Lactosyl-sepharose binding proteins from pancreatic cancer cells show differential expression in primary and metastatic organs

Abstract

In normal cells, glycan binding proteins mediate various cellular processes upon recognition and binding to respective ligands. In tumor cells, these proteins have been associated with metastasis. Lactosyl-sepharose binding proteins (LSBPs) were isolated and identified in a workflow involving lactosyl affinity chromatography and label-free quantification mass spectrometry (LFQ MS). A binding study with monosaccharides was performed by microscale thermophoresis and nuclear magnetic resonance spectroscopy. Influence of galactose on LSBPs’ binding to the lactosyl resin was investigated by competitive affinity chromatography followed by LFQ MS. An analysis of amino acids with sugar binding motifs was searched using bioinformatics tools. The expression profiles of these proteins at the mRNA level, as determined by a chip array from a pancreatic ductal adenocarcinoma (PDAC) liver metastasis model, were used for evaluating their potential role in cancer progression. Proteomics data and their respective genes were analyzed by MaxQuant and Ingenuity Pathway Analysis. In total, 1295 LSBPs were isolated and identified from Suit2-007 human pancreatic adenocarcinoma cells. Interaction studies revealed that these proteins exhibit low to moderate affinity for monosaccharide sugars. Some of these LSBPs even showed reduced affinity after calcium depletion. Among the isolated proteins were annexins and galectins in addition to other families, with no history of binding lactosyl residues. A subset of LSBPs exhibited differential profiles in the pancreas, liver, and lung environments. These modulations may be related to tumor progression. In conclusion, we show that PDAC cells contain LSBPs, a subset of which binds galactose with calcium dependency. The differential expression of these proteins in a rat model highlights their value for diagnosis and as potential drug targets for PDAC therapy. Future work will be required to validate these findings in patient samples.

Impact statement

Interaction of glycan binding proteins with aberrantly expressed glycans in tumor environment is crucial for metastasis. Here, we established a work flow for investigating the presence of a subset of these proteins in PDAC cells, which bind to a lactosyl-sepharose resin. The resin had been designed to isolate proteins with lectin-like properties. The corresponding lactosyl-sepharose binding proteins (LSBPs) show affinity for galactose and other monosaccharides. A subset of the LSBPs shows also calcium dependency. The importance of these proteins is highlighted by their differential expression profiles in PDAC cells growing in primary (pancreas) and metastatic (liver and lung) organ sites. Based on their affinity for the lactosyl-resin and monosaccharides, LSBPs hold potential for PDAC diagnosis and as drug targets. This work has set the stage for further investigation of the occurrence and the role of LSBPs in patient samples using the newly established workflow.