Blarina toxin, a mammalian lethal venom from the short-tailed shrew Blarina brevicauda: Isolation and characterization Masaki Kita*, Yasuo Nakamura†‡, Yuushi Okumura†, Satoshi D. Ohdachi§, Yuichi Oba¶, Michiyasu Yoshikuni ʈ, Hiroshi Kido†, and Daisuke Uemura**†† *Research Center for Materials Science, ¶Graduate School of Bioagricultural Sciences, and **Graduate School of Science, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8602, Japan; †Institute for Enzyme Research, University of Tokushima, Kuramoto-Cho 3-18-15, Tokushima 770-8503, Japan; §Institute of Low Temperature Science, Hokkaido University, Kita-19 Nishi-8, Kita-ku, Sapporo 060-0819, Japan; and ʈLaboratory of Reproductive Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki 444-8585, Japan Communicated by Satoshi Omura, Kitasato Institute for Life Sciences, Tokyo, Japan, April 8, 2004 (received for review December 24, 2003) Venomous mammals are rare, and their venoms have not been tion) are toxic. Therefore, it has been suggested that the most characterized. We have purified and characterized the blarina toxin toxic ingredient is a high molecular weight water-soluble protein, (BLTX), a lethal mammalian venom with a tissue kallikrein-like not a carbohydrate or nucleoprotein (1). However, because the activity from the submaxillary and sublingual glands of the short- Blarina venom loses its toxicity rapidly after the victim’s death, tailed shrew Blarina brevicauda. Mice administered BLTX i.p. de- its purification and more precise identification have not been veloped irregular respiration, paralysis, and convulsions before successfully completed. dying. Based on the amino acid sequence of purified protein, we In this study, we purify and characterize the B. brevicauda cloned the BLTX cDNA. It consists of a prosequence and an active venom, blarina toxin (BLTX). This venom exerted a kallikrein- form of 253 aa with a typical catalytic triad of serine proteases, like proteolytic activity similar to that of a lizard venom (16). with a high identity with tissue kallikreins. BLTX is an N-linked microheterogeneous glycoprotein with a unique insertion of 10 Materials and Methods residues, L106TFFYKTFLG115. BLTX converted kininogens to kinins, Chemicals. Peptidyl-4-methylcoumaryl-7-amide (MCA) sub- which may be one of the toxic pathogens, and had dilatory effects strates and kinins were obtained from Peptide Research Insti- on the blood vessel walls. The acute toxicity and proteolytic tute (Osaka). Human low molecular weight kininogen (LK), activity of BLTX were strongly inhibited by aprotinin, a kallikrein RNAlater, inhibitors for the enzyme assay and deglycosylation inhibitor, suggesting that its toxicity is due to a kallikrein-like reaction, porcine pancreatic kallikrein (PPK), and thromboxane activity of the venom. A2 analogue (U46619) were purchased from Sigma. Human high molecular weight kininogen (HK) was provided by Biogenesis enomous mammals are rare (1), and their venoms have not (Bournemouth, U.K.). Peptide-N4-(N-acetyl-␤-glucosami- Vbeen characterized. Toxic constituents produced by other nyl)asparagine amidase (N-glycosidase) (EC 3.5.1.52) and O- vertebrates, such as snake, lizard, and frog, have been well glycopeptide endo-D-galactosyl-N-acetyl-␣-galactosamino hy- studied. Several bird species (the genus Pitohui) are also known drolase (O-glycosidase) (EC 3.2.1.97) were supplied by Roche to contain steroidal alkaloid toxin as a chemical defense (2). Diagnostics. Plasma kallikrein specific inhibitor and other chem- However, only a few members of the order Insectivora produce icals were purchased from Wako Pure Chemical (Osaka). toxic compounds, including the Haitian solenodon (Solenodon paradoxus), the European water shrew (Neomys fodiens), the Harvesting and Handling of Tissue Specimens. Fifty B. brevicauda Mediterranean shrew (Neomys anomalous), and the American shrews (28 males and 22 females) were trapped with Sherman short-tailed shrew (Blarina brevicauda) (3–6). Mammals in other traps (SFA type; H. B. Sherman Traps, Tallahassee, FL) baited orders, such as Monotremata (platypuses and echidnas), are also with peanut butter and oats, within the Fresh Air Camp and suspected to have venom (7). Envenoming by a platypus causes the E. S. George Reserve, University of Michigan (Livingstone immediate excruciating pain that evolves toward prolonged Country, MI), between July 22 and July 26, 2002, and Sep- hyperalgesia. Defensin-like peptides (8) and the C-type natri- tember 9 and September 13, 2003. The animals were deposited uretic peptides (9) have been isolated from this venom. However, in the mammal collection of one of the authors (S.D.O.) at the precise mechanism of the excruciating pain caused by Hokkaido University. Tissue samples were excised immedi- platypus toxin in humans remains unclear. ately after the animals were trapped and were stored in Among mammals, the short-tailed shrew B. brevicauda (Say, acetone at Ϫ20°C