Duck-Billed Platypus Venom Peptides Induce Ca²⁺ Influx in Neuroblastoma Cells

Abstract

The duck-billed platypus (Ornithorhynchus anatinus) is one of the few venomous Australian mammals. We previously found that its crude venom potently induces Ca(2+) influx in human neuroblastoma IMR-32 cells. Guided by this bioassay, we identified 11 novel peptides, including the heptapeptide H-His-Asp-His-Pro-Asn-Pro-Arg-OH (1). Compounds 1-4 and 5-11 coincided with the 6-9 N-terminal residues of Ornithorhynchus venom C-type natriuretic peptide (OvCNP) and the 132-150 part of OvCNP precursor peptide, respectively. Heptapeptide 1, which is one of the primary components of the venom fluid (approximately 200 ng/microL), induced a significant increase in [Ca(2+)](i) in IMR-32 cells at 75 microM. To the best of our knowledge, this is the first example of the isolation of the N-terminal linear fragments of CNPs in any mammal.

Venom evolution through gene duplications

“…Strikingly, some toxin peptides show a successive pattern of posttranslational proteolysis, termed 'rosary-like', in reference to the cleaving of extensive repetitive domain units from a precursor mRNA. Toxins derive from the proteolysis of multiple tandem domain genes have been identified in viper sarafotoxins, kininogens from the giant fire-bellied toad and in platypus, reptile (beaded lizards and gila monsters) and snake C-type natriuretic peptide precursors (Ducancel et al, 1993;Fry et al, 2010aFry et al, , 2010bHiguchi et al, 1999;Kita et al, 2009;Lai et al, 2001). Helofensins, comprising of four tandemly repeated beta-defensin domains are found in helodermatid lizards but do not undergo domain-specific posttranslational cleavage but rather act as a single functional unit to inhibit electrical stimuli (Fry et al, 2010a(Fry et al, , 2010bKomori et al, 1988).…”

Proteomics and Deep Sequencing Comparison of Seasonally Active Venom Glands in the Platypus Reveals Novel Venom Peptides and Distinct Expression Profiles

“…Five different types of molecules were identified: hyaluronidase, C-type natriuretic peptides, nerve growth factor (OvNGF), L-to-D-amino acidresidue isomerase, and defensin-like peptides. Only two of these components have been fully sequenced: natriuretic peptides and defensin-like peptides (10,14,15). These polypeptides and enzymes likely work together to cause swelling, lowered blood pressure, and pain.…”

“…These polypeptides and enzymes likely work together to cause swelling, lowered blood pressure, and pain. C-type natriuretic mRNA is posttranslationally cleaved to produce peptides that form cation channels in lipid bilayer membranes (16), relax smooth muscles, release mast cell histamine (10), and cause calcium influx into neuroblastoma cells (15). The function of venom defensin-like peptides is unclear; although they have a threedimensional structure similar to those of sea anemone sodium channel neurotoxins and antimicrobial peptides (defensins), they do not modulate sodium channel function or display antimicrobial activity (14,12).…”

Bioorganic Studies on the Key Natural Products from Venomous Mammals and Marine Invertebrates

“…26 Unlike the fast and temporary increase in [Ca 2+ ] i (internal calcium ion) caused by KCl, the calcium uptake caused by 1 was slow and continuous, which was identical to the result with crude venom. We used differentiated IMR-32 cells that expressed L-and N-type voltage-gated calcium channels (VGCCs) for the calcium uptake assay.…”

“…Through the use of MALDI-TOF/TOF MS analysis, the primary structure of 1 was established to be HHis AspHisProAsnProArgOH, which coincided with the N- terminal domain residues of platypus C-type natriuretic peptide. 26 CNPs are vasorelaxant peptide hormones that are widely expressed in vivo, including the central nervous system. 27 OvCNPs cause the relaxation of rat uterine smooth muscle, promote histamine release from mast cells, and form fast cation channels in lipid bilayers.…”