title: Synthesis of a novel photoaffinity derivative of 1-deoxynojirimycin for active site-directed labeling of glucosidase I.
abstract: Glucosidase I releases the distal alpha1,2-glucosyl residue in the Glc(3)Man(9)GlcNAc(2) precursor immediately after its transfer from the dolichol-P-P-linked intermediate in the endoplasmic reticulum and triggers the processes for the posttranslational remodeling, folding, and maturation of N-linked glycoproteins. The enzyme has been purified and characterized from several eukaryotic systems. Its cDNA and the gene have also been cloned. The enzyme is a target for the development of drugs for several pathological conditions. A structural analysis on the biochemically purified enzyme has been hampered because of its low abundance and unstable character. The recombinant enzyme has not been obtained in quantity and characterized. Glucosidase I is strongly inhibited by the glucose analog 1-deoxynojirimycin (DNM). To gain an insight into the architecture of the active site of the enzyme, we here report the synthesis of a photoactive derivative of DNM, viz. 4-(rho-azidosalicylamido)butyl-5-amido-pentyl-1-DNM (ASBA-P-DNM). With an IC(50) of 0.42 micro M, it is nearly nine times stronger inhibitor than DNM (IC(50) = 3.5 micro M). On photolysis, the bound [(125)I]ASBA-P-DNM specifically labels the native enzyme, which yields a 24-kDa peptide after treatment with V8 protease, apparently representing the region around its active site. Thus ASBA-P-DNM should serve as a novel reagent to conduct structure-function analysis on glucosidase I.
authors.affiliation: Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA.
authors.forename: Andrew V
authors.initials: AV
authors.lastname: Romaniouk
authors.forename: Anne
authors.initials: A
authors.lastname: Silva
authors.forename: Jie
authors.initials: J
authors.lastname: Feng
authors.forename: Inder K
authors.initials: IK
authors.lastname: Vijay
mesh_terms: D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D007456:Iodine Isotopes; D008969:Molecular Sequence Data; D015394:Molecular Structure; D019920:Photoaffinity Labels / Q000138:chemical synthesis* / Q000737:chemistry; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D007456:Iodine Isotopes; D019920:Photoaffinity Labels; D017485:1-Deoxynojirimycin; C042059:glucosidase I; D000520:alpha-Glucosidases