Investigation of the lectin-like binding domains in pertussis toxin using synthetic peptide sequences. Identification of a sialic acid binding site in the S2 subunit of the toxin.
Synthetic peptides corresponding to selected sequences in the S2 and S3 subunits of pertussis toxin were prepared and evaluated for their ability to inhibit the binding of biotinylated pertussis toxin and three biotinylated sialic acid specific plant lectins to fetuin and asialofetuin. The screening results indicated that two regions in the S2 subunit corresponding to amino acids 78-98 and 123-154 inhibited pertussis toxin binding to fetuin at submillimolar concentrations, while S3 sequences corresponding to amino acids 87-108 and 134-154 inhibited pertussis toxin-biotin binding to asialofetuin albeit with lower affinity. These results confirm earlier findings, which suggest that the S2 subunit is responsible for binding sialylated glycoconjugates. This was further confirmed by the ability of S2 peptides to inhibit the binding of the lectins from Maackia amurensis and wheat germ to fetuin. Two additional peptides from the S2 subunit of pertussis toxin corresponding to sequences 9-23 and 1-23 were found to contain within their sequences a 6-amino acid fragment which has strong homology with a sequence in wheat germ agglutinin that has been shown to be a component of the sialic acid binding site as determined by x-ray crystallography. One of these sequences from S2 (9-23) was biotinylated and evaluated for its ability to bind to carbohydrate. Through a series of experiments using fetuin, asialofetuin, asialoagalactofetuin, and simple saccharides, the biotinylated peptide was shown to bind with high affinity to sialic acid-containing glycoconjugates indicating that these sequences within the S2 subunit of pertussis toxin also play an important role in binding sialic acid.
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay; D007700:Kinetics; D037102:Lectins / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D037342:Pertussis Toxin; D012794:Sialic Acids / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000737:chemistry / Q000378:metabolism*; D000509:alpha-Fetoproteins / Q000378:metabolism
D037102:Lectins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010455:Peptides; D012794:Sialic Acids; D010566:Virulence Factors, Bordetella; D000509:alpha-Fetoproteins; D037342:Pertussis Toxin; D019158:N-Acetylneuraminic Acid