Glucosylated free oligosaccharides are biomarkers of endoplasmic reticulum {alpha}-glucosidase inhibition
Résumé
The inhibition of endoplasmic reticulum (ER) α-glucosidases I and II by imino sugars including N-butyl-deoxynojirimycin (NB-DNJ), 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 ER-associated degradation (ERAD), a consequence of which is the production of cytosolic free oligosaccharides (FOS). 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 (anthranilic 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 free oligosaccharides. In mouse and human urine, glucosylated free oligosaccharides were detected as a result of transrenal excretion and provide unique and quantifiable biomarkers of ER-glucosidase inhibition.
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