- Email: [email protected]
cDNA cloning and protein sequence analysis of a K49-PLA2 myotoxin: a highly conserved class of proteins
Abstracts
292
noncytolytic peptides is the production of fatty acids. In the case of the PLA, myotoxins, fatty acid production is the direct result of the toxin PLA, activity, whereas with the cytolytic peptides the fatty acids are derived from tissue phospholipase C and, possibly, tissue PLA,. Despite very different structures, the myotoxic agents share many mechanisms of action. Lipid metabolism may be an important factor increasing the rapidity and extent of myotoxicity. Fletcher,
J. E. and Jiang,
M.-S. (1993) Toxicon 31, 669495.
Myotoxicity of venoms and toxins. P. Gopalakrishnakone University of Singapore, Singapore 0511).
(Venom
and
Toxin
Research
Group,
National
The term myotoxicity is used in a wider sense when a venom or toxin acts on skeletal muscle and causes changes in the muscle as shown by biochemical, pharmacological or morphological evidence. There are two types of PLA, toxin which cause muscle damage: crotoxin, taipoxin, notexin and mojave toxin produce muscle degeneration followed by regeneration when injected locally, whereas PLA, from Enhydrina schistosa and PLA, from Australian elapids of the species Pseudechis, produce systemic myotoxicity, characterized by myoglobinuria and generalized muscle damage. Muscle damage can be observed by light and electron microscopic methods. The changes include dilatation of sarcoplasmic reticulum, vacuolation, oedema within 4 hr of injection of toxin, followed by disruption and hypercontraction of the fibres with inflammatory changes by about 6 hr. The inflammatory reaction characterized by infiltration by phagocytic cells was maximally seen between 12 and 24 hr. Evidence of regeneration stated by 36 hr and complete regeneration was seen by 7-10 days. The mechanism(s) which these toxins cause muscle damage is not fully understood. However, the disruption of sarcolemmal membrane, increase in influx of Car+ into sarcoplasm, and Ca2+- activated neutral proteases have a definitive role in this process. The possible mechanism(s) for muscle degeneration, as well as structure-function activity of the PLA, myotoxins in reference to enzymatic activity, neurotoxicity and myotoxicity will be discussed. Scorpion venoms which cause myodegeneration will also be presented for the first time. cDNA cloning and protein sequence analysis of a K49-PLA, myotoxin: a highly conserved class of proteins. H. S. Selistre de Arauio.‘,j S. P. White’ and C. L. Ownbv’ (Departments of ‘Physiological Sciences and rBiochemistry . and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, U.S.A.; and ‘Department de Ciencias Fisiologicas, Universidade Federal de SBo Carlos, SHo Carlos, SP 13565, Brazil). _.
_
A few catalytically inactive snake venom phospholipase A, (PLA,) myotoxins have been described, indicating that the PLA, activity is not necessary for induction of myotoxicity. However, the myotoxic site in these molecules is still unknown. To aid the understanding of the complex structure-function relationship of the myotoxins, we have cloned and sequenced the cDNA for ACL myotoxin from an Agkistrodon contortrix laficinctus venom gland cDNA library. The library was made in the UNILAP’” vector and screened with a degenerate oligonucleotide probe which was designed from the N-terminal sequence of the purified ACL myotoxin. One of the isolated clones (ACLPREMTI) is 734 bp in length and has an open reading frame of 414 bp. It codes for a K49 PLA, with 121 amino acid residues, of which the first 20 match exactly the N-terminal sequence of the purified ACL myotoxin. ACLPREMTI has a domain organization that is very conserved in the type II PLA, gene family in Vipcridae snakes. Comparison of the predicted sequence of ACL myotoxin and other K49 and D49 PLA, proteins shows that K49 PLA, myotoxins have several very conserved residues not present in D49 enzymes. Also, sequence comparison and molecular modeling of ACL myotoxin show the presence of at least two different molecular sites in type II PLA,s. These results may help to explain the difference in activities between distinct classes of PLA,s. HSSA has a fellowship
from CNPq,
Brazil
Partial biochemical characterization of a myotoxin from the Duvernoy’s secretion of Philodryas olfersii. J. Prado-Franceschi.’ S. Hyslop,’ J. C. Cogo,’ A. L. Andrade,’ A. P. Reichk3 M. T. Assakura,’ M. A. CruzHofling’ and L. Rodrigues-Simioni’ (Departments of ‘Pharmacology and *Histology, UNICAMP, Campinas, SP, Brazil; and 3Laboratory of Biochemistry and Biophysics, Instituto Butantan, SLo Paulo, SP, Brazil). The Duvernoy’s secretion of the xenodontine colubrid Philodryas olfersii (green snake) possesses a variety of biological activities including fibrin(ogen)olytic, hemorrhagic and edematogenic actions (Assakura et al., 1992, 1994). In the present work, the protein responsible for the myotoxic activity previously observed in the above secretion was purified to homogeneity by chromatography on Sephadex G-100 SF. In SDS-PAGE, the toxin migrates as a single band with a mol. wt of 20,000. The myotoxin contains 182 amino acid residues (approx. 20% acidic) and has a blocked N-terminal amino acid. The pl of the protein is 4.8. In vitro, the myotoxin stimulates the release of creatine kinase from the chick biventer cervicis preparation in a manner similar to the crude Duvernoy’s secretion. Histological examination showed that the myotoxin causes severe muscle necrosis
292
noncytolytic peptides is the production of fatty acids. In the case of the PLA, myotoxins, fatty acid production is the direct result of the toxin PLA, activity, whereas with the cytolytic peptides the fatty acids are derived from tissue phospholipase C and, possibly, tissue PLA,. Despite very different structures, the myotoxic agents share many mechanisms of action. Lipid metabolism may be an important factor increasing the rapidity and extent of myotoxicity. Fletcher,
J. E. and Jiang,
M.-S. (1993) Toxicon 31, 669495.
