Fig. 5.

Probing the contribution of sulfotransferases to Siglec sialoglycan recognition. (A) Sulfotransferases knocked in into HEK^WT cells and predicted sulfated glycan structures are shown. Heat map shows Siglec Fc binding to the KI cells as MFI values normalized to HEK^WT. (B) Dot plots show Siglec-3/7/8/15 binding to HEK^WT and HEK^{KI CHST1} cells treated with PBS or sialidase. Data of three independent experiments are presented as average MFI ± SEM. (C) Representative histograms show binding of Siglec-3/7/8/15 to CHO^WT and CHO^{KI CHST1} cells. Cells stained only with anti-human IgG AF647 indicate background fluorescence. (D) Radar charts show Siglec-3 (Left) and Siglec-8 (Right) binding to HEK^WT and HEK^{KI CHST1} cells with additional KO of O-GalNAc glycans (KO COSMC), N-glycans (KO MGAT1), glycolipids (KO B4GALT5), N-glycans and glycolipids (KO MGAT1, KO B4GALT5), or α2-3Sia on N-glycans (single or double KO ST3GAL4/6). (E) Siglec binding to glycan arrays. Recombinant Siglec-8 and Siglec-3 Fc chimera were tested for binding to two sialoglycan arrays: a 123-glycan array printed on glass slides (Left) and an 11-glycan sialoglycolipid array on 384-well plates. The glycan structures tested are listed in SI Appendix. Binding is expressed in arbitrary fluorescence units for the printed array and as colorimetric enzyme activity (ΔA405/min × 1,000) for the glycolipid array. Each point is the average of quadruplicate determinations. As indicated, a replicate glycolipid array was pretreated with 50 mU/mL Vibrio cholerae sialidase for 90 min at 37 °C and washed prior to addition of the Siglec Fc chimeras. (F) Radar chart shows Siglec-7 (Left) and Siglec-15 (Right) binding to HEK^WT and HEK^{KI CHST1} cells with additional KO of O-GalNAc glycans (KO COSMC), core2 (KO GCNT1), N-glycans (KO MGAT1), glycolipids (KO B4GALT5), or N-glycans and glycolipids (KO MGAT1, KO B4GALT5). Representative MFI values of three independent experiments are shown, and deleted glycoconjugates are depicted in gray.