Selective toxin-lipid membrane interactions of natural, haemolytic Scyphozoan toxins analyzed by surface plasmon resonance.
A comparison of the molecular interaction of natural Scyphozoan lysins with their bioactivity in a haemolytic assay was performed by establishing an efficient, automatable and reproducible procedure for the measurement of protein-membrane interactions. The toxin-membrane interactions were analyzed utilising a chip-based technology with immobilized liposomes as artificial cell membranes. The technique was established with streptolysin O as a cholesterol-selective model toxin and its cholesterol-selectivity has been proven. The haemolytic potency of protein fractions derived from the venom of the jellyfish Aurelia aurita and Cyanea capillata was tested and EC50 values of 35.3mug/mL and 43.1mug/mL against sheep and 13.5mug/mL and 8.8mug/mL against rabbit erythrocytes were measured. Cell membrane binding as a first step in the haemolytic process was analyzed using the Biacore((R)) technology. Major cell membrane lipids (cholesterol, sphingomyelin and phosphatidylcholine) were immobilized as pure liposomes and in binary mixtures. A preference for cholesterol and sphingomyelin of both jellyfish species was demonstrated. The specificity of the method was proven with a non-haemolytic A. aurita protein fraction that did not express a lipid binding. Additionally, an inactivated C. capillata lysine with negligible haemolytic activity showed a remaining but reduced adsorption onto lipid layers. The binding level of the lytic venom fraction of these dominant boreal jellyfish species increased as a function of protein concentration. The binding strength was expressed in RU50 values ranging from 12.4mug/mL to 35.4mug/mL, which were in the same order of magnitude as the EC50 values in the haemolytic assay.
Helmholz
Heike
H
GKSS Forschungszentrum Geesthacht GmbH, Institute for Coastal Research, Department: Marine Bioanalytical Chemistry, Max-Planck-Street 1, D-21502 Geesthacht, Germany. heike.helmholz@gkss.de
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism; D003064:Cnidarian Venoms / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006461:Hemolysis / Q000187:drug effects; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D037401:Scyphozoa / Q000737:chemistry*; D012756:Sheep; D013109:Sphingomyelins / Q000737:chemistry / Q000378:metabolism; D013301:Streptolysins / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*; D013997:Time Factors
D001426:Bacterial Proteins; D003064:Cnidarian Venoms; D008081:Liposomes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D013109:Sphingomyelins; D013301:Streptolysins; C025964:streptolysin O; D002784:Cholesterol