observed under a phase contrast and a fluorescent optosis-inducing activity from C. mackerel visceral extracts. Human microscope. C, Cells were fixed in 70% ethanol, treated with RNase A, HL-60 cells were treated with C, various concentrations of purified AIP for stained with propidium iodide, and subjected to a flow cytometric analysis. 12 h, or with D, 20 ng/ml of purified AIP for the times indicated. The percentage of viable cells was determined by MTS assay.

645-bp product (residues 38–252) (Fig. 4A) from the total RNA of capsules containing larva, in which AIP transcripts were mainly those from uninfected fish (five samples) (Fig. 3A–D). Further- detected. This PCR product was then used to isolate the full-length more, AIP was detected in extracts from tissues infected with lar- 2025-bp cDNA from a cDNA library constructed from the mRNA vae (data not shown), particularly in capsules that surrounded the of capsules containing larva. The cDNA contained an open reading larvae on the surface of the tissue (see below). 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 se- AIP is a novel reticuloplasmin with potent apoptosis-inducing quences were found within the open reading frame. SignalP and activity through its H2O2-producing function PSORT analyses indicated the presence of a signal peptide se- The N terminus and four V8 protease-digested peptides of the quence, but not one that would be cleavable in mammals. How- purified AIP were sequenced. On the basis of the sequences (see ever, N terminus of purified, mature AIP started at position 31 cDNA cloning in Materials and Methods), degenerate PCR prim- amino acid (Glu), indicating that the signal peptide is cleaved out ers were designed, and RT-PCR was used to amplify a single in fish. Five potential N-glycosylation sites were observed in the 1494 AIP, A NOVEL INFECTION-DEPENDENT APOPTOSIS-INDUCING PROTEIN

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 (9–11). 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 man- ner 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 apopto-

sis, although it does possess H2O2-producing activity (data not Downloaded from 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 demon- strated (6, 7). LAO is also not an ER rumen protein and does not FIGURE 3. Induction of AIP depends on infection with a larval nem- induce apoptosis in HL-60 cells at concentrations lower than 2.5 atode, A. simplex. Visceral extracts from five fishes infected with larval ␮g/ml even by 24-h treatment (6), while treatment with 20 ng/ml http://www.jimmunol.org/ nematodes (I1 to I5) and five uninfected fishes (U1 to U5) were concen- of AIP induced apoptosis in HL-60 cells within 2 h (Fig. 2). In trated by ultrafiltration with a 100-kDa molecular mass cutoff membrane these respects, snake venom LAO appears to be more similar to and subjected to assays for cytolytic activity or immunoblot analysis. A, Fig 1 protein than to AIP. HL-60 cells were incubated for 12 h with each visceral extract at the same protein concentration with the same dilution series in a 96-well microplate. The relative activities presented were calculated by comparing each me- AIP predominantly localizes in the inner cavity of capsule dian cytolytic dose with that of I5 as diluted fold. Median cytolytic dose of surrounding the larvae in vivo and is efficiently secreted into the I5 was taken to be equal to 1. Visceral extracts from uninfected fishes showed no apoptosis-inducing activity at the maximal concentration used medium as a functional protein with apoptosis-inducing activity

(1 mg/ml); their relative activities were indicated as 0. The percentage of in vitro through calcium perturbation by guest on September 23, 2021 viable cells was determined by MTS assay. B, A total of 200 ␮g of each Larval nematodes are found in the abdominal cavity on the surface extract was resolved by SDS-PAGE, and subjected to immunoblot analysis of visceral organs or in the peritoneal cavity, as well as in the ␮ using anti-AIP mAbs. C, A total of 500 g of each extract was immuno- intestine of infected C. mackerel. The host forms a capsule around precipitated with anti-AIP mAb and immunoblotted with anti-AIP. D, One the larvae to prevent their migration from the abdominal region milligram of each extract was incubated with Con A-Sepharose, and the bound materials were eluted with ␣-methylmannoside, resolved by SDS- into various viscera. Apoptosis-inducing activity or AIP was 400 PAGE, and immunoblotted with anti-AIP. 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 amino acid sequence of AIP, which may explain the difference B). As revealed by Southern blot analysis, AIP is a ﬁsh-derived

