The possible involvement of specific regions/loops of cardiotoxin from Naja sputatrix venom in mediating its cytolytic activity is evaluated using a new cytolytic assay. In this assay, the amount of chloramphenicol acetyltransferase (CAT) that is released upon lysis of the cellular membranes by the cytotoxin has been measured as an index of cytolysis. This newly developed CAT system is more sensitive than the traditional haemolysis method utilizing red blood cells or the lactate dehydrogenase assay for cytolysis. Series of chimaeric toxin molecules have been constructed by swapping the loops between highly hydrophilic neurotoxin and highly hydrophobic cardiotoxin molecules from Naja sputatrix, which are known to exhibit structural similarity (three-finger conformation) but to have different functional properties. Comparison of the cytolytic activities of the recombinant chimaeric toxins demonstrated the possible involvement of all three loops of cardiotoxin in its cytolytic potency. However, the first two loops of the protein appear to make the major contribution to its lytic activity. cDNAs encoding cardiotoxin and the chimaeric toxins, when expressed in transfected cultured Chinese hamster ovary cells, resulted in cell lysis, indicating that these cDNAs can be developed as useful cytolytic agents.
- chimaeric toxins
- chloramphenicol acetyltransferase
- homology modelling
- sequential PCR mutagenesis
- three-finger toxins
Abbreviations used: CAT, chloramphenicol acetyltransferase; CHO, Chinese hamster ovary; CTX, cardiotoxin; GST, glutathione S-transferase; LDH, lactate dehydrogenase; MEM, minimal essential medium; NTX, neurotoxin; prefixes in chimaeras denote the source of loops I—III, e.g. CNC-CTX contains loops I and III of CTX-4b (denoted by the Cs in CNC) and loop II of NTX-2 (denoted by the N in CNC).
- The Biochemical Society, London ©2002