Elsevier

Biochimie

Volume 84, Issue 7, July 2002, Pages 675-680
Biochimie

cDNA sequence and molecular modeling of a nerve growth factor from Bothrops jararacussu venomous gland

https://doi.org/10.1016/S0300-9084(02)01429-3Get rights and content

Abstract

The complete nucleotide sequence of a nerve growth factor precursor from Bothrops jararacussu snake (Bj-NGF) was determined by DNA sequencing of a clone from cDNA library prepared from the poly(A) + RNA of the venom gland of Bjararacussu. cDNA encoding Bj-NGF precursor contained 723 bp in length, which encoded a prepro-NGF molecule with 241 amino acid residues. The mature Bj-NGF molecule was composed of 118 amino acid residues with theoretical pI and molecular weight of 8.31 and 13,537, respectively. Its amino acid sequence showed 97%, 96%, 93%, 86%, 78%, 74%, 76%, 76% and 55% sequential similarities with NGFs from Crotalus durissus terrificus, Agkistrodon halys pallas, Daboia (Vipera) russelli russelli, Bungarus multicinctus, Naja sp., mouse, human, bovine and cat, respectively. Phylogenetic analyses based on the amino acid sequences of 15 NGFs separate the Elapidae family (Naja and Bungarus) from those Crotalidae snakes (Bothrops, Crotalus and Agkistrodon). The three-dimensional structure of mature Bj-NGF was modeled based on the crystal structure of the human NGF. The model reveals that the core of NGF, formed by a pair of β-sheets, is highly conserved and the major mutations are both at the three β-hairpin loops and at the reverse turn.

Introduction

Nerve growth factors (NGFs) belong to the family of neurotrophic factors and are by far the best characterized neurotrophic polypeptides. NGFs play an important role in ontogenesis and regulate the development and maintenance of neurons derived from embryonic sensory neurons and neural cells of the vertebrate peripheral sympathetic nerve [1], [2]. Recently, it has been evidenced that NGFs have a similar effect on the populations of cholinergic neurons in the central nervous system. Besides inducing the typical fiber outgrowth of cultured cells, cobra venom and human NGFs exhibit also non-neuronal effects, such as the induction of plasma extravasation and histamine release from whole blood cells [3], [4].

Several NGFs were isolated and biologically characterized from Elapidae and Viperidae snake venoms, such as Naja naja [5], Nnatra [3], [6], [7], [8], Nn. siamensis [8], Vipera lebetina [9], Vberus berus [10], Vrusselli russelli [11], Bitis arietans [12], Bungarus multicinctus [13], [14] and Agkistrodon halys pallas [15]. Although NGFs are also present in the venoms of a large variety of other snakes, their study has lagged relatively behind [2], [16], [17].

Snake venom NGFs can be classified into three groups: (a) dimeric forms with an Mr of approximately 25,000 or 13,000 per subunit; (b) higher Mr glycoprotein forms (Mr ∼ 35,000); and (c) the NGFs from Bmulticinctus and Bitis arietans, which seem to be composed of homodimers whose subunits are covalently linked by disulfide bonds to one another [2], [12]. The only NGF cDNAs so far resolved, namely, those from Nnaja, Bmulticinctus and Ahalys, showed similar amino acid sequences to NGFs of other animals [14], [15].

The structures of mouse and mature human NGFs have been solved [18], [19] in the native form and bound with receptors; both structures are structurally very similar. However, no snake NGF has been solved so far and only a little structural information is presently available.

In order to gain further insights into the structure–function relationships of these NGFs, we have determined the first nucleotide sequence of an NGF from a South American Crotalinae snake belonging to the genus Bothrops and named it Bj-NGF (Bothrops jararacussu NGF).

Section snippets

Total RNA extraction and cDNA cloning

Total RNA was extracted after excision of the Bothrops jararacussu snake venom gland using the Trizol method (Gibco BRL). A fraction of the total mRNA (poly A+) was isolated using the Quick Preparation mRNA Purification Kit (Pharmacia). The cDNA pool was obtained using the time saver cDNA Synthesis Kit (Pharmacia).

cDNA cloning was carried out using pUC 19 derived plasmid vector with a modified polylinker and was named pUC 99 (Fig. 1). The plasmid vector pUC 99 was modified in the Bam HI and Pst

Results

The cDNA fragment that appeared as a single band of predicted size by electrophoresis analysis (results not shown) was sequenced. This cDNA of 726 bp codified a prepro-NGF of 241 and a mature Bj-NGF with 119 amino acid residues (Fig. 2) and an estimated isoelectric point and Mr of 8.31 and 13,537, respectively.

The combined Swiss-Prot and NCBI programs allowed the alignment of mature Bj-NGF and other NGFs. Fig. 3A shows six Cys residues in the mature NGFs, at highly conserved positions (Cys14,

Discussion

In the present study, we have isolated the cDNA fragment coding for an NGF precursor from Bjararacussu snake and determined the complete amino acid sequences (241 residues) of prepro-NGF and (118 residues) of the mature Bj-NGF. The structure of the predicted Bj-NGF precursors resembles that of the NGF precursor from the B. multicinctus, N. naja and A. hpallas, with a highly conserved C-terminal region in the mature NGF protein [14], [15], [24].

In the non-Bj-NGF moiety, a putative signal

Acknowledgements

The authors express their gratitude to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Proc. No. 99/00386-7) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support.

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    Accession No.: the sequence data reported in this paper will appear in the GenBank under accession No. AY007318.

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