between the Mr determined by SDS-PAGE and that predicted from protein (Fig. 5C). Therefore, AIP is produced by larva-infected fish the amino acid sequence. and appears to be concentrated in the capsule surrounding the lar- AIP contains the typical ␤␣␤ dinucleotide-binding fold com- vae. Interestingly, cytolytic activity was not found in viscera ex- monly found in flavin adenine dinucleotide- and NADPH-binding tracts from sardines, even when infected with larvae (data not proteins (3, 4). Indeed, purified AIP showed an absorption spec- shown). In sardines, localization of Anisakis larvae is restricted to trum resembling that of flavoprotein (data not shown). This ob- the muscular and glandular parts of the esophagus and intestine or servation strongly suggests that binding of flavin might be required intestinal wall, and larvae have never been found on visceral or- for AIP function. Interestingly, the carboxyl-terminal region of gans or in the abdominal cavity of these fish, suggesting that the AIP was found to possess a KDEL sequence, which has been pos- early third stage larvae have not the ability to penetrate through the tulated to be both necessary and sufficient for proteins to be re- digestive tract of fishes. These results implied that AIP induction tained in the lumen of ER. Indeed, AIP was predominantly de- is associated with the capacity of host fish to encapsulate larvae tected in the ER of murine fibroblast cells transfected with the AIP after they have penetrated the abdominal cavity. gene (data not shown). AIP was dominantly found in the fluid rather than in the cells of The amino acid sequence of AIP displays 41% overall identity the capsule, as revealed by immunoblotting and the apoptosis as- to those of two flavoproteins, the predicted IL-4-induced mouse B say using HL-60 cells (Fig. 5, A and B), suggesting that AIP is cell gene (Fig 1) protein (5) of unknown function and snake venom secreted from the ER. Proteins that contain the C-terminal ER L-amino acid oxidase (LAO) (6, 7) (Fig. 4B). We therefore inves- retention signal (KDEL) are found in the ER lumen and are called

tigated whether AIP could catalyze H2O2 production. AIP puriﬁed reticuloplasmins. The major reticuloplasmins have been proposed from larva-infected ﬁsh or expressed in COS-7 cells transfected to function as molecular chaperones during protein assembly and with the AIP gene oxidized L-amino acids, especially L-lysine (Fig. degradation and for calcium storage (12–14). These proteins are

