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Glycobiology Advance Access published online on January 12, 2004
Glycobiology, doi:10.1093/glycob/cwh044
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Submitted on July 2, 2003
Revised on August 12, 2003
Accepted on November 14, 2003

© 2004 Oxford University Press

ORIGINAL ARTICLES

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

Andrew V. Romaniouk 1, Anne Silva 1, Jie Feng 1, and Inder K. Vijay 1*

1 Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA

* To whom correspondence should be addressed. E-mail: ivijay{at}umd.edu.

Abstract

Glucosidase I releases the distal {alpha}1,2-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 post-translational 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-deoxynojirimycin (ASBA-P-DNM). With an IC50 of 0.42 µM, it is nearly 9 times stronger inhibitor than DNM (IC50 = 3.5 µM). Upon 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.


glucosidase I, 1-deoxynojirimycin, photoactive probe
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