N-terminal truncation mutagenesis of equinatoxin II, a pore-forming protein from the sea anemone Actinia equina.
The role of the N-terminal segment 1-33 of equinatoxin II, a 20 kDa pore-forming protein from the sea anemone Actinia equina, was studied by N-truncation mutagenesis. A part of this segment was classified as being amphiphilic and membrane seeking. Wild-type equinatoxin II and its mutants lacking 5, 10 and 33 amino acid residues, respectively, were produced in Escherichia coli using T7 RNA polymerase-based expression vector. Soluble recombinant proteins were isolated from bacterial lysates and assayed for their inhibition by sphingomyelin, binding to red blood cells and hemolytic activity. The N-terminal deletion of 33 amino acids resulted in an insoluble protein, while mutants lacking 5 and 10 residues expressed increased relative avidity for sphingomyelin and red blood cell membranes. Their specific hemolytic activity was decreased, however, with increasing truncation. The results suggest that the N-terminus, which has been found to be conserved in sea anemone pore-forming toxins, contributes to the solubility of the equinatoxin II, but it is not essential for binding to lipid membranes. It is very likely that the N-terminus play a role in the formation of functional pores.
Anderluh
G
G
Department of Biology, Biotechnical Faculty, University of Ljubljana, Slovenia.
Pungercar
J
J
Krizaj
I
I
Strukelj
B
B
Gubensek
F
F
Macek
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D003064:Cnidarian Venoms / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D017931:DNA Primers / Q000235:genetics; D004912:Erythrocytes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006461:Hemolysis / Q000187:drug effects; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D012615:Sea Anemones / Q000235:genetics*; D013109:Sphingomyelins / Q000494:pharmacology
D003064:Cnidarian Venoms; D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013109:Sphingomyelins; C012307:equinatoxin