Bv8, a small protein from frog skin and its homologue from snake venom induce hyperalgesia in rats

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Abstract

From skin secretions of Bombina variegata and Bombina bombina, we isolated a small protein termed Bv8. The sequence of its 77 amino acids was established by peptide analysis and by cDNA cloning of the Bv8 precursor. Bv8 stimulates the contraction of the guinea-pig ileum at nanomolar concentrations. The contraction is not inhibited by a variety of antagonists. Injection of a few micrograms of Bv8 into the brain of rats elicits, as assessed by the tail-flick test and paw pressure threshold, a marked hyperalgesia which lasts for about 1 h. Bv8 is related to protein A, a component of the venom of the black mamba. After i.c.v. injection, protein A is even more active than Bv8 in inducing hyperalgesia.

Introduction

Over the past 30 years, numerous studies have demonstrated that skin secretions of amphibia contain peptides with diverse biological activities (Bevins and Zasloff, 1990; Lazarus and Attila, 1993). Many of these peptides are related to mammalian hormones or neurotransmitters. In addition, in recent years, a large variety of antimicrobial peptides has been isolated from the same source (Barra and Simmaco, 1995).

The peptides produced in the skin of Bombina species were among the first to be investigated (Kiss and Michl, 1962). These are mainly bombesin (Erspamer et al., 1970; Taché et al., 1988), a peptide related to gastrin releasing hormone from mammals, and the bombinins (Gibson et al., 1991; Simmaco et al., 1991) and bombinins H (Mignogna et al., 1993), two heterogeneous groups of antimicrobial peptides. In addition, a trypsin inhibitor related to protease inhibitors from nematodes has recently been isolated from this source (Mignogna et al., 1996).

Here, we describe the isolation and characterization of a small protein from Bombina skin secretions. This amphibian protein is related to protein A, a non-toxic constituent of the venom of the black mamba (Joubert and Strydom, 1980). The new protein is termed Bv8 (Bombina variegata protein with a molecular mass of 8 kDa). Some of the pharmacological activities of Bv8 and protein A are described.

Section snippets

Materials

Lys-C and Asp-N endoproteinases were purchased from Boehringer, 4-vinylpyridine from Aldrich-Chemie, solvents for high-pressure liquid chromatography (HPLC) from Carlo Erba Reagenti, and other sequence grade chemicals from Perkin-Elmer. Restriction enzymes were obtained from Boehringer Mannheim, Bethesda Research Laboratories and MBI Fermentas; [32P]-dATP was purchased from New England Nuclear; oligonucleotides were synthesized on a Beckman Oligo 1000 synthesizer. IRD-41 labeled sequencing

Isolation and structural studies

Bv8 was purified from Bombina skin secretions as described under Methods. Peak 3 in Fig. 1 is Bv8. The amino acid sequence of the reduced and pyridylethylated peptide could be determined up to residue 49 by automated Edman degradation. Several proteolytic fragments generated by digestion with Lys-C or Asp-N endoprotease were also sequenced (see Fig. 2A). These experiments demonstrated that Bv8 contains 77 amino acids including 10 cysteines. Its molecular mass determined on a Finnigan LCQ ion

Discussion

From skin secretions of two Bombina species, we have isolated a small protein containing 77 amino acids, of which 10 are cysteines. The amino acid sequence has been established by automated Edman degradation and analysis of proteolytic fragments as well as independently by cDNA cloning. Possible homologues of Bv8 with the amino-terminal sequence AVITGA×ERDVQ… have also been detected in skin secretions of Rana temporaria and Rana esculenta (unpublished experiments). This frog skin product is

Acknowledgements

We thank Dr. Günter Lepperdinger for help with the fractionation of the mamba venom, Dr. Manfred Sippl (University of Salzburg) and Dr. Stefano Pascarella (University of Rome) for their findings on the similarity between Bv8 and co-lipases, Christa Tippelt for help with the collection of skin secretions and the fractionation procedures and Elisa Giannini for help in testing compounds on isolated organs. This work was supported in part by grants from the Italian Ministero dell'Università e della

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