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 References This article cites 25 articles, 10 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/165/3/1491.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 23, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 1492 AIP, A NOVEL INFECTION-DEPENDENT APOPTOSIS-INDUCING PROTEIN 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).