Expression, activity and cytotoxicity of pertussis toxin S1 subunit in transfected mammalian cells.
Pertussis toxin (PT) comprises an active subunit (S1), which ADP-ribosylates the alpha subunit of several mammalian G proteins, and the B oligomer (S2-S5), which binds glycoconjugate receptors on cells. In a previous report, expression of S1 in Cos cells resulted in no observable cytotoxicity, and it was hypothesized that either S1 failed to locate its target proteins or the B oligomer was also necessary for cytotoxicity. To address this, we stably transfected S1 with and without a signal peptide into mammalian cells. Immunofluorescence analysis confirmed the function of the signal peptide. Surprisingly, we found that S1 was active in both transfectants, as determined by clustering of transfected Chinese hamster ovary (CHO) cells and ADP-ribosylation of G proteins. Constructs with a cysteine-to-serine change at residue 201 or a truncated S1 (residues 1-181) were also active when transfected into cells. Constructs with an inactive mutant S1 had no activity, confirming that the observed results were due to the activity of the toxin subunit. We conclude that S1 is active when expressed in mammalian cells without the B oligomer, that secretion into the endoplasmic reticulum does not prevent this activity and that the C-terminal portion of S1 is not required for its activity in cells.
Castro
M G
MG
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore 21201, USA.
McNamara
U
U
Carbonetti
N H
NH
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001886:Bordetella pertussis / Q000235:genetics; D016466:CHO Cells / Q000378:metabolism; D019556:COS Cells; D006224:Cricetinae; D003545:Cysteine / Q000235:genetics; D005057:Eukaryotic Cells; D019204:GTP-Binding Proteins / Q000378:metabolism; D009154:Mutation; D037342:Pertussis Toxin; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D012694:Serine / Q000235:genetics; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D010566:Virulence Factors, Bordetella; C083388:pertussis toxin, S1 subunit; D000246:Adenosine Diphosphate Ribose; D012694:Serine; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D003545:Cysteine