Synthesis of a novel photoaffinity derivative of 1-deoxynojirimycin for active site-directed labeling of glucosidase I.
Synthesis of a novel photoaffinity derivative of 1-deoxynojirimycin for active site-directed labeling of glucosidase I.
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DOI：
10.1093/glycob/cwh044
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发表时间：
2004-04
期刊：
影响因子：
4.3
通讯作者：
A. Romaniouk;Anne Silva;Jie Feng;I. K. Vijay
中科院分区：
文献类型：
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作者：
A. Romaniouk;Anne Silva;Jie Feng;I. K. Vijay
葡萄糖苷酶I在Glc（3）Man（9）GlcNAc（2）前体从内质网中的长胆固醇-P-P-连接中间体转移后立即释放其远端α 1，2-葡萄糖基残基，并触发N-连接糖蛋白的翻译后重塑、折叠和成熟过程。该酶已被纯化，并从几个真核系统的特点。它的cDNA和基因也已克隆。该酶是开发用于多种病理状况的药物的靶标。由于其丰度低和性质不稳定，对生物化学纯化的酶的结构分析受到阻碍。重组酶尚未大量获得和表征。葡萄糖苷酶I被葡萄糖类似物1-脱氧野尻霉素（DNM）强烈抑制。为了深入了解酶的活性位点的结构，我们在这里报告的光活性衍生物的DNM，即4-（rho-azidosalicylamido）丁基-5-氨基戊基-1-DNM（ASBA-P-DNM）的合成。IC（50）为0.42 μ M，其抑制剂比DNM（IC（50）= 3.5 μ M）强近9倍。在光解时，结合的[（125）I]ASBA-P-DNM特异性标记天然酶，其在用V8蛋白酶处理后产生24-kDa肽，显然代表其活性位点周围的区域。因此ASBA-P-DNM有望成为一种新型的糖苷酶I结构-功能分析试剂。
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.