title: Photoaffinity labeling of a bacterial sialidase with an aryl azide derivative of sialic acid.
abstract: A photoreactive radioiodinatable derivative of 2-deoxy-2,3-didehydro-5-N-acetylneuraminic acid (NeuAc2en), 5-N-acetyl-9-(4-azidosalicoylamido)-2-deoxy-2,3-didehydroneuram inic acid (ASA-NeuAc2-en) has been synthesized and used to label the active site of Clostridium perfringens sialidase. Like NeuAc2en, its aryl azide derivative is a strong competitive inhibitor of sialidase (Ki approximately 15 microM). The absorbance spectrum of ASA-NeuAc2en shows a characteristic aryl azide peak, which disappears upon photolysis with UV light. When its radioiodinated counterpart 5-N-acetyl-9-(4-iodoazidosalicoylamido)-2-deoxy-2,3-didehydrone uraminic acid ([125I]IASA-NeuAc2en) was photolyzed in the presence of C. perfringens sialidase a 72-kDa protein was labeled. Labeling occurred specifically in the active site since it was inhibited in the presence of NeuAc2en. Chemical cleavage of the photoaffinity-labeled 72-kDa protein demonstrates that specifically labeled peptides involved in the formation of the active site can easily be determined. ASA-NeuAc2en is a valuable new tool for the identification and structural/functional analysis of sialidases and other proteins, recognizing this sialic acid derivative.
authors.affiliation: Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands.
authors.forename: G T
authors.initials: GT
authors.lastname: van der Horst
authors.forename: G M
authors.initials: GM
authors.lastname: Mancini
authors.forename: R
authors.initials: R
authors.lastname: Brossmer
authors.forename: U
authors.initials: U
authors.lastname: Rose
authors.forename: F W
authors.initials: FW
authors.lastname: Verheijen
mesh_terms: D000345:Affinity Labels; D001386:Azides / Q000138:chemical synthesis / Q000494:pharmacology; D001665:Binding Sites; D003016:Clostridium perfringens / Q000201:enzymology*; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D007457:Iodine Radioisotopes; D015394:Molecular Structure; D008970:Molecular Weight; D009439:Neuraminidase / Q000037:antagonists & inhibitors; D010446:Peptide Fragments; D010777:Photochemistry; D012794:Sialic Acids / Q000138:chemical synthesis / Q000494:pharmacology; D013053:Spectrophotometry
chemical_list: D000345:Affinity Labels; D001386:Azides; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D012794:Sialic Acids; C064632:5-N-acetyl-9-(4-azidosalicoylamido)-2-deoxy-2,3-didehydroneuraminic acid; D009439:Neuraminidase; D003488:Cyanogen Bromide