Myotoxicity of venoms and toxins. P. Gopalakrishnakone University of Singapore, Singapore 0511).
(Venom
and
Toxin
Research
Group,
National
The term myotoxicity is used in a wider sense when a venom or toxin acts on skeletal muscle and causes changes in the muscle as shown by biochemical, pharmacological or morphological evidence. There are two types of PLA, toxin which cause muscle damage: crotoxin, taipoxin, notexin and mojave toxin produce muscle degeneration followed by regeneration when injected locally, whereas PLA, from Enhydrina schistosa and PLA, from Australian elapids of the species Pseudechis, produce systemic myotoxicity, characterized by myoglobinuria and generalized muscle damage. Muscle damage can be observed by light and electron microscopic methods. The changes include dilatation of sarcoplasmic reticulum, vacuolation, oedema within 4 hr of injection of toxin, followed by disruption and hypercontraction of the fibres with inflammatory changes by about 6 hr. The inflammatory reaction characterized by infiltration by phagocytic cells was maximally seen between 12 and 24 hr. Evidence of regeneration stated by 36 hr and complete regeneration was seen by 7-10 days. The mechanism(s) which these toxins cause muscle damage is not fully understood. However, the disruption of sarcolemmal membrane, increase in influx of Car+ into sarcoplasm, and Ca2+- activated neutral proteases have a definitive role in this process. The possible mechanism(s) for muscle degeneration, as well as structure-function activity of the PLA, myotoxins in reference to enzymatic activity, neurotoxicity and myotoxicity will be discussed. Scorpion venoms which cause myodegeneration will also be presented for the first time. cDNA cloning and protein sequence analysis of a K49-PLA, myotoxin: a highly conserved class of proteins. H. S. Selistre de Arauio.‘,j S. P. White’ and C. L. Ownbv’ (Departments of ‘Physiological Sciences and rBiochemistry . and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, U.S.A.; and ‘Department de Ciencias Fisiologicas, Universidade Federal de SBo Carlos, SHo Carlos, SP 13565, Brazil). _.
_
A few catalytically inactive snake venom phospholipase A, (PLA,) myotoxins have been described, indicating that the PLA, activity is not necessary for induction of myotoxicity. However, the myotoxic site in these molecules is still unknown. To aid the understanding of the complex structure-function relationship of the myotoxins, we have cloned and sequenced the cDNA for ACL myotoxin from an Agkistrodon contortrix laficinctus venom gland cDNA library. The library was made in the UNILAP’” vector and screened with a degenerate oligonucleotide probe which was designed from the N-terminal sequence of the purified ACL myotoxin. One of the isolated clones (ACLPREMTI) is 734 bp in length and has an open reading frame of 414 bp. It codes for a K49 PLA, with 121 amino acid residues, of which the first 20 match exactly the N-terminal sequence of the purified ACL myotoxin. ACLPREMTI has a domain organization that is very conserved in the type II PLA, gene family in Vipcridae snakes. Comparison of the predicted sequence of ACL myotoxin and other K49 and D49 PLA, proteins shows that K49 PLA, myotoxins have several very conserved residues not present in D49 enzymes. Also, sequence comparison and molecular modeling of ACL myotoxin show the presence of at least two different molecular sites in type II PLA,s. These results may help to explain the difference in activities between distinct classes of PLA,s. HSSA has a fellowship
from CNPq,
Brazil
Partial biochemical characterization of a myotoxin from the Duvernoy’s secretion of Philodryas olfersii. J. Prado-Franceschi.’ S. Hyslop,’ J. C. Cogo,’ A. L. Andrade,’ A. P. Reichk3 M. T. Assakura,’ M. A. CruzHofling’ and L. Rodrigues-Simioni’ (Departments of ‘Pharmacology and *Histology, UNICAMP, Campinas, SP, Brazil; and 3Laboratory of Biochemistry and Biophysics, Instituto Butantan, SLo Paulo, SP, Brazil). The Duvernoy’s secretion of the xenodontine colubrid Philodryas olfersii (green snake) possesses a variety of biological activities including fibrin(ogen)olytic, hemorrhagic and edematogenic actions (Assakura et al., 1992, 1994). In the present work, the protein responsible for the myotoxic activity previously observed in the above secretion was purified to homogeneity by chromatography on Sephadex G-100 SF. In SDS-PAGE, the toxin migrates as a single band with a mol. wt of 20,000. The myotoxin contains 182 amino acid residues (approx. 20% acidic) and has a blocked N-terminal amino acid. The pl of the protein is 4.8. In vitro, the myotoxin stimulates the release of creatine kinase from the chick biventer cervicis preparation in a manner similar to the crude Duvernoy’s secretion. Histological examination showed that the myotoxin causes severe muscle necrosis