Analysis of Bothrops jararacussu venomous gland transcriptome focusing on structural and functional aspects1: I—gene expression profile of highly expressed phospholipases A2
Introduction
Through the course of the history of civilization, venomous snakes have represented a unique and particular fascination for humans. Snake venoms are rich source of pharmacologically active compounds with diverse spectrum of biological activities [1]. Venoms are usually composed of a complex mixture of proteins, enzymes, peptides and inorganic components. Study of snake venoms has yielded a vast body of important information on biological systems and insights into medical problems [1], [2], [3].
Indeed, some venom components (phospholipase A2, PLA2; metalloproteases, disintegrins, serine-proteases, L-amino acid oxidases (LAAO), nerve growth factor (NGF) and others) are thought to be promising as agents in the treatment of diseases and medical complications [4], [5]. It includes the use of important therapeutic agents such as the angiotensin converting enzyme (ACE) to treat high blood pressure. The prototype ACE inhibitor captopril was developed from the sequence study of small peptides isolated from the venom of the viper snake Bothrops jararaca [6], [7].
Local tissue damage, such as hemorrhage, myonecrosis and edema are among the most dramatic effects of envenomation by Crotalinae and Viperinae snakes. Snakes belonging to the genus Bothrops are largely distributed from South to North America. Among the bioactive proteins from these venoms, the PLA2s and metalloproteases are outstanding as major components [8], [9]. A great number of these enzymes have been purified and characterized, however, little is known concerning the genes encoding those enzymes.
Analysis of expressed sequence tags (ESTs) is an efficient approach for gene discovery, expression profiling [10], [11], [12] and development of resources useful for functional genomics studies. Moreover, gene cataloguing and profiling of the venom gland of Bothrops jararacussu is an essential requisite to provide molecular reagents for functional genomic studies needed for the elucidation mechanisms of action of toxins and the discovery of their antagonists. In order to gain further insights toward the structure–function relationships of those proteins, we constructed a high quality cDNA library to identify genes and their putative products. Phylogenetic analysis of the PLA2s from B. jararacussu and described PLA2s from other Bothrops species were also carried out. Moreover, molecular modeling studies were performed to point out highly conserved structural domains.
Section snippets
RNA extraction and cDNA cloning
Two days before excision of B. jararacussu venomous gland, the venom was exhaustively extracted. Total RNA was extracted from the venomous gland of B. jararacussu using the Trizol method (Gibco BRL). The fraction of total mRNA (poli A+) was isolated using the Quick Preparation mRNA Purification Kit (Amersham Pharmacia Biotech). The cDNA pool was obtained using the Time Saver cDNA Synthesis Kit (Amersham Pharmacia Biotech). All the cDNA fragments were then linked to Eco RI and Not I adaptors
Quality assessment of the cDNA library
A cDNA venomous gland library comprising 846 independent clones was constructed for ESTs analysis. The length of cloned cDNAs were estimated by agarose gel electrophoresis analysis, of PCR amplified products, resulting in a distribution between 200 and 2000 bp with highest density between 500 and 1000 bp (data not shown).
Assigning putative function to ESTs
An efficient and informative approach to characterize a transcriptome is to analyze ESTs. As part of the transcriptome study, RNA transcripts analysis, of B. jararacussu, we
Discussion
Snake venoms are constituted by complex mixtures of proteins and bioactive peptides playing different roles in the local damage and systemic injury processes that characterize the level of toxicity. B. jararacussu venom exhibits high level of myotoxic and low level of hemorrhagic effects mainly induced by PLA2s and metalloproteases, respectively.
In this work we analyzed ESTs, using a cDNA library from venomous gland of B. jararacussu, a typical snake from the southeast of Brazil, to identify
Acknowledgements
The authors express their gratitude to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Universidade de Ribeirão Preto (UNAERP) for financial support. We thank Dr. Richard J. Ward and Dr. Raghuvir K. Arni for the BthTX-I coordinates.
References (38)
- et al.
Snake venom proteins acting on hemostasis
Biochimie
(2000) Angiotensin converting enzyme: history and relevance
Semin. Perinatol.
(2000)- et al.
