Localisation of a receptor-recognition domain on the S3 subunit of pertussis toxin by peptide mapping.
Overlapping 10-amino-acid peptides, which consecutively span the amino acid sequence of the S3 subunit of pertussis toxin, were synthesised on polyethylene pins and screened for their ability to bind the glycoprotein fetuin. Fetuin binding was localised to a single peptide comprising amino acids 46-55. A free peptide, (E)S3c, of longer sequence (S3 amino acids 44-58) was also found to bind alpha-1-acid glycoprotein, mixed brain gangliosides and fetuin. (E)S3c also recognised asialofetuin but with a lower apparent affinity relative to fetuin. The single tryptophan residue of the peptide yielded a fluorescence-emission maximum of 355 nm. In the presence of either ganglioside or the phospholipid L-alpha-lysolecithin, but not N-acetylneuramin-lactose or lactosylceramide, the emission intensity of (E)S3c was enhanced and the emission maximum blue-shifted to 340 nm by ganglioside, or to 345 nm by L-alpha-lysolecithin. Monosialogangliosides, disialogangliosides, and trisialogangliosides, when fluorescence-titrated, were each found to bind the peptide with a similar dissociation constant of 4.4 +/- 2.8 microM. These findings demonstrate that region 44-58 of the pertussis-toxin S3 subunit is likely to be involved in the recognition of both glycosylated and phospholipid constituents of target-cell membranes.
Tallett
A
A
Division of Biologics and Biotechnology, Centre for Applied Microbiology and Research, Porton Down, Salisbury, England.
Seabrook
R N
RN
Irons
L I
LI
Robinson
A
A
Van Heyningen
S
S
Atkinson
T
T
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology; D001665:Binding Sites; D004797:Enzyme-Linked Immunosorbent Assay; D005732:Gangliosides / Q000378:metabolism / Q000494:pharmacology; D006017:Glycolipids / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008969:Molecular Sequence Data; D009961:Orosomucoid / Q000378:metabolism; D010449:Peptide Mapping; D037342:Pertussis Toxin; D013050:Spectrometry, Fluorescence; D010566:Virulence Factors, Bordetella / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D000509:alpha-Fetoproteins / Q000378:metabolism
D000906:Antibodies; D000941:Antigens; D005732:Gangliosides; D006017:Glycolipids; D006023:Glycoproteins; D008244:Lysophosphatidylcholines; D009961:Orosomucoid; D010566:Virulence Factors, Bordetella; D000509:alpha-Fetoproteins; D037342:Pertussis Toxin