Identification and characterization of the carbohydrate ligands recognized by pertussis toxin via a glycan microarray and surface plasmon resonance.
Binding of pertussis toxin (PTx) was examined by a glycan microarray; 53 positive hits fell into four general groups. One group represents sialylated biantennary compounds with an N-glycan core terminating in alpha2-6-linked sialic acid. The second group consists of multiantennary compounds with a canonical N-glycan core, but lacking terminal sialic acids, which represents a departure from the previous understanding of PTx binding to N-glycans. The third group consists of Neu5Acalpha2-3(lactose or N-acetyllactosamine) forms that lack the branched mannose core found in N-glycans; thus, their presentation is more similar to that of O-linked glycans and glycolipids. The fourth group of compounds consists of Neu5Acalpha2-8Neu5Acalpha2-8Neu5Ac, which is seen in the c series gangliosides and some N-glycans. Quantitative analysis by surface plasmon resonance of the relative affinities of PTx for terminal Neu5Acalpha2-3 versus Neu5Acalpha2-6, as well as the affinities for the trisaccharide Neu5Acalpha2-8Neu5Acalpha2-8Neu5Ac versus disaccharide, revealed identical global affinities, even though the amount of bound glycan varied by 4-5-fold. These studies suggest that the conformational space occupied by a glycan can play an important role in binding, independent of affinity. Characterization of N-terminal and C-terminal binding sites on the S2 and S3 subunits by mutational analysis revealed that binding to all sialylated compounds was mediated by the C-terminal binding sites, and binding to nonsialylated N-linked glycans is mediated by the N-terminal sites present on both the S2 and S3 subunits. A detailed understanding of the glycans recognized by pertussis toxin is essential to understanding which cells are targeted in clinical disease.
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524, USA.
Scott H
SH
Millen
Daniel M
DM
Lewallen
Andrew B
AB
Herr
Suri S
SS
Iyer
Alison A
AA
Weiss
D000606:Amino Sugars; D002240:Carbohydrate Sequence; D002241:Carbohydrates; D008024:Ligands; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism; D037342:Pertussis Toxin / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D012794:Sialic Acids; D020349:Surface Plasmon Resonance; D014312:Trisaccharides
D000606:Amino Sugars; D002241:Carbohydrates; D008024:Ligands; D011134:Polysaccharides; D012794:Sialic Acids; D014312:Trisaccharides; C000458:N-acetyllactosamine; D037342:Pertussis Toxin; D019158:N-Acetylneuraminic Acid