Elsevier

Biochimie

Volume 86, Issue 3, March 2004, Pages 211-219
Biochimie

Analysis of Bothrops jararacussu venomous gland transcriptome focusing on structural and functional aspects1: I—gene expression profile of highly expressed phospholipases A2

https://doi.org/10.1016/j.biochi.2004.02.002Get rights and content

Abstract

Snake venom glands are a rich source of bioactive molecules such as peptides, proteins and enzymes that show important pharmacological activity leading to in local and systemic effects as pain, edema, bleeding and muscle necrosis. Most studies on pharmacologically active peptides and proteins from snake venoms have been concerned with isolation and structure elucidation through methods of classical biochemistry. As an attempt to examine the transcripts expressed in the venom gland of Bothrops jararacussu and to unveil the toxicological and pharmacological potential of its products at the molecular level, we generated 549 expressed sequence tags (ESTs) from a directional cDNA library. Sequences obtained from single-pass sequencing of randomly selected cDNA clones could be identified by similarities searches on existing databases, resulting in 197 sequences with significant similarity to phospholipase A2 (PLA2), of which 83.2% were Lys49-PLA2 homologs (BOJU-I), 0.1% were basic Asp49-PLA2s (BOJU-II) and 0.6% were acidic Asp49-PLA2s (BOJU-III). Adjoining this very abundant class of proteins we found 88 transcripts codifying for putative sequences of metalloproteases, which after clustering and assembling resulted in three full-length sequences: BOJUMET-I, BOJUMET-II and BOJUMET-III; as well as 25 transcripts related to C-type lectin like protein including a full-length cDNA of a putative galactose binding C-type lectin and a cluster of eight serine-proteases transcripts including a full-length cDNA of a putative serine protease. Among the full-length sequenced clones we identified a nerve growth factor (Bj-NGF) with 92% identity with a human NGF (NGHUBM) and an acidic phospholipase A2 (BthA-I-PLA2) displaying 85–93% identity with other snake venom toxins. Genetic distance among PLA2s from Bothrops species were evaluated by phylogenetic analysis. Furthermore, analysis of full-length putative Lys49-PLA2 through molecular modeling showed conserved structural domains, allowing the characterization of those proteins as group II PLA2s. The constructed cDNA library provides molecular clones harboring sequences that can be used to probe directly the genetic material from gland venom of other snake species. Expression of complete cDNAs or their modified derivatives will be useful for elucidation of the structure–function relationships of these toxins and peptides of biotechnological interest.

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.

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    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).

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