Biochemical, pharmacological and structural characterization of two PLA2 isoforms Cdr-12 and Cdr-13 from Crotalus durissus ruruima snake venom.
Cdr-12 and Cdr-13 isoforms of PLA2, a D49 protein, were purified from Crotalus durissus ruruima venom after one chromatographic step, reverse phase HPLC on micro-Bondapack C-18. The molecular mass by SDS-PAGE of Cdr-12 and Cdr-13 isoforms of PLA2 was 14333.49 Da and 14296.42 Da, respectively and confirmed by MALDI-TOF mass spectrometry. The amino acid composition showed that both isoforms Cdr-12 and Cdr-13 have a high content of Lys, Tyr, Gly, Arg, and 14 half-Cys residues, typical of a basic PLA2. The isoforms Cdr-12 and Cdr-13 had a sequence of amino acids of 122 amino acid residues, being Cdr-12: SLLQFNKMIK FETRKNAIPF YAFYGCYCGW GGQGRPKDAT DRCCIVHDCC YGKLAKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGYMIY PDSRCREPSE TC and pI value 8.37 and Cdr-13: SLVQFEKMIK EETGKNAVPF YAFYGCYCGW GGRGRPKDAT DRCCIVHDCC YEKLVKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGKMIY PDSRCREPSE TC with a pI value of 8.13 This sequence shows high identity values when compared to other D49 PLA2s isolated from venoms of crotalics snakes. Skeletal muscle preparations from the young chicken have been previously used in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. In mice, the PLA2 isoforms Cdr-12 and Cdr-13 induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. In vitro, Cdr-12 and Cdr-13 isoforms of PLA2, caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and produced cytotoxicity in murine C2C12 skeletal muscle myotubes and lack cytolytic activity upon myoblasts in vitro. Thus, the combined structural and functional information obtained identify Cdr-12 and Cdr-13 isoforms as members of the PLA2 family, which presents the typical bioactivities described for such proteins.
Ponce-Soto
Luis Alberto
LA
Department of Biochemistry, Institute of Biology, State University of Campinas, P.O. Box 6109, Zip code 13083-970, Campinas, SP, Brazil. poncesoto@yahoo.com.ar
Baldasso
Paulo Aparecido
PA
Romero-Vargas
Frey Francisco
FF
Winck
Flávia V
FV
Novello
José Camillo
JC
Marangoni
Sergio
S
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D005591:Chemical Fractionation; D002645:Chickens; D003435:Crotalid Venoms / Q000737:chemistry / Q000201:enzymology* / Q000302:isolation & purification / Q000633:toxicity; D017839:Crotalus; D003964:Diaphragm / Q000187:drug effects; D007526:Isoelectric Point; D007527:Isoenzymes; D051379:Mice; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000473:pathology; D032446:Myoblasts / Q000187:drug effects / Q000378:metabolism; D009336:Necrosis / Q000473:pathology; D010741:Phospholipases A / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity*; D054467:Phospholipases A2; D010791:Phrenic Nerve / Q000187:drug effects; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003435:Crotalid Venoms; D007527:Isoenzymes; D010741:Phospholipases A; C523284:Cdr-12 protein, Crotalus durissus ruruima; C523285:Cdr-13 protein, Crotalus durissus ruruima; D054467:Phospholipases A2