Journal
GLYCOBIOLOGY
Volume 14, Issue 4, Pages 301-310Publisher
OXFORD UNIV PRESS INC
DOI: 10.1093/glycob/cwh044
Keywords
glucosidase I; 1-deoxynojirimycin; photoactive probe
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Funding
- NIGMS NIH HHS [GM59943] Funding Source: Medline
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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 IC50 of 0.42 muM, it is nearly nine times stronger inhibitor than DNM (IC50 = 3.5 muM). On photolysis, the bound [I-125]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.
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