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.
Millen
Scott H
SH
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524, USA.
Lewallen
Daniel M
DM
Herr
Andrew B
AB
Iyer
Suri S
SS
Weiss
Alison A
AA
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