Elsevier

Gene

Volume 292, Issues 1–2, 12 June 2002, Pages 225-231
Gene

Nucleotide sequence of phospholipase A2 gene expressed in snake pancreas reveals the molecular evolution of toxic phospholipase A2 genes

https://doi.org/10.1016/S0378-1119(02)00682-0Get rights and content

Abstract

We have cloned two phospholipase A2 (PLA2) DNA complementary to RNA that contained nucleotide sequences encoding pancreas loop by the reverse transcription-polymerase chain reaction cloning procedure using messenger RNA isolated from Laticauda semifasciata pancreas. Additionally, a gene clone encoding PLA2 with the pancreatic loop sequence was isolated from a L. semifasciata genomic library. Subsequent sequence analysis revealed that PLA2 clones encoding group IB″ PLA2. Comparative analysis of group IA and IB″ PLA2 genes revealed that the exon-intron organization is conserved in the genes of both groups. The invaded sequences in the second intron were very similar to those of the L. semifasciata group IA gene. This observation suggested that the integration of the invaded sequences occurred before the divergence of groups IA and IB″ during the evolution of PLA2 gene. The comparative analysis revealed that the arising of group IA PLA2 occurred by the deletion and substitution of nucleotide sequences in exon III region during the process of accelerated evolution.

Introduction

Phospholipase A2 (PLA2; EC 3.1.1.4) catalyzes the hydrolysis of the acyl ester bond at the sn-2 position of phosphoglycerides (Tischfield, 1997). PLA2 enzymes are found as both intracellular (high molecular weight) and secretory (low molecular weight) forms. Snake venom PLA2s are low molecular weight, well known as secreted type PLA2 and often contained a large number of isozymes. PLA2s are commonly classified into 12 groups and many subgroups. This number is growing as evidenced by many recent reports of novel PLA2 isozymes (Dennis, 1997, Six and Dennis, 2000, Valentin and Lambeau, 2000, Ho et al., 2001). Danse et al. (1997) proposed the division of group I into five subgroups and the division of group II into six subgroups. According to their classification, PLA2s in snake venoms belong to groups IA, IA′, IB′, IB″, IIA, IIA′ and IIB. Group IB′ and IB″ PLA2s have been isolated from various venom sources, including Oxyuranus scutellatus scutellatus (taipoxin γ chain, Fohlman et al., 1976). Pseudonaja textilis (textilotoxin D chain, Pearson et al., 1991), Micropechis ikaheka (MiPLA-l, MiPLA-2, MiPLA-3 and MiPLA-4, Gao et al., 1999, Gao et al., 2001), Notechis scutatus scutatus (HTe and HTg, Francis et al., 1995) and O. scutellatus scutellatus (OS1, Lambeau et al., 1990). Group IA, IA′, IIA and IIA′ PLA2 genes, DNA complementary to RNAs (cDNAs) and the physiological functions of the encoding proteins, have been well investigated. There have, however, been few reports on the nucleotide sequences of group IB′ and IB″ PLA2s. Initial reports on the cloning of secretory PLA2s dealt with investigations of group IB PLA2 cDNAs from pancreas (dog, rat and porcine) or lung (human) (Ohara et al., 1986, Seilhamer et al., 1986). During past decade, the mammalian group IB PLA2, which response appears to be mediated through the release of free fatty acids within the cell, has been well investigated (Tohkin et al., 1993, Fonteh et al., 1998). We have already cloned group IA PLA2 (neurotoxin) genes and cDNAs from sea snake, Laticauda semifasciata (Fujimi et al., 2002). Comparative analysis of group IA and IB″ genes from the same animal source would contribute to our understanding of the evolution and divergence of snake PLA2 genes. To elucidate the molecular evolution of snake PLA2s, group IB″ PLA2 cDNAs and genomic clones from L. semifasciata were isolated and their nucleotide sequences were analyzed.

This is the first report detailing the cloning of a snake pancreatic PLA2 gene and cDNAs. The results of the present investigation will help us to understand the gene evolution and functional divergence of secretory PLA2s.

Section snippets

Cloning of cDNAs encoding PLA2 with pancreatic loop sequence from L. semifasciata

Total RNA was extracted from the pancreas (0.5 g) of L. semifasciata using the acid guanidine phenol chloroform method (Chomczynski and Sacchi, 1987). Poly(A)+ RNA was purified using Dynabeads Oligo (dT)25 (Dynal, Norway) from total RNA. Single stranded cDNA (cDNA pool) was synthesized using reverse transcriptase RAV-2 (Takara, Japan) with an anchored oligo (dT) primer (5′-CTGATCTAGAGGTACCGGATCCT20-3′) according to the manufacturer's protocol. Complementary DNAs encoding PLA2 with pancreatic

Nucleotide sequences and structures of group IB″ cDNAs

Two pancreatic PLA2 cDNA clones (LsGIB″pkP2 and LsGIB″pkP5) were isolated and sequenced. The size of the clones was about 900 bp (Fig. 1). The difference in length relative to group IA was mostly due to the presence of an inserted sequence of around 300 bp in the 3′ untranslated region (3′ UTR). No homologous sequences to this region were found using a BLAST search. It was revealed that there was a sequence encoding pancreatic loop. This sequence contributed marginally to the size of cDNA. The

Discussion

Two cDNA clones (LsGIB″pkP2 and LsGIB″pkP5) encoding group IB″ PLA2s were isolated from a cDNA pool of L. semifasciata pancreas. These PLA2s might function as digestion enzymes in the tissue because significant regions for enzyme activity (Ca2+ loop and active site) are highly similar to mammalian group IB PLA2s (Valentin and Lambeau, 2000), and all cysteine residues are also conserved to mammalian group IB PLA2s (Fig. 2). The predicted amino acid sequences did not contain a discernible

Acknowledgements

This work was partially supported by grants from The Ministry of Education, Science and Culture of Japan.

References (30)

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The nucleotide sequence data reported here have been submitted to the DDBJ sequence data bank. LsPLA2GL1-1, A062439; LsPLA2GL5-1, A062440; LsPLA2GL16-1, AB078346; LsGIB″pkP2 (recorded as LsPLA2pkP2 in DDBJ), AB078347; LsGIB″pkP5 (recorded as LsPLA2pkP5 in DDBJ), AB078348.

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