Journal Article

Synthesis of a novel photoaffinity derivative of 1-deoxynojirimycin for active site-directed labeling of glucosidase I

Andrew V. Romaniouk, Anne Silva, Jie Feng and Inder K. Vijay

in Glycobiology

Published on behalf of Society for Glycobiology

Volume 14, issue 4, pages 301-310
Published in print April 2004 | ISSN: 0959-6658
Published online January 2004 | e-ISSN: 1460-2423 | DOI: https://dx.doi.org/10.1093/glycob/cwh044
Synthesis of a novel photoaffinity derivative of 1-deoxynojirimycin for active site-directed labeling of glucosidase I

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Glucosidase I releases the distal α1,2-glucosyl residue in the Glc3Man9GlcNAc2 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-(ρ-azidosalicylamido)butyl-5-amido-pentyl-1-DNM (ASBA-P-DNM). With an IC50 of 0.42 µM, it is nearly nine times stronger inhibitor than DNM (IC50 = 3.5 µM). On photolysis, the bound [125I]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.

Keywords: glucosidase I; 1-deoxynojirimycin; photoactive probe; AA-P-DNM, 4-azidoanilido-N-pentyl-1-deoxynojirimycin; AE-DNM, N-(2-aminoethanoyl)-1-deoxynojirimycin; AP-DNM, N-(2-aminopropanoyl)-1-deoxynojirimycin; ASBA, ρ-azido-salicyl-butyl-amine; ASBA-P-DNM,4-(ρ-azidosalicylamido)butyl-5-amido-pentyl-1-deoxynojirimycin; CM-DNM, N-2-carboxymethyl-1-deoxynojirimycin; CP-DNM,N-5-carboxypentyl-1-deoxynojirimycin; DMSO, dimethyl sulfoxide; DMF, N,N′-Dimethylformamide; DNM, 1-deoxynojirimycin; EDC, 1-ethyl-3-(3-dimethylaminoprpyl)-carbodiimide; ER, endoplasmic reticulum; MES, 2-(4-morpholine)-Ethane Sulfate Acid; NHS, N-hydroxysuccinimide; NMR, nuclear magnetic resonance; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TCA, trichloroacetic acid; TLC, thin-layer chromatography

Journal Article.  6314 words.  Illustrated.

Subjects: Carbohydrates

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