Site-specific alterations in the B oligomer that affect receptor-binding activities and mitogenicity of pertussis toxin.
Pertussis toxin plays a major role in the pathogenesis of whooping cough and is considered an important constituent of vaccines against this disease. It is composed of five different subunits associated in a molar ratio 1S1:1S2:1S3:2S4:1S5. The S1 subunit is responsible for the ADP-ribosyltransferase activity of the toxin. The B moiety, composed of S2 through S5, recognizes and binds to the target cell receptors and has some ADP-ribosyltransferase-independent activities such as mitogenicity. Site-directed mutagenesis of subunits S2 and S3 allowed us to identify amino acid residues involved in receptor binding. Of all the modifications generated, the deletion of Asn 105 in S2 and of Lys 105 in S3 resulted in the more drastic reduction of binding to haptoglobin and CHO cells, respectively. A holotoxin carrying both deletions presented a mitogenicity reduced to an undetectable level. The combination of these B oligomer mutations with two substitutions in the S1 subunit led to the production of a toxin analog with reduced ADP-ribosyltransferase-dependent and -independent activities including mitogenicity. As shown by immunoprecipitation with various monoclonal antibodies, the mutant holotoxin was correctly assembled and antigenically similar to the native toxin. This toxin analog induced toxin-neutralizing antibodies at the same level as the holotoxin carrying only mutations in the S1 subunit, and may therefore be considered a useful candidate for the development of a new generation vaccine against whooping cough.
Lobet
Y
Y
Research Department, SmithKline Beecham Biologicals, Rixensart, Belgium.
Feron
C
C
Dequesne
G
G
Simoen
E
E
Hauser
P
P
Locht
C
C
D036002:ADP Ribose Transferases / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001886:Bordetella pertussis / Q000235:genetics*; D016466:CHO Cells / Q000187:drug effects; D006224:Cricetinae; D007376:Interleukin-2 / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008934:Mitogens / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D037342:Pertussis Toxin; D013329:Structure-Activity Relationship; D010566:Virulence Factors, Bordetella / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*
D007376:Interleukin-2; D008934:Mitogens; D010566:Virulence Factors, Bordetella; D036002:ADP Ribose Transferases; D037342:Pertussis Toxin