Cloning and characterization of novel snake venom proteins that block smooth muscle contraction

Abstract

In this study, we isolated a 25-kDa novel snake venom protein, designated ablomin, from the venom of the Japanese Mamushi snake (Agkistrodon blomhoffi). The amino-acid sequence of this protein was determined by peptide sequencing and cDNA cloning. The deduced sequence showed high similarity to helothermine from the Mexican beaded lizard (Heloderma horridum horridum), which blocks voltage-gated calcium and potassium channels, and ryanodine receptors. Ablomin blocked contraction of rat tail arterial smooth muscle elicited by high K + -induced depolarization in the 0.1-1 lM range, but did not block caffeine-stimulated contraction. Furthermore, we isolated three other proteins from snake venoms that are homologous to ablomin and cloned the corresponding cDNAs. Two of these homologous proteins, triflin and latisemin, also inhibited high K + -induced contraction of the artery. These results indicate that several snake venoms contain novel proteins with neurotoxin-like activity.Keywords: snake venom; neurotoxin; helothermine; cysteinerich secretory proteins; ablomin.Over the past 30 years, a plethora of toxins have been isolated from poisonous organisms, such as snakes, scorpions, spiders, and micro-organisms. These natural toxins use a variety of approaches to arrest the homeostatic mechanisms of other living organisms, including disruption of intracellular signal transduction and cytoskeleton organization [1][2][3][4], and activation or inhibition of blood coagulation factors [5][6][7][8][9][10]. Toxins that block synaptic transmission, called neurotoxins, are widely distributed in venoms. These toxins include the conotoxins from cone snails, agatoxins from spiders, and scorpion toxins [11][12][13][14][15][16]. These toxins exert their potentially lethal effects by specifically and potently blocking a variety of ion channels, including those that conduct Na + , K + , and Ca 2+ . Therefore, neurotoxins have been employed as useful tools to investigate the structure and function of these ion channels [17][18][19][20]. A large number of neurotoxin families have also been found in the venom of Elapidae snakes. These toxins, the a-neurotoxins [21] (represented by a-bungarotoxin [22,23], a-cobratoxin [24][25][26][27], and erabutoxin [28,29]) potently and specifically prevent nicotinic acetylcholine receptor activation. A second family of snake venom neurotoxins, the dendrotoxins, are homologous to Kunitz-type serine protease inhibitors and act primarily by blocking neuronal K + channels [30,31]. In contrast to the neurotoxin-rich venom from Elapidae snakes, the venom from other deadly snakes, including Viperidae and Colubridae snakes, contain surprisingly few neurotoxins, although some neurotoxic phospholipases have been discovered [32][33][34][35][36].In this report, we describe the isolation of a novel protein, ablomin, from the venom of the Japanese Mamushi snake (Agkistrodon blomhoffi, a member of the Viperidae family). When applied to arterial smooth muscle preparations from rat-tails, ablomin blocks K + -stimulated c...

Cysteine‐rich secretory proteins are not exclusively expressed in the male reproductive tract

“…All members of this family share a common CAP domain (also known as a sperm-coating protein, SCP, domain or a PR-1 domain), which is characterized by the presence of two signature motifs that are involved in the formation of a cleft-like structure forming a putative active site containing (in the CRISPs) three intra-molecular disulphide bonds (Henriksen et al, 2001;Shikamoto et al, 2005). CAPs are typically secreted and found in an extraordinary range of species in both bacteria and eukaryotes including, for example, yeast, fungi, plants, cone snails, drosophila, lampreys, snakes, mice, and humans (Pfitzner and Goodman, 1987;Schuren et al, 1993;Miosga et al, 1995;Murphy et al, 1995;Schreiber et al, 1997;Yamazaki et al, 2002b;Milne et al, 2003;Ito et al, 2007)). The presence of such an evolutionarily diverse, yet conserved, structure is suggestive of a common function, although the identification of this function remains a challenge.…”

“…The CRISP domain (also known as the cysteine-rich domain) contains two sub-regions: a hinge region composed of two crossed disulphide bonds, which forms a ridged link to the CAP domain and a carboxyl ion channel regulatory region (ICR) Shikamoto et al, 2005; with structural similarity to peptide toxins from the sea anenome (Dauplais et al, 1997). The addition of purified endogenous CRISPs from a range of reptile venoms, and more recently recombinant mouse CRISP2 CRISP domain, have defined the CRISP domain as having ion channel regulatory activity (Brown et al, 1999;Yamazaki et al, 2002b;. As yet, the mechanism behind ion channel specificity is not known.…”

“…As yet, the mechanism behind ion channel specificity is not known. However, individual CRISPs have been identified as having K ϩ , Ca 2ϩ , cyclic nucleotide-gated (CNG), and ryanodine receptor (RyR) ion channel regulatory activity (Morrissette et al, 1995;Yamazaki et al, 2002a;Yamazaki et al, 2002b;Wang et al, 2005). To date, the only mammalian CRISP that has been tested for ion channel activity is mouse CRISP2, which we have shown inhibited Ca 2ϩ movement through RyR2, activated RyR1, when added to the cytoplasmic aspect, and inhibited both subtypes at negative applied voltages when added to the luminal aspect .…”

β-Microseminoprotein binds CRISP-3 in human seminal plasma

“…The C-terminal domain of CRISP-3 has structural similarity to potassium-channel inhibitors [5] and several reptile CRISPs can inhibit different ion-channels [4]. Recently, CRISP-1 in rats was shown to inhibit capacitation of spermatozoa and thus prevent premature sperm activation [17].…”

“…It belongs to a family of CRISPs found in mammals and reptiles, which is characterized by 16 highly conserved cysteine residues of a total of 220-230 amino acids and 35-85% identity in the primary structure [2][3][4]. Recently, the crystal structure of a snake venom CRISP was elucidated [5].…”