Skip to main content
. Author manuscript; available in PMC: 2023 Jul 1.
Published in final edited form as: Nat Chem. 2022 Oct 10;15(1):33–42. doi: 10.1038/s41557-022-01059-z

Fig. 5. Sigelc-7(SFY) cross-linked with azido-GD3 in vitro and with sialoglycan on cell surface.

Fig. 5.

a) ESI-MS spectrum of intact Siglec-7v(104SFY) confirmed SFY incorporation. b) Cross-linking of azido-GD3 with Siglec-7v with SFY incorporated at indicated Lys sites. c) Sigelc-7(SFY) cross-linked with azido-GD3 but not azido-lac. d) Crystal structure of Siglec-7v in complex of GT1b (PDB: 2HRL), showing Lys104, Lys127, and Gln129 in magenta stick, at which SFY incorporation led to cross-linking of azido-GD3. e-f) Flow cytometric quantification of Siglec-7v protein bound on SK-MEL-28 cell surface. After washing, more Siglec-7v(127SFY) bound with sialoglycan on SK-MEL-28 cell surface than WT Siglec-7v (e). When the cells were pretreated with sialidase to remove cell surface sialoglycan (f), the binding markedly decreased and the difference became small or nonsignificant. The line and error bar represent mean ± SEM; n = 3 independent batches of Siglec-7v proteins. * p < 0.05; ** p < 0.01, *** p < 0.001; NS, not significant; two-sided t test. P values in panel e: 0.000002 for 0.6 μM, <0.000001 for 3 μM, and <0.000001 for 12 μM. P values in panel f: 0.0069 for 0.6 μM, 0.074 for 3 μM, and 0.029 for 12 μM.