Alanine scanning mutagenesis identifies surface amino acids on domain II of Pseudomonas exotoxin required for cytotoxicity, proper folding, and secretion into periplasm.
Pseudomonas exotoxin A (PE) is a single polypeptide chain that contains 613 amino acids and is arranged into three major structural domains. Domain Ia is responsible for cell recognition, domain II for translocation of PE across the membrane, and domain III for ADP-ribosylation of elongation factor 2. Recombinant PE can be produced in Escherichia coli and is efficiently secreted into the periplasm when an OmpA signal sequence is present. To investigate the role of the amino acids located on the surface of domain II in the action of the toxin against mammalian cells, we substituted alanine for each of the 27 surface amino acids present in domain II. Surprisingly, all 27 mutant proteins had some alteration in cytotoxicity when tested on human A431 or MCF7 cells or mouse L929 cells. Native PE has a compact structure and therefore is relatively protease resistant and very little ADP-ribosylation activity is detected in the absence of the denaturing agents like urea and dithiothreitol. Several of the mutations resulted in altered protease sensitivity of the toxin. Seven of the mutant molecules exhibited ADP-ribosylation activity without urea and dithiothreitol, indicating they are partially unfolded. Out of these seven mutants, six had increased cytotoxic activity on at least one of the target cell lines and the other retained its native cytotoxic potency.
Kasturi
S
S
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Kihara
A
A
FitzGerald
D
D
Pastan
I
I
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000409:Alanine; D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002918:Chymotrypsin / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose; D000409:Alanine; D001427:Bacterial Toxins; D002918:Chymotrypsin; D005098:Exotoxins; D011994:Recombinant Proteins; D014357:Trypsin; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A