Purification and cloning of platelet activating proteins from green pit virer venom (Cryptelytrops albolabris)

Abstract

Green pit viper (GPV) is the most common cause of snakebites in Bangkok. C-type lectin-like protein (CLP) obtained from the venom acts on haemostatic system. This study is aimed to clone, purifying a high-molecular weight CLP and characterize its effects on human platelets. Based on the partial cDNA library of Cryptelytrops albolabris, we had cloned full-length cDNA encoding the CLP subunit by 5’-RACE and 3’RACE method. The cDNA sequence of α subunit was 477 bp that was translated into 23 amino acid residue signal peptide and a 135 amino acid residue mature protein. The cDNA sequence of β subunit was 447 bp and translated into 23 amino acid residue signal peptide and a 125 amino acid residue mature protein. The novel C. albolabris CLP was isolated from crude venom using gel filtration column and followed by ion-exchange chromatography. The purified C. albolabris CLP was electrophoresed on SDS-PAGE showing the apparent molecular mass of 120 kDa (native condition) and displayed 2 bands of 14 and 17 kDa, (reduced condition) suggesting a tetramer of heterodimers (αβ)4. Liquid chromatography-tandem mass spectrometry analysis of the peptides found a perfect match with the conceptually-translated sequence in the green pit viper venom gland cDNA library. The peptide contained extra cysteine residues that probably formed inter-chain disulfide bonds. CLP induced human platelet aggregation in the absence of any cofactor with EC50 of 0.25 nM. Antibodies against both GPIb and GPVI inhibited platelet aggregation induced by C. albolabris CLP. Furthermore, C. albolabris CLP was found to activate tyrosine phosphorylation in human platelet. Up to now, this is the first report of the novel CLP and its cDNA cloning from C. albolabris.