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. 2021 Dec 16;59(10):957–964. doi: 10.1136/jmedgenet-2021-108061

Figure 4.

Figure 4

Investigation of the functional consequences of the ARSK variants. ARSK-WT but not ARSK-Arg84Cys desulfates synthetic 2-sulfoglucuronate-N-acetylglucosamine (G2A0) disaccharides (A, B). (A) G2A0 treated with cell lysates expressing ARSK-WT resulted in a minor peak at 26.5 mL representing the 2-O-sulfated educt (m/z 670.15) and a major peak at 28 mL retention volume representing the desulfated product (m/z 590.19), indicating the loss of a sulfate group (highlighted in yellow). Analysis with C18-reversed-phase chromatography. (B, C) Incubation of AMAC-labelled G2A0 disaccharide with ARSK-Arg84Cys cell lysates or with cell lysates of untransfected cells resulted in a main AMAC-peak at 26 mL (m/z 670.15). The G0A0-mediated fluorescence signal remained the minor peak in both samples. Of note, this minor peak in untransfected as well as in ARSK-Arg84Cys transfected cells results most likely from the activity of the endogenous ARSK of the HT1080 cells. The ubiquitous peak in the right of the chromatogram (>30 mL of retention volume) was not analysed in more detail as it was also present in unreacted samples. Western blot analysis (D): Comparable expression levels for ARSK-WT and ARSK-Arg84Cys but lack of ARSK-Leu187Ter in HT1080 cell lysates. This indicates comparable stability of ARSK-WT and ARSK-Arg84Cys and probable nonsense mediated mRNA-decay as a consequence of the Leu187Ter variant. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. WT, wild type.