4C). Then, we examined the effect of scavengers of H2O2 on the transferred into the ER with the help of their signal peptide and are The Journal of Immunology 1495 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 4. Molecular characterization of AIP. A, Predicted amino acid sequence of AIP. The nucleotide sequence of AIP cDNA contains an open reading frame that would code for a protein of 524 aa with the Kozak consensus at the initiation codon. Regions with similarity to previously defined functional domains are boxed. Sequences corresponding to those obtained from N terminus and V8 protease-digested peptides are underlined. Potential N-glycosylation sequences are indicated in boldface. The sequence reported in this study has been deposited in the European Molecular Biology Laboratory (EMBL) database under accession number AJ400871. B, Multiple alignment of the predicted AIP protein with svLAO and Fig 1 protein. Sequences were aligned by Clustal method. Identical and similar residues are shaded in black and light, respectively. The flavin adenine dinucleotide-binding motif is boxed. svLAO, snake venom LAO (7); Fig 1, the reduced amino acid sequence of Fig 1 (5). C, AIP is a novel lysyl oxidase. Hydrogen peroxide productions were measured by peroxidase/OPD method, as described in Materials and Methods. Fish AIP, purified from larva-infected fish; Cos-7 AIP, expressed in COS-7 cells transfected with pEF-AIP; Hypro, hydroxyproline; Orn, ornithine; Spe, spermine; Put, putrescine; 5-HT, 5-hyroxytryptamine; Hista, histamine. D, ϩ ϩ AIP-induced apoptosis is mediated by H2O2. HL-60 cells were treated by 20 ng/ml AIP for 12 h with (AIP catalase) or without (AIP ) 1000 U/ml catalase. retained there by continuous retrieval from a post-ER compart- cells was found to induce membrane blebbing, cell rounding, and ment, which relies on a pH-dependent interaction of the retention cell detachment from the plates, which are characteristic features signal with a specific receptor (15, 16). Some proteins with the of apoptosis (data not shown). These observations indicated that KDEL retention signal were reported to be exported from the ER overexpression of AIP within cells does not result in apoptosis. to plasma membranes or extracellular medium under normal con- However, AIP was efficiently secreted into the medium as a func- ditions or by an activation-dependent mechanism (17–19). To in- tional protein with apoptosis-inducing activity when the trans- vestigate whether AIP can be secreted as a functional molecule, we fected murine fibroblast cells were treated with the calcium iono- constructed pSecAIP, which contained the murine Ig ␬-chain phore A23187 (Fig. 5, E and F). We also found a progressive leader sequence in place of the signal sequence of AIP, to fully increase in the amount of endogenous Ig heavy chain binding pro- function for translocation into ER of mammalian cells. NIH3T3 tein (BiP, also known as Grp78) in the medium of the same iono- cells were cotransfected with the pSec-AIP and Escherichia coli phore-treated cells (Fig. 5F). Such treatments with calcium iono- lacZ gene, and stained for ␤-galactosidase. Transfected cells were phores have been shown to cause secretion of reticuloplasmins, identified by their blue color, and apoptotic cells were determined including BiP, endoplasmin, protein disulfide isomerase, and cal- microscopically. The typical morphological properties of apoptotic reticulin, suggesting a role for calcium ions in the retention system cells were not observed even more than 48 h after transfection (17). Taken together, these results suggest that AIP may be se- (Fig. 5D), while 12-h AIP treatment (20 ng/ml) of control NIH3T3 creted by a similar mechanism into the capsule cavity from, as yet, 1496 AIP, A NOVEL INFECTION-DEPENDENT APOPTOSIS-INDUCING PROTEIN Downloaded from

