Fig. 4: Membrane-bound Neu2 enhances desialylation of α2,6-linked sialic acids on membrane.

a, b Neu2-transfected MIAPaCa2 and AsPC1 cells exhibited decreased α2,6-linked sialic acids. Status of sialic acids on the cell surface was demonstrated through the binding of FITC-conjugated two sialic acid-binding lectins (SNA and MALII). Mock and Neu2-transfected (1 × 10⁵) MIAPaCa2 and AsPC1 cells were washed and resuspended in lectin binding buffer. Cells were incubated separately with FITC-conjugated SNA for 1 h. FITC positivity was acquired by FACS. Data are represented as mean ± S.D. from three independent experiments. c, d Elevated level of Neu2 expression in membrane fraction in Neu2-overexpressed MIAPaCa2 and AsPC1 cells. Cell lysate was prepared from Neu2-transfected cells; membrane fraction was isolated by ultracentrifugation. Representative immunoblots experiment confirmed increased Neu2 expression in membrane fraction of transfected PDAC cells than mock. E-cadherin was used to show the purity of membrane preparation and also used as loading control. e Cell lysate exhibited enhanced association of Fas with Neu2 in Neu2-overexpressed cells. Cell lysates from mock and Neu2-overexpressed MIAPaCa2 and AsPC1 cells were incubated with anti-Fas antibody for overnight and immunoprecipitated. The immuno-complex was resolved by SDS-PAGE. Subsequently, it was identified by anti-Neu2 antibody and detected by ECL. Representative immunoblots of co-immunoprecipitation experiments of cell lysate in PDAC confirmed increased binding of Fas with Neu2 in Neu2-overexpressed cells. IgG was used as negative control. f Enhanced association of membrane-bound Neu2 with Fas in Neu2-overexpressed cells. Co-immunoprecipitation experiments with membrane fractions of mock and Neu2-overexpressed MIAPaCa2 and AsPC1 cells confirmed enhanced association of Neu2 with Fas. IgG was used as negative control. g, h Decreased sialylation of Fas in Neu2-transfected cells. Cell lysate from mock and Neu2-overexpressed MIAPaCa2 and AsPC1 cells were incubated with anti-Fas antibody for overnight and immunoprecipitated. Immuno-complex was resolved, and subsequently incubated with biotinylated-α2,6-linked sialic acid binding lectin (SNA) then developed by avidin-HRP antibody and detected by ECL. Lectin affinity study of Neu2-transfected MIAPaCa2 and AsPC1 cells confirmed decreased association of Fas with SNA. i Schematic representation of Neu2-mediated regulation for desialylation of Fas and downstream signaling for inducing extrinsic-mediated apoptosis by activated Fas