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Purification and Cloning of an Apoptosis-Inducing Protein Derived from Fish Infected with Anisakis simplex, a Causative Nematode of Human Anisakiasis¹

Sang-Kee Jung^2,*,, Angela Mai^*,, Mitsunori Iwamoto^*, Naoki Arizono, Daisaburo Fujimoto^§, Kazuhiro Sakamaki and Shin Yonehara^2,

^* 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

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 sequence) 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.

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