as described (3). Tissues were also imme- 1923) is well known to produce a potent venom in its saliva, which diately stored in RNAlater for mRNA extraction, according to is toxic to mammals, such as mice, voles, rabbits, and cats (1, 3). the manufacturer’s instructions. All animals were treated in Human accounts of bites from Blarina describe a local burning sensation around the tooth puncture marks and subsequent swelling (10). In general, soricine shrews consume large amounts Abbreviations: BLTX, blarina toxin; MCA, 4-methyl-coumaryl-7-amide; LK, low molecular of food to meet their high metabolic demands (11). Although weight kininogen; HK, high molecular weight kininogen; PPK, porcine pancreatic kal- they belong to the order Insectivora, B. brevicauda do not eat likrein; Bis-Tris, [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane; BK, bradykinin; GTX, gila toxin. insects and invertebrates exclusively but also vertebrates, even Data deposition: The sequence reported in this paper has been deposited in the GenBank larger than themselves, such as murid rodents and frogs (1, database (accession no. AB111919). 11–13). Therefore, this shrew species may use its venom to ‡Present address: Department of Parasitology, Kurume University, Asahi-machi 67, Kurume paralyze and catch larger preys. 830-0011, Japan. The stability of crude extracts of Blarina submaxillary glands ††To whom correspondence should be addressed at: Department of Chemistry, Graduate has been studied (14, 15). Its toxicity was lost rapidly at pH Ͼ7 School of Science, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8602, Japan. E-mail: and gradually at room temperature. Products from precipitation [email protected]. of the crude extracts with ammonium sulfate (33–80% satura- © 2004 by The National Academy of Sciences of the USA 7542–7547 ͉ PNAS ͉ May 18, 2004 ͉ vol. 101 ͉ no. 20 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0402517101 Downloaded by guest on September 30, 2021 accordance with the ethical guidelines of the International 12.5% polyacrylamide gel, and the gels were stained with a Silver Association for the Study of Pain (17). Stain Kit, Protein. Purification of Venom. All procedures were performed at 4–6°C Acute Toxicity Assay. Lethality was tested by i.p. injection into and all separation systems and columns were purchased from male ddY and C57BL mice weighing between 9.9 and 10.1 g Amersham Biosciences. Fifteen submaxillary and sublingual (Japan SLC, Hamamatsu, Japan). The injectates were diluted up gland specimens were homogenized with 0.85% NaCl and to 0.5 ml with 0.85% NaCl. For the inhibition of acute toxicity centrifuged as described (3). The supernatant was loaded twice by protease inhibitors, crude extracts of Blarina salivary glands on a Hiroad 26͞60 Superdex 200pg column connected to an (0.2 individuals) or purified BLTX were incubated with various FPLC system and eluted with 0.85% NaCl at a flow rate of 1 inhibitors and EDTA for 30 min at 0°C before injection. ml⅐minϪ1, with monitoring of absorbance at 280 nm. The fraction with toxic activity was dialyzed against 20 mM [bis(2- Enzyme Assays. Enzyme activity using peptidyl-MCA substrates hydroxyethyl)amino]tris(hydroxymethyl)methane (Bis- was analyzed with a fluorescence spectrophotometer (model Tris)⅐HCl (pH 7.0), then applied to a Mono Q HR 5͞5 column 650–10 MS, Hitachi, Tokyo) by measuring the released amount connected to the same system as above, which had been previ- of 4-amino-7-methylcoumarin, with excitation and emission ously equilibrated with the same buffer. A linear gradient of wavelengths of 370 nm and 460 nm, respectively, at 37°C, as NaCl from 0.1 to 0.2 M was applied for 100 min at a flow rate described (18). For inhibition studies, BLTX was preincubated of 0.5 ml⅐minϪ1, with monitoring at 280 nm. After the active with various inhibitors for 5 min at 37°C before measurements fraction was diluted 5-fold with 20 mM Bis-Tris⅐HCl (pH 7.0), it of activity. was applied twice to a Mini Q PE 4.6͞50 column connected to The conversion of kininogens to small peptides was measured ⅐ Ϫ1 ⅐ a SMART system and equilibrated with 20 mM Bis-Tris⅐HCl by incubating 0.2 mg ml LK and HK in 50 mM Tris HCl buffer, (pH 7.0) containing 60 mM NaCl. After equilibrium was pH 9.0, with purified BLTX or PPK (14 ng) at 37°C, with a reaction mixture volume of 20 ␮l. At 0, 10, 30, and 60 min, 5 ␮l reached, a linear gradient of NaCl from 0.06 to 0.1 M was applied ␮ for 120 min at a flow rate of 0.3 ml⅐minϪ1, with monitoring at 280 of the reaction mixture was withdrawn and added to 5 lof denaturing solution, as described (16). The samples were boiled nm. The protein concentration was measured with a Bio-Rad ͞ protein assay kit with BSA as a standard. SDS͞PAGE of BLTX for 5 min, SDS PAGE was performed by using a precast 10% was performed by using a precast 10–20% polyacrylamide polyacrylamide gel, and the gels were stained with a Silver Stain Kit, Protein.