Alonzi Dominic S, Neville David C A, Lachmann Robin H, Dwek Raymond A, Butters Terry D
Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Biochem J. 2008 Jan 15;409(2):571-80. doi: 10.1042/BJ20070748.
The inhibition of ER (endoplasmic reticulum) alpha-glucosidases I and II by imino sugars, including NB-DNJ (N-butyl-deoxynojirimycin), causes the retention of glucose residues on N-linked oligosaccharides. Therefore, normal glycoprotein trafficking and processing through the glycosylation pathway is abrogated and glycoproteins are directed to undergo ERAD (ER-associated degradation), a consequence of which is the production of cytosolic FOS (free oligosaccharides). Following treatment with NB-DNJ, FOS were extracted from cells, murine tissues and human plasma and urine. Improved protocols for analysis were developed using ion-exchange chromatography followed by fluorescent labelling with 2-AA (2-aminobenzoic acid) and purification by lectin-affinity chromatography. Separation of 2-AA-labelled FOS by HPLC provided a rapid and sensitive method that enabled the detection of all FOS species resulting from the degradation of glycoproteins exported from the ER. The generation of oligosaccharides derived from glucosylated protein degradation was rapid, reversible, and time- and inhibitor concentration-dependent in cultured cells and in vivo. Long-term inhibition in cultured cells and in vivo indicated a slow rate of clearance of glucosylated FOS. In mouse and human urine, glucosylated FOS were detected as a result of transrenal excretion and provide unique and quantifiable biomarkers of ER-glucosidase inhibition.
包括NB-DNJ（N-丁基-脱氧野尻霉素）在内的亚氨基糖对内质网（ER）α-葡萄糖苷酶I和II的抑制作用，会导致葡萄糖残基保留在N-连接寡糖上。因此，正常的糖蛋白通过糖基化途径的运输和加工被阻断，糖蛋白被导向进行内质网相关降解（ERAD），其结果是产生胞质游离寡糖（FOS）。用NB-DNJ处理后，从细胞、小鼠组织以及人血浆和尿液中提取FOS。开发了改进的分析方案，先采用离子交换色谱法，然后用2-氨基苯甲酸（2-AA）进行荧光标记，再通过凝集素亲和色谱法进行纯化。通过高效液相色谱法（HPLC）分离2-AA标记的FOS提供了一种快速且灵敏的方法，能够检测源自内质网输出的糖蛋白降解产生的所有FOS种类。在培养细胞和体内，源自糖基化蛋白降解的寡糖的生成迅速、可逆，且与时间和抑制剂浓度相关。在培养细胞和体内的长期抑制表明糖基化FOS的清除率较低。在小鼠和人类尿液中，由于经肾排泄而检测到糖基化FOS，它们是内质网葡萄糖苷酶抑制作用的独特且可定量的生物标志物。
