A subgroup of lactosyl-Sepharose binding proteins requires calcium for affinity and galactose for anti-proliferation

Abstract

Lactosyl-Sepharose binding proteins (LSBPs) were recently described in human pancreatic ductal adenocarcinoma (PDAC) Suit2-007 cells regarding their lectin-like properties and role in metastasis. This study further investigated how calcium and galactose influence the binding of LSBPs to the lactosyl resin as well as their anti-proliferative effect in Suit2-007 cells. Altered binding of LSBPs to the lactosyl resin was evaluated by affinity chromatography and mass spectrometry. Calcium binding EF-hand proteins were aligned and identified with a motif derived from the Uniprot protein database. The antiproliferative effects of LSBPs and monosaccharides were determined by MTT assay. In addition, LSBPs and galactose effects were investigated by chip array and tumor take in nude rats. LSBPs reduced Suit2-007 cells' proliferation with an IC50 of 125 μg/mL. Coincubation of LSBPs with EGTA decreased the number of LSBPs binding to the lactosyl resin by ~50%. Ca2+ ˗sensitive LSBPs included subgroups of galactose-sensitive (10%) and EF-hand calcium binding motifs containing (2.5%) proteins. In vitro, the combination of LSBPs with monosaccharides including galactose synergistically decreased cell proliferation compared to single agents (p < 0.05). In addition, LSBPs in combination with galactose prevented the tumor growth of Suit2-007 cells in nude rats, as opposed to single treatments. At mRNA level, the combination treatment modulated 5% of Ca2+ ˗sensitive LSBPs and downregulated 216 genes, 18% of which were up-regulated during PDAC progression. This study highlights the importance of calcium and galactose in modulating the affinity and anti-proliferative activity of LSBPs and their potential application as therapeutic agents for metastatic PDAC.