FIGURE 5. AIP is localized to capsules surrounding the larvae and is secreted from the capsular cells of larva-infected fish. A, Cytolytic analysis of AIP http://www.jimmunol.org/ localization in the capsules. Capsules with Anisakis were washed, decapsulated in PBS, and separated into the fractions of cells and fluid after removing larvae. Cells in the capsules and the associated Anisakis larvae were washed and lysed, and soluble fractions were collected for apoptosis assay. HL-60 cells were treated with the same concentration of each sample for 12 h. The relative activities were calculated as diluted fold by comparing each median cytolytic dose with that of I1, which was taken to be equal to 1, as detailed in Fig. 3A. B, Immunoblot analysis of AIP localization in capsules. Cells, fluid, and Anisakis larvae in capsules were separated as described above, resolved by SDS-PAGE, and immunoblotted with anti-AIP. I1, 200 ␮g per lane; larva, 50 ␮g per lane; cells, 4 ␮g per lane; fluid, 4 ␮g per lane. C, AIP is derived from fish. Genomic DNA isolated from the brain of fish and from Anisakis larvae were digested with EcoRI, resolved on a 1% agarose gel, transferred to nylon membrane, and hybridized with a 1068-bp BamHI-SacI fragment of AIP cDNA (right panel). Numbers indicate ␮g genomic DNA per lane. Left panel, Shows ethidium bromide staining of the same gel. D, Overexpression of AIP in cells does not result in apoptosis. NIH3T3 cells were cotransfected with CMV ␤-galactosidase and pEF-empty, pEF-AIP, or pSec-AIP with lipofectin. by guest on September 23, 2021 Fifty hours after transfection, cells were fixed and stained with 5-bromo-4-chloro-3-indolyl ␤-D-galactoside, and the percent viability of the blue cells that appeared flat and without any sign of apoptosis was determined. E and F, Exogenous AIP is secreted as a functional protein from NIH3T3 cells treated with a calcium ionophore. NIH3T3 cells were transfected with pSec-empty or pSec-AIP. Thirty hours after transfection, the cells were treated for the indicated times (h) with or without 2 ␮M A23187. E, Samples of culture medium (CM) were assayed for cytolytic activity using HL-60 cells. Also, purified AIP (10 ng/ml) was used as a positive control. F, Whole cell lysates (cell) and CM were analyzed by SDS-PAGE and immunoblot analysis with anti-AIP (upper panel) and anti-BiP (lower panel) (Santa Cruz Biotechnology, Santa Cruz, CA). unidentified capsular cells, and that the secreted AIP confers re- References sistance to larval nematodes. The ingestion of larval nematode, A. simplex, was reported to 1. Thompson, C. B. 1995. Apoptosis in the pathogenesis and treatment of disease. Science 267:1456. cause a parasitic disease known as anisakiasis in humans (20, 21). 2. Mizushima, S., and S. Nagata. 1990. pEF-BOS, a powerful mammalian expres- Several species of larval nematodes including Anisakis larvae un- sion vector. Nucleic Acids Res. 18:5322. dergo extensive migration within their intermediate hosts, and the 3. Wierenga, R. K., P. Terpstra, and W. G. J. Hol. 1986. Prediction of the occurrence of the ADP-binding ␤␣␤-fold in proteins, using an amino acid sequence finger- migrated visceral larvae cause a severe inflammatory disease char- print. J. Mol. Biol. 187:101. acterized by hepatomegaly, eosinophilia, and hypergammaglobu- 4. Dailey, T. A., and H. A. Dailey. 1998. Identification of an FAD superfamily linemia (22–24). Encapsulation of the larvae by the host certainly containing protoporphyrinogen oxidases, monoamine oxidases, and phytoene de- saturase. J. Biol. Chem. 273:13658. prevents their migration and growth, and may function as a major 5. Chu, C. C., and W. E. Paul. 1997. Fig 1, an interleukin 4-induced mouse B cell defense system against larval infection. We purified infection-spe- gene isolated by cDNA representational difference analysis. Proc. Natl. Acad. cific AIP and cloned the corresponding gene. AIP induction in fish Sci. USA 94:2507. is the result of an interaction between parasite and host, and is 6. Torii, S., M. Naito, and T. Tsuruo. 1997. Apoxin I, a novel apoptosis-inducing factor with L-amino acid oxidase activity purified from Western diamondback mainly restricted into the capsules surrounding larval nematode. rattlesnake venom. J. Biol. Chem. 272:9539. Recently, it has been reported that catalase is needed to extend 7. Raibekas, A. A., and V. Massey. 1998. Primary structure of the snake venom L-amino acid oxidase shows high homology with the mouse B cell interleukin lifespan in Caenorhabditis elegans by protecting nematodes from 4-induced Fig 1 protein. Biochem. Biophys. Res. Commun. 248:476. oxidative damages (25). The formation of capsules by the host may 8. Sher, A., and R. L. Coffman. 1992. Regulation of immunity to parasites by T cells serve to confine larvae to an area in which AIP expression can and T cell-derived cytokines. Annu. Rev. Immunol. 10:385. strongly suppress their vitality and ability to invade host tissues. In 9. Urban, J. F., I. M. Katona, W. E. Paul, and F. D. Finkelman. 1991. Interleukin 4 is important in protective immunity to a gastrointestinal nematode infection in addition, because AIP may play a crucial role in determining re- mice. Proc. Natl. Acad. Sci. USA 88:5513. sistance to nematode infection, we hope that this study will lead to 10. Else, K. J., F. D. Finkelman, C. R. Maliszewski, and R. K. Grencis. 1994. Cy- a better understanding of host defense system against larval nem- tokine mediated regulation of chronic intestinal helminth infection. J. Exp. Med. 179:347. atodes and provide a conceptual basis for the development of novel 11. Finkelman, F. D., T. Shea-Donohue, J. Goldhill, C. A. Sullivan, S. C. Morris, and more efficient means for combating this type of infection. K. B. Madden, W. C. Gause, and J. F. Urban, Jr. 1997. Cytokine regulation of The Journal of Immunology 1497

