Analysis of sequences required for the cytotoxic action of a chimeric toxin composed of Pseudomonas exotoxin and transforming growth factor alpha.
Chimeric toxins composed of transforming growth factor alpha fused to mutant forms of Pseudomonas exotoxin bind to the EGF receptor and kill cells bearing these receptors. In early experiments, the binding domain of Pseudomonas exotoxin was deleted and replaced with TGF alpha to make TGF alpha-PE40. This chimeric toxin required proteolytic processing within the target cell to be converted to its active form (Siegall et al. (1989) FASEB J. 3, 2647-2652). Subsequently, recombinant toxins that do not require proteolytic processing were constructed by inserting TGF alpha near the carboxyl terminus of domain III and deleting toxin residues up to the processing site at position 280. In addition, the carboxyl terminus of this toxin was converted from REDLK to KDEL to increase its activity. Recombinant toxins of this type, termed PE37/TGF alpha/KDEL, are about 100-fold more potent than TGF alpha-PE40. To determine if other sequences can be removed from such chimeric toxins to make a smaller molecule that can penetrate tissues better, we have carried out a deletion analysis of sequences present within domains II and Ib. We find that all of domain Ib and a portion of domain II can be deleted without significant loss of cytotoxic activity, but larger deletions extending further into domain II lose cytotoxic activity. We also find that inserting a small linking peptide (Gly)4Ser between residual sequences in domain II and domain III, in molecules with diminished cytotoxic activity, enhances cytotoxicity suggesting that one role of domain Ib is to prevent undesirable interactions between domains II and III.(ABSTRACT TRUNCATED AT 250 WORDS)
Kihara
A
A
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Pastan
I
I
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001427:Bacterial Toxins; D001483:Base Sequence; D001665:Binding Sites; D016923:Cell Death / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D066246:ErbB Receptors / Q000378:metabolism; D005098:Exotoxins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D005260:Female; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009368:Neoplasm Transplantation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology*; D017421:Sequence Analysis; D013329:Structure-Activity Relationship; D016211:Transforming Growth Factor alpha / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose; D000970:Antineoplastic Agents; D001427:Bacterial Toxins; D066246:ErbB Receptors; D005098:Exotoxins; D011993:Recombinant Fusion Proteins; D016211:Transforming Growth Factor alpha; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A