Fig. 1: Glycoengineered cell-based high-throughput screening identifies lead compounds altering O-glycan reporter protein glycosylation.

a Schematic presentation of the glycoengineering strategy yielding HEK293 (HEK) cells that stably secrete Tn-LDLR (HEK^{KO COSMC}) or T-MUC1 (HEK^{KO GCNT1, ST3GAL1/2}) reporter proteins, respectively, for cell-based HTP screening of 1952 compounds in a 96-well plate format by a VVA lectin ELLA (enzyme-linked lectin assay) detecting loss/gain of the Tn epitope on the reporter proteins. Secreted protein reporters include 6x-His-tag (His), FLAG-tag (F) and Myc-tag (M). b, c Dot plots show binding of VVA to LDLR (b) or MUC1 (c) reporter proteins secreted by glycoengineered cells treated with the 1952 compounds organized per 96-well plate, each containing 80 compounds and 3 DMSO vehicle controls. VVA binding is presented as fold-change binding normalized to DMSO control (black line). Active compounds were selected based on cut-off values (gray lines) for VVA binding to Tn-LDLR ( > 4-fold decrease) and to T-MUC1 ( > 3-fold increase). c, e Representative western blots show changes in molecular weight of the Tn-LDLR (c) and T-MUC1 (d) reporters secreted from HEK^WT cells treated with 10 µM of each lead compound or DMSO control for 24 h. Reporters were detected by anti-6x His-tag antibodies. f, g Western blots show the MUC1 reporter produced in HEK^WT (f) and HEK^{KO COSMC} (g) cells incubated for 24 h with 0–10 µM NSC80997 or NSC255112 and detected by anti-6xHis-tag antibody (upper panel) and VVA (lower panel).