host defense against parasitic gastrointestinal helminths: lessons from studies 19. Terada, K., P. Manchikalapudi, R. Noiva, H. O. Jauregui, R. J. Stockert, and with rodent models. Annu. Rev. Immunol. 15:505. M. L. Schilsky. 1995. Secretion, surface localization, turnover, and steady state 12. Gething, M. J., and J. Sambrook. 1992. Protein folding in the cell. Nature 355:33. expression of protein disulfide isomerase in rat hepatocytes. J. Biol. Chem. 270: 13. Nigam, S. K., A. L. Goldgerg, S. Ho, M. F. Rohde, K. T. Bush, and 20410. M. Y. Sherman. 1994. A set of endoplasmic reticulum proteins possessing prop- 2ϩ 20. Deardorff, T. L., T. Fukumura, and R. B. Raybourne. 1986. Invasive anisakiasis: erties of molecular chaperones includes Ca -binding proteins and members of a case report from Hawaii. Gastroenterology 90:1047. the thioredoxin superfamily. J. Biol. Chem. 269:1744. 14. Hubbard, M. J. 1996. Abundant calcium homeostasis machinery in rat dental 21. Bouree, P., A. Paugam, and J. C. Petithory. 1995. Anisakidosis: report of 25 cases enamel cells: up-regulation of calcium store proteins during enamel mineraliza- and review of the literature. Comp. Immunol. Microbiol. Infect. Dis. 18:75. tion implicates the endoplasmic reticulum in calcium transcytosis. Eur. J. Bio- 22. Fox, A. S., K. R. Kazacos, N. S. Gould, P. T. Heydemann, C. Thomas, and chem. 239:611. K. M. Boyer. 1985. Fetal eosinophilic meningoencephalitis and visceral larva 15. Wilson, D. W., M. J. Lewis, and H. R. B. Pelham. 1993. pH-dependent binding migrans caused by the raccoon ascarid Baylisascaris procyonis. N. Engl. J. Med. of KDEL to its receptor in vitro. J. Biol. Chem. 268:7465. 312:1619. 16. Scheel, A. A., and H. R. B. Pelham. 1996. Purification and characterization of the 23. Alonso, A., A. Daschner, and A. Moreno-Ancillo. 1997. Anaphylaxis with Ani- human KDEL receptor. Biochemistry 35:10203. sakis simplex in the gastric mucosa. N. Engl. J. Med. 337:350. 17. Booth, C., and G. L. E. Koch. 1989. Perturbation of cellular calcium induces secretion of luminal ER proteins. Cell 59:729. 24. Kayes, S. G. 1997. Human toxocariasis and the visceral larva migrans syndrome: 18. Yoshimori, T., T. Semba, H. Takemoto, S. Akagi, A. Yamamoto, and Y. Tashiro. correlative immunopathology. Chem. Immunol. 66:99. 1990. Protein disulfide-isomerase in rat exocrine pancreatic cells is exported from 25. Taub, J., J. F. Lau, C. Ma, J. H. Hahn, R. Hoque, J. Rothblatt, and M. Chalfie. the endoplasmic reticulum despite possessing the retention signal. J. Biol. Chem. 1999. A cytosolic catalase is needed to extend adult lifespan in C. elegans daf-C 265:15984. and clk-1 mutants. Nature 399:162. Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021