Molecular cloning and characterization of alboaggregin D, a novel platelet activating protein, from Green pit viper (Cryptelytrops albolabris) venom.
Viper venoms are abundant sources of proteins affecting hemostasis. This study aimed to clone and purify a high-molecular-weight C-type lectin-like protein (snaclec) from Green pit viper (Cryptelytrops albolabris) venom, as well as to characterize its effects on human platelets. Based on the partial sequences from the C. albolabris venom gland library, we cloned full-length cDNAs encoding the snaclec subunits using 5'RACE and 3'RACE methods. The cDNA sequence of the α subunit contained 477 base pairs (bp) that were translated into 23 amino acid residue signal peptide and a 135-residue mature protein. The cDNA sequence of the β subunit contained 447bp that were translated into 23-residue signal peptide and a 125-residue mature protein. Compared with known sequences of dimeric snaclecs, these peptides contained extra cysteines that probably formed a high-order multimer. In parallel, a snaclec was isolated from C. albolabris crude venom using gel filtration followed by ion-exchange chromatography. The purified C. albolabris snaclec on SDS-PAGE showed the apparent molecular mass of 120kDa under native condition and 2 bands of 14 and 17 kD under reduced condition suggesting a tetramer of heterodimers (αβ)(4). Liquid chromatography-tandem mass spectrometry analysis of the peptides found perfect matches with the conceptually translated sequences from the cDNA library. This protein was unique from any other snaclecs previously purified from C. albolabris and named alboaggregin D. It induced human platelet aggregation in the absence of any cofactor with the EC(50) of 0.25nM and caused tyrosine phosphorylation in human platelets. Antibodies against either platelet glycoprotein (GP) Ib or GPVI could inhibit alboaggregin D-induced platelet aggregation. This snaclec may be useful for dissecting the mechanisms of platelet activation.
Department of Medicine, Division of Hematology, Chulalongkorn University, Bangkok 10330, Thailand.
Ponthip
P
Mekchay
Ponlapat
P
Rojnuckarin
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000737:chemistry*; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D037181:Lectins, C-Type / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects; D030162:Reptilian Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D015398:Signal Transduction / Q000187:drug effects; D053719:Tandem Mass Spectrometry; D017819:Viperidae / Q000235:genetics*
D003435:Crotalid Venoms; D018076:DNA, Complementary; D037181:Lectins, C-Type; D030162:Reptilian Proteins