Glucosylated free oligosaccharides are biomarkers of endoplasmic- reticulum α-glucosidase inhibition

Abstract

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.

Normalizing glycosphingolipids restores function in CD4+ T cells from lupus patients

“…(C) Cumulative quantitative data for each GSL species identified on the HPLC plots. GSL expression was calculated from the peak HPLC areas after applying an experimentally derived response factor (18) by relating the area of the HPLC peak to the cell number of the sample. Two-tailed Mann-Whitney U test; **P = 0.008; *P ≤ 0.05.…”

Serum glycomarkers of endoplasmic reticulum and lysosomal-endosomal system stress in human healthy aging and diseases

“…2, III) and are marked with the same numbers on the chromatograms. Of these, 9 were structural counterparts (Glc 0-3 Man 3-9 GlcNAc 1-2 ) of the intracellular high-mannose FOS as deciphered before [17]. As in the case of serum FOS from healthy volunteers the second peak was structurally distinct from recognised intracellular FOS.…”

“…A comparison with an external standard -the partially hydrolyzed dextran that can be seen in dotted lines on the chromatogram -allowed molecular weight calculation of each species in glucose units (GU, the upper sсale) and then matching them up with the intracellular FOS which structures were well-known. Among intracellular FOS specified before [17], 11 glucosylated or non-glucosylated high-mannose counterparts (Glc 0-3 Man 3-9 GlcNAc 1-2 ) were found for 12 identified serum species and subspecies. There was no identified counterpart for the only species -the second peak.…”

“…Native plasma was precipitated with 10% trichloracetic acid (TCA) and centrifuged for 10 min at 3000 g to deproteinize, partially sterilize, and stabilize before transportation without lyophilisation. The further purification comprised filtration through a syringe with Millex-LH, 0.45 µm, filter (Millipore Corp., USA) or Amicon Ultra-0.5 ml Centrifugal Filter Devices (Millipore Corp., USA) to remove remaining protein, according to Alonzi et al [17].…”

“…The column was washed twice with 1 ml of acetonitrile-water mixture (95:5, v/v). The labelled oligosaccharides were eluted twice with 0.75 ml of Milli-Q™ water and stored at -20 °C [17].…”