Molecular cloning of a γ-phospholipase A2 inhibitor from Lachesis muta muta (the bushmaster snake)
Introduction
Phospholipases A2 (E.C. 3.1.1.4; PLA2) are commonly occurring enzymes that catalyze specifically the hydrolysis of the sn-2 acylester bond in 1,2-diacyl-3-sn-phosphoglycerides in a calcium dependent reaction. According to their biochemical characteristics and cellular origin, these enzymes have been classified as cytosolic (cPLA2) or secretory (sPLA2) phospholipases A2 (Glaser et al., 1993). Snake venoms are a well known source of sPLA2 which are responsible for a wide variety of distinct pharmacological effects such as neurotoxicity, cardiotoxicity, myonecrosis, anticoagulant and platelet effects upon envenoming (Kini and Evans, 1989).
In the last decade, purification of a growing number of endogenous PLA2 inhibitors (PLIs) from the blood plasma of snake species from all continents has been reported (Dunn and Broady, 2001, Faure, 2000, Fortes-Dias, 2002 for recent reviews). The main role ascribed to PLIs has been a physiological protection of snakes against an accidental leaking of venom from their venom glands. In South American snakes, a PLI was isolated and characterized from the blood plasma of the tropical rattlesnake, Crotalus durissus terrificus (Crotalinae, Viperidae) (Fortes-Dias et al., 1991, Fortes-Dias et al., 1994, Perales et al., 1995). That protein, named CNF, is a member of a large class of PLA2 inhibitors, recently grouped as γPLIs (Ohkura et al., 1997).
CNF is a 160 kDa glycoprotein composed of 6–8 identical subunits of approximately 24 kDa with a broad range of PLA2 specificity like other γPLIs. CNF acts by replacing the subunit CA in the crotoxin complex (CACB) from C. d. terrificus venom, inhibiting the PLA2 activity of the subunit CB. Besides CB, CNF is able to inhibit basic and acidic PLA2s from L. m. muta (Fortes-Dias et al., 1999) and B. jararacussu (Fortes-Dias, unpublished data) venoms, even more efficiently.
Evolutionary studies on the Viperidae family have demonstrated that Lachesis, comprising a single species Lachesis muta, is the genus most closely related to Crotalus (Brattstrom, 1964). However, the venom from L. muta venom is much more complex than that from C. d. terrificus, being composed of a variety of biologically active components including high levels of phospholipases A2 (Valiente et al., 1992, Fuly et al., 1993, Fuly et al., 1997, Fortes-Dias et al., 1999).
Taking into consideration: (1) The closer evolutionary relationship between Crotalus and Lachesis; (2) The capability of CNF to inhibit L. muta PLA2; (3) The significant levels of PLA2 in L. muta venom and (4) The important physiological role of PLIs in snakes, we decided to search for the presence of a CNF-homologue transcript in L. m. muta liver.
Section snippets
Materials and methods
L. m. muta liver. The liver of one adult specimen of L. m. muta was removed immediately after its death from natural causes. That snake had been captured in Costa Rica, bred at the Dallas Zoo (Texas, USA) and donated to the Herpetarium of Fundação Ezequiel Dias (Belo Horizonte, Brazil), where it was registered as Lmt 9501.
RNA extraction. Total RNA was extracted from the liver of L. m. muta using Trizol® (GibcoBRL, Rockville, MD, USA) according to the manufacturer's instructions. Briefly,
Results
Amplification of cDNA from L. m. muta liver with CNF primers resulted in an amplicon with about 640 bp (data not shown), which was cloned in a plasmid vector. After checking for the presence of the insert in the recombinant plasmid DNA, two clones (Lmm1 and Lmm2) were selected for DNA purification and nucleotide sequencing. Comparison of these sequences with CNF cDNA showed 7 and 24 nucleotide (nt) substitutions in Lmm1 and Lmm2, respectively, including one in the non-coding region (Fig. 1).
Discussion
Two transcripts corresponding to CNF-homologues (LNFs) have been isolated from the liver of a single L. m. muta snake. Each clone isolated coded for a distinct isoform, which contrasts with C. d. terrificus, where a single nt substitution was found after complete sequencing of eight clones (Fortes-Dias et al., 1994). The presence of isoforms, however, seems to be a common feature in γPLIs, especially for heteromeric inhibitors such as those described in the Elapidae family (Dunn and Broady, 2001
Acknowledgements
We are grateful to G.A. Cotta, biologist from the Herpetarium of Fundação Ezequiel Dias, for providing the snake used in our study. DNA sequencing was performed by one of us (Fortes-Dias) in a short-term stay (FAPEMIG CAM 90403/00) at the Départment d'Ingénierie et d'Études des Protéines (CEA, Saclay) and we thank Pr. A. Ménez and Dr F. Ducancel for the support and suggestions. We are also indebted with Dr M. Richardson for kindly reviewing the language. This work was supported by the Fundação
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2017, International Journal of Biological MacromoleculesCitation Excerpt :Members of the PLIγ I subgroup have a heteromeric composition and two subunits (A and B) with less than 33% identity to each other and inhibit groups I, II, and III PLA2s. Members of the PLIγ II subgroup preferentially inhibits those PLA2s of group II and are constituted of a single subunit [7,13,15,16]. However, recently the possibility of a second subunit in a PLIγ II from Crotalus durissus terrificus has been raised [17].
An α-type phospholipase A<inf>2</inf> inhibitor from Bothrops jararacussu snake plasma: Structural and functional characterization
2008, BiochimieCitation Excerpt :Venomous and non-venomous snakes display PLA2 inhibitory proteins (PLIs) in their blood plasma in order to protect themselves against toxins from snake venoms, which could, eventually, reach the circulatory system [14,15]. Many studies have been carried out in search for these natural PLA2 inhibitors, including those from snake plasmas of Atropoides nummifer, Elaphe quadrivirgata, Lachesis muta muta and Vipera russelii [16–19]. This paper reports the isolation of a myotoxin inhibitor protein from Bothrops jararacussu plasma (αBjussuMIP) and its biochemical, structural and functional characterization.
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2008, ToxiconCitation Excerpt :Nevertheless, these inhibitors have been reported in two species of non-venomous, non-ophiophagus snake species, Python reticulatus (γPLI) and Elaphe quadrivirgata (α-, β- and γPLIs) (Okumura et al., 1999; Thwin et al., 2002). In spite of the rich diversity of the fauna in our country, specially regarding ophidian species, not more than three PLIs have been described in Brazilian snakes so far: an αPLI from Bothrops moojeni (Soares et al., 2003) and two γPLIs from Crotalus durissus terrificus (the South American rattlesnake) and Lachesis muta (the bushmaster snake) (Fortes-Dias et al., 1994, 2003; Perales et al., 1995). The γPLIs are known to be potent inhibitors of IIA-PLA2 from viperid snake venoms.
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