Cell targeting of a pore-forming toxin, CytA delta-endotoxin from Bacillus thuringiensis subspecies israelensis, by conjugating CytA with anti-Thy 1 monoclonal antibodies and insulin.
The cytolytic protein toxin CytA was linked to two monoclonal antibodies (mAb) directed against the mouse or the rat Thy 1 antigen. The purified CytA-mAb conjugates were not toxic to either target or nontarget cells. The conjugates did bind specifically to target cells since they agglutinated the target cells but not nontarget cells. When the conjugates were treated with dithiothreitol, the released CytA was toxic to all cells tested. These results suggested that the attachment of CytA to a molecule such as the mAb prevented it from forming a pore. Another conjugate was made by linking CytA to insulin. The purified insulin-CytA conjugate bound to and intoxicated cells bearing a high number of insulin receptors. Furthermore, the conjugate was far less toxic to cells expressing a low number of insulin receptors and not toxic to a known CytA target cell line from Aedes aegypti. However, CytA released from the conjugate by reduction was toxic to all cells tested. These results suggested that the cytotoxicity exhibited by CytA in the conjugate form against cells bearing insulin receptors was mediated through insulin and that, in the conjugate form, CytA no longer shows its broad in vitro cytolytic activity. The difference in toxicity between CytA-mAb conjugates and insulin-CytA conjugate is discussed in relation to size of the ligands, the number, distribution, and mobility of the target molecules, and intracellular trafficking.
al-yahyaee
S A
SA
Department of Biochemistry, University of Cambridge, United Kingdom. men0804@squ.edu
Ellar
D J
DJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001413:Bacillus thuringiensis; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins; D001667:Binding, Competitive; D002460:Cell Line; D009033:Culicidae; D019286:Endopeptidase K / Q000378:metabolism; D004731:Endotoxins / Q000378:metabolism*; D006460:Hemolysin Proteins; D006461:Hemolysis; D018796:Immunoconjugates; D007328:Insulin / Q000378:metabolism*; D007518:Isoantibodies / Q000378:metabolism*; D051379:Mice; D051381:Rats; D013601:T-Lymphocytes / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D018796:Immunoconjugates; D007328:Insulin; D007518:Isoantibodies; C035678:anti-Thy antibody; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D019286:Endopeptidase K