Bolevenine, a toxic protein from the Japanese toadstool Boletus venenatus
Graphical abstract
Bolevenine, a toxic protein, was isolated from the Boletaceae mushroom and its Mr, pI, and N-terminal sequence were characterized.
Introduction
Most of the Boletaceae mushrooms have long been thought to be edible; however, recently, a few species, such as Boletus satanas (distributed in Europe and America), Xanthoconium affine (synonym, Boletus affinis; Japanese name, utsuroi-iguchi; distributed in Japan, North America, and Madagascar), and Tylopilus sp. (Japanese name, mikawakuroamiashi-iguchi), were found to be exceptions. Of these, B. satanas induces gastroenteritis in humans and its responsible proteinaceous toxin, bolesatine, has been isolated by a French group (Kretz et al., 1989). X. affine can also result in death of cows after ingestion, and the responsible toxin has been found to be the protein bolaffinine (Razanamparany et al., 1986). The Tylopilus sp. had also been toadstool of some health concern, and recently a Japanese group demonstrated that the responsible toxin for acute toxicity on mice was the 2-butyl-1-azacyclohexene iminium salt (Watanabe et al., 2002).
In Japan, in addition to the X. affine and Tylopilus sp., Boletus venenatus (Japanese name, dokuyamadori or tahei-iguchi) is known to be toxic. Ingestion of the mushroom causes a severe gastrointestinal syndrome, such as nausea, repetitive vomiting, diarrhea, and stomachache. Severe poisoning brings about dehydration; fortunately, most of the patients, however, usually recover in a few days. Regarding B. venenatus, there has been confusion between similar species, but recent taxonomic studies have made it possible to identify this species (Nagasawa, 1995). This situation prompted us to attempt to identify the toxic component of the Japanese Boletaceae mushroom. Guided by its lethal effect on mice, we isolated a toxic protein and have called it bolevenine.
Section snippets
Purification
The lethal effect on mice was used as the toxicity index for the isolation of the toxic component. Fruiting bodies were extracted with water and MeOH, and each extract was injected into mice. Only the aqueous extract exhibited toxicity. Next, the aqueous extract underwent ultrafiltration, and the resulting filtrate and retentate were tested for toxicity. Only the retentate showed toxic activity, which after heating at 70 °C for 20 min, disappeared. The toxicity of the retentate under various pH
General
All separation procedures were carried out at 4 °C. Each fraction was monitored by UV spectra (U-2001, Hitachi) at 280 nm otherwise stated.
Materials
The fruiting bodies of Boletus venenatus were collected during 2002–2003 in the Nagano and Gifu Prefectures, Japan, and stored at −30 °C until use.
Purification
Fruiting bodies of B. venenatus (250 g) were cut into pieces, soaked in water (500 ml), and extracted overnight. The mixture was filtered through filter paper (No. 5A, Kiriyama) under suction and the filtrate was
Acknowledgements
We are grateful to Mr. Takashi Suda and Mr. Toshiro Masai for collecting the mushrooms. We thank Dr. Shuichi Matsumura (Keio University) for technical assistance with SDS–PAGE. We are indebted to Mr. Takashi Hamada (Kyoto Pharmaceutical University) and Mr. Jyunji Ichita (Aomori Advanced Industrial Technology Center) for MALDI-TOFMS measurements. This research was partially supported by a Grant-in-Aid from the 21st Century COE program “KEIO Life Conjugate Chemistry” (M.M., Y.S.), Frontier
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