Snake venom metalloproteinases: their role in the pathogenesis of local tissue damage
Biochimie
(2000) - et al.
Identification of novel genes from Entamoeba histolytica by expressed sequence tag analysis
Gene
(1996) - et al.
A survey of gene expression and diversity in the venom glands of the pitviper snake Bothrops insularis through the generation of expressed sequence tags (ESTs)
Gene
(2002) - et al.
cDNA sequence and molecular modeling of a nerve growth factor from Bothrops jararacussu venomous gland
Biochimie
(2002) RIBBONS
Methods Enzymol.
(1997)- et al.
Snake venom proteases affecting hemostasis and thrombosis
Biochim. Biophys. Acta
(2000) - et al.
Isolation and characterization of a new clotting factor from Bothrops jararacussu (jararacuçu) venom
Toxicon
(1997) - et al.
Purification, characterization and crystallization of jararacussin-I, a fibrinogen-clotting enzyme isolated from the venom of Bothrops jararacussu
Toxicon
(2002)
Fractionation of Bothrops jararacussu snake venom: partial chemical characterization and biological activity of bothropstoxin
Toxicon
Structural and functional characterization of myotoxin I, a Lys49 phospholipase A2 homologue from Bothrops moojeni (Caissaca) snake venom
Arch. Biochem. Biophys.
Myotoxic phospholipases A2 in Bothrops snake venoms: effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu
Biochimie
Structural and functional characterization of BnSP-7, a Lys49 myotoxic phospholipase A2 homologue from Bothrops neuwiedi pauloensis venom
Arch. Biochem. Biophys.
Refolding and purification of Bothropstoxin-I, a Lys49-phospholipase A2 homologue, expressed as inclusion bodies in Escherichia coli
Protein Exp. Purif.
Effects of Mn2+ on actions of bothropstoxin-I, a Lys49 myotoxic phospholipase A2 homologue from Bothrops jararacussu snake venom
Int. J. Biochem. Cell Biol.
Structural characterization and phylogenetic relationships of myotoxin II from Atropoides (Bothrops) nummifer snake venom, a Lys49 phospholipase A2 homologue
Int. J. Biochem. Cell Biol.
Phospholipase A2—a structural review
Toxicon
Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A2 from Bothrops jararacussu snake venom
Biochem. Pharmacol.
Cited by (84)
Generation and In-planta expression of a recombinant single chain antibody with broad neutralization activity on Bothrops pauloensis snake venom
2020, International Journal of Biological MacromoleculesFirst report on BaltCRP, a cysteine-rich secretory protein (CRISP) from Bothrops alternatus venom: Effects on potassium channels and inflammatory processes
2019, International Journal of Biological MacromoleculesCitation Excerpt :However, the roles of CRISPs during envenoming are still poorly understood. Evaluation by omic techniques revealed that CRISPs correspond to a small portion of the composition of Bothrops venoms: 1.6% for B. jararaca [49], 1% for B. jararacussu [50], 0.6% for B. insularis [51] and 0.2% for B. atrox [52]. Proteomic analysis of B. alternatus venom revealed that it consists mainly of metalloproteases and serine proteases [53], and the analysis of the transcriptome of B. alternatus venom gland showed that only 0.4% of the transcripts corresponded to CRISPs [54].
Nanobodies as novel therapeutic agents in envenomation
2018, Biochimica et Biophysica Acta - General SubjectsCitation Excerpt :Bothrops toxin I and II (BthTX I and II) are members of PLA2 group that cause depolarization and they interfere with Ca2+ influx in cells, which in turn leads to a series of events causing cell death [130,131]. BthTX I and BthTX II can take up 40 to 50% of the dry weight of this snake venom [132,133]. Prado et al. showed that a Nb against PLA2 of the venom effectively neutralizes this toxin [134].
- 1
All sequence data reported in this paper will appear in the GenBank database under the following accession numbers: BOJU-I (AY 185200), BOJU-II (AY 185206), BOJU-III (AY 145836), BOJUMET-I (AY 55005), BOJUMET-II (AY 25584), BOJUMET-III (AY 258153), C-type lectin (AY 251283), serine-proteases (AY 251282).