Duck-Billed Platypus Venom Peptides Induce Ca 2+ Influx in Neuroblastoma Cells Masaki Kita,* ,† David StC. Black, ‡ Osamu Ohno, § Kaoru Yamada, § Hideo Kigoshi, † and Daisuke Uemura* ,§,| Graduate School of Pure and Applied Sciences, UniVersity of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571, Japan, School of Chemistry, The UniVersity of New South Wales, UNSW Sydney, NSW 2052, Australia, Department of Biosciences and Informatics, Keio UniVersity, Hiyoshi 3-14-1, Yokohama 223-8522, Japan, and Institute for AdVanced Research, Nagoya UniVersity, Furo-cho, Chikusa, Nagoya 464-8602, Japan Received September 24, 2009; E-mail: mkita@chem.tsukuba.ac.jp, uemura@bio.keio.ac.jp The duck-billed platypus, Ornithorhynchus anatinus, is one of the few venomous Australian mammals. 1 Envenoming by a male platypus causes immediate excruciating pain in humans, which evolves toward a long-lasting hyperalgesia. Several constituents of the venom fluid or its crural gland have been identified, including defensin-like peptides, 2 C-type natriuretic peptides (OvCNPs), 3 NGF, and hyaluronidase. OvCNP causes the relaxation of rat uterine smooth muscle, promotes histamine release from mast cells, and forms fast cation channels in lipid bilayers. 4 Recently, an L-to-D- peptide isomerase was identified by which D-Leu 2 in OvCNPs and D-Met 2 in defensins are formed. 5 Furthermore, the platypus genome project (completed in 2008) revealed that reptile and platypus defensin-like peptides have been co-opted independently from the same gene families. 6 Notably, only a few members of the most ancient mammals, such as shrews and a solenodon (InsectiVora) as well as a platypus (Monotremata), have been shown to produce toxic venom. 7 Thus, identification of the unique bioactive substances in these venoms should help to clarify their evolutionary properties and ecological roles. Recently, we reported that crude platypus venom potently induces Ca 2+ influx in human neuroblastoma IMR-32 cells. 8 Guided by this bioassay, we have identified 11 novel peptides 1-11 (Figure 1). Here we describe the purification and characterization of these peptides found in the venom. On the basis of the finding that platypus venom contains variously sized molecules, we first chose gel-permeation HPLC for purifica- tion (Figure 2). As expected, the low-molecular-weight components, especially fraction (fr.) IX, potently induced Ca 2+ influx, and most proteins were eluted in fr. II-V. 9 Reversed-phase HPLC (RP- HPLC) purification of fr. IX gave heptapeptide 1 as a primary component (∼200 ng/μL of venom fluid). With regard to fr. VI, five major peaks were detected between 30 and 80 min, from which hexapeptide 2, peptides 5-8, and peptides 9-11 were purified as major constituents. Furthermore, direct RP-HPLC purification of the crude venom afforded peptides 3 and 4. 10 Through the use of MALDI-TOF/TOF MS analysis, the primary structure of 1 was established to be H-His-Asp-His-Pro-Asn-Pro- Arg-OH. Similarly, 2-4 were found to be analogues of 1 that coincided with the 6-9 N-terminal residues of OvCNP. Meanwhile, peptides 5-9 coincided with the 132-150 part of OvCNP precursor peptide (preOvCNP), a translated sequence from the predicted mRNA in the platypus genome (GenomeNet Database, XM_001515107). Peptides 1-9 were all chemically synthesized, and their retention times were identical to those of the natural compounds. Moreover, the Leu 2 residues in peptides 3 and 4 were D-form, not L-form, as in OvCNP-39b. 10 The y-ion fragment patterns in the MS/MS data suggested that the 12 C-terminal residues in peptides 10 and 11 were identical to those in 9, but their molecular mass units were 17 less and 1 more than those of preOvCNP(132-150), respec- tively. As a result, we concluded that 10 had a cyclic amide structure at the Ala 1 -Asn 2 moiety by deamidation, 11 while the amide moiety of the Asn 2 residue was hydrolyzed to an aspartic acid residue in 11. Synthetic heptapeptide 1 induced a significant increase in [Ca 2+ ] i in IMR-32 cells at 75 μM (Figure 3). 12 Nonapeptide 4 also showed weak Ca 2+ influx at 500 μM, but hexapeptide 2 did not at the same † University of Tsukuba. ‡ The University of New South Wales. § Keio University. | Nagoya University. Figure 1. Sequence alignment of CNPs and the venom peptides 1-11. Conserved cysteine residues are highlighted in yellow. D-Amino acids are indicated in red. A schematic representation of the position of venom peptides in preOvCNP is shown at the lower right. Figure 2. Purification of the venom peptides. (a) Gel-permeation HPLC of the venom fluid (5 μL). The Ca 2+ influx effect (blue column) and peptidase activity (Pro-Phe-Arg-MCA, red 9) for each fraction are shown. (b, c) RP-HPLC traces obtained using a C 30 semimicro column in which (b) 2/5 amount of fr. IX and (c) 1/20 amount of fr. VI were loaded; 9 markers show purified peptides. Published on Web 11/24/2009 10.1021/ja908148z  2009 American Chemical Society 18038 9 J. AM. CHEM. SOC. 2009, 131, 18038–18039