BE-I-PLA2, a novel acidic phospholipase A2 from Bothrops erythromelas venom: isolation, cloning and characterization as potent anti-platelet and inductor of prostaglandin I2 release by endothelial cells.
A novel acidic Asp49 phospholipase A(2) was isolated from Bothrops erythromelas (jararaca malha-de-cascavel) snake venom by four chromatographic steps. BE-I-PLA2 present a molecular weight of 13,649.57 Da as estimated by mass spectrometry. N-terminal and four internal peptides were sequenced, covering around one-third of the complete toxin sequence. The complete BE-I-PLA2 cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 457 bp and encodes a protein with significant sequence similarity to many other phospholipase A(2) from snake venoms. When tested in platelet rich plasma, the enzyme showed a potent inhibitory effect on aggregation induced by arachidonic acid and collagen, but not ADP. On the other hand, BE-I-PLA2 did not modify aggregation in washed platelet. Furthermore, no action of BE-I-PLA2 on the principal platelets receptors was observed. Chemical modification with p-bromophenacyl bromide abolished the enzymatic activity of BE-I-PLA2, but its anti-platelet activity was only partially inhibited. In human umbilical-cord veins endothelial cells, BE-I-PLA2 was neither apoptotic nor proliferative but stimulated endothelial cells to release prostaglandin I(2), suggesting an increase of its potential anti-platelet activity in vivo. Further studies are required in order to determine the exact mechanism of action of BE-I-PLA2 in the inhibition of platelet aggregation.
de Albuquerque Modesto
Jeanne Claine
JC
Laboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo SP, Brazil. clainealbuquerque@hotmail.com
Spencer
Patrick J
PJ
Fritzen
Márcio
M
Valença
Renata C
RC
Oliva
Maria Luíza Vilela
ML
da Silva
Marcia Bezerra
MB
Chudzinski-Tavassi
Ana Marisa
AM
Guarnieri
Miriam Camargo
MC
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D017837:Bothrops / Q000235:genetics*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011464:Epoprostenol / Q000096:biosynthesis / Q000378:metabolism*; D005260:Female; D054501:Group II Phospholipases A2; D054513:Group IV Phospholipases A2; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D054467:Phospholipases A2; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D030162:Reptilian Proteins; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
D003435:Crotalid Venoms; D018076:DNA, Complementary; D010975:Platelet Aggregation Inhibitors; D030162:Reptilian Proteins; D011464:Epoprostenol; D010741:Phospholipases A; C512871:BE-I-PLA2 protein, Bothrops erythromelas; D054501:Group II Phospholipases A2; D054513:Group IV Phospholipases A2; D054467:Phospholipases A2