Nucleotide sequence of phospholipase A2 gene expressed in snake pancreas reveals the molecular evolution of toxic phospholipase A2 genes☆
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)
- et al.
Basic local alignment search tool
J. Mol. Biol.
(1990) - et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction
Anal. Biochem.
(1987) The growing phospholipase A2 superfamily of signal transduction enzymes
Trends Biochem. Sci.
(1997)- et al.
Mechanisms that account for the selective release of arachidonic acid from intact cells by secretory phospholipase 2
Biochim. Biophys. Acta
(1998) - et al.
Amino acid sequence of a new type of toxic phospholipase A2 from the venom of the Australian tiger snake (Notechis scutatus scutatus)
Arch. Biochem. Biophys.
(1995) - et al.
A comparative analysis of invaded sequences from group IA phospholipase A2 genes provides evidence about the divergence period of genes groups and snake families
Toxicon
(2002) - et al.
Purification, properties, and amino acid sequence of a hemoglobinuria-inducing phospholipase A2, MiPLA-1, from Micropechis ikaheka venom
Arch. Biochem. Biophys.
(1999) - et al.
Purification and properties of three new phospholipase A2 isoenzymes from Micropechis ikaheka venom
Biochim. Biophys. Acta
(2001) - et al.
Structural aspects of interfacial adsorption. A crystallographic and site-directed mutagenesis study of the phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus
J. Biol. Chem.
(1997) - et al.
A novel group of phospholipase A2s preferentially expressed in type 2 helper T cells
J. Biol. Chem.
(2001)
Multiple sequence alignment with Clustal X
Trends Biochem. Sci.
Identification and purification of a very high affinity binding protein for toxic phospholipases A2 in skeletal muscle
J. Biol. Chem.
Accelerated evolution of Trimeresurus okinavensis venom gland phospholipase A2 isozyme-encoding genes
Gene
Studies on the subunit structure of textilotoxin, a potent presynaptic neurotoxin from the venom of the Australian common brown snake (Pseudonaja textilis). 2. The amino acid sequence and toxicity studies of subunit D
Biochim. Biophys. Acta
The expanding superfamily of phospholipase A2 enzymes: classification and characterization
Biochim. Biophys. Acta
Cited by (22)
Comparative venomics and preclinical efficacy evaluation of a monospecific Hemachatus antivenom towards sub-Saharan Africa cobra venoms
2021, Journal of ProteomicsCitation Excerpt :Based on clinical observations in envenomed individuals and in preclinical toxicity studies, the lethal effect of these venoms appears to be caused predominantly by neurotoxic and cardiotoxic (H. haemachatus and N. annulifera) or cytotoxic/cardiotoxic (CTxs) (N. mossambica and N. nigricollis) 3FTxs [22,23,54,65,66]. On the other hand, Elapidae venom group I PLA2 molecules are evolutionary derived from non-toxic pancreatic-type PLA2 [67–69] and exert a multiplicity of toxic activities, including neurotoxic, myotoxic, and antiplatelet activities [70]. PLA2s exerting myotoxic, cardiotoxic and anticoagulant activities have been isolated from the venom of N. nigricollis [71–73].
Toxinology provides multidirectional and multidimensional opportunities: A personal perspective
2020, Toxicon: XCitation Excerpt :Fujimi et al. analyzed several gene sequences encoding PLA2 from Laticauda semifasciata to determine the relationship between pancreatic and venom PLA2 genes (Fujimi et al., 2002a, b). They found insertions in the promoter and the first intron of group IA (venom) PLA2 gene compared with group IB” (pancreatic) PLA2 gene (Fujimi et al., 2002a, 2004) (Fig. 2). The 411-bp insert in the promoter region has two E box and one GC box binding sites and interrupts promoter region of pancreatic PLA2 gene (Fig. 2B).
- ☆
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.