Figure 7.

Sia α2-8 Sia α2-3 structure is essential Siglec-7 binding to ligand glycans

(A) The glioblastoma cell line LN319 was transfected with pcDNA3.1 ST8Sia6 or pcDNA3.1 empty vector using Lipofectamine 2000. Those cells were selected using G418 and cloned using a limiting dilution method. The binding of Siglec-7-Fc to the LN319 ST8Sia6 gene transfectant and mock. Thus, ST8Sia6 further enhanced the binding of Siglec-7-Fc to the cell lines, as shown in colorectal carcinomas DLD-1.

(B) α2-3 specific sialidase SiaS can cut terminal α2-3-linked sialic acid, but it cannot cut non-terminal (inside) α2-3-linked sialic acid. However, pan sialidase (SiaP) removes all linkage modes of Sia.

(C) Biotinylated membrane proteins of LN319 and of ST8Sia6 transfectant LN319 were detected using avidin-HRP. Upper panel indicates biotinylated membrane proteins from mock and ST8Sia6-LN319 transfectants with pan sialidase (SiaP) or α2-3 Sia specific sialidase (SiaS) treatment and subsequent IB. Biotin-labeled products were applied for IP with recombinant Siglec-7-Fc and analyzed using IB with avidin-HRP (ABC) to detect all biotinylated molecules. Middle panel indicates IB of IP products with anti-PDPN antibody. Lower panel indicates PDPN molecular size changed due to sialylation and sialidases.

(D) DLD-1 and the ST transfectants, SW837 and K562 were treated with SiaP or SiaS for 1 h at 37°C. The binding of Siglec-7-Fc was analyzed using FACS and the mean fluorescence intensity is shown in the graph.