N-Glycoproteins Have a Major Role in MGL Binding to Colorectal Cancer Cell Lines: Associations with Overall Proteome Diversity

Abstract

Colorectal cancer (CRC) is the second-leading cause of cancer death worldwide due in part to a high proportion of patients diagnosed at advanced stages of the disease. For this reason, many efforts have been made towards new approaches for early detection and prognosis. Cancer-associated aberrant glycosylation, especially the Tn and STn antigens, can be detected using the macrophage galactose-type C-type lectin (MGL/CLEC10A/CD301), which has been shown to be a promising tool for CRC prognosis. We had recently identified the major MGL-binding glycoproteins in two high-MGL-binding CRC cells lines, HCT116 and HT29. However, we failed to detect the presence of O-linked Tn and STn glycans on most CRC glycoproteins recognized by MGL. We therefore investigated here the impact of N-linked and O-linked glycans carried by these proteins for the binding to MGL. In addition, we performed quantitative proteomics to study the major differences in proteins involved in glycosylation in these cells. Our results showed that N-glycans have a significant, previously underestimated, importance in MGL binding to CRC cell lines. Finally, we highlighted both common and cell-specific processes associated with a high-MGL-binding phenotype, such as differential levels of enzymes involved in protein glycosylation, and a transcriptional factor (CDX-2) involved in their regulation.

Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2)

“…Of note, the AGR2 O-glycopeptide was found at higher levels in one of the three cell lines (LS174T) compared to the others (HT29 and LS174T). This corresponds to the overall higher level of AGR2 that was found in this cell line [23], indicating that the AGR2 O-glycosylation per se is not different between the CRC cell lines.…”

“…To test this, we examined LC-MS/MS data from TMT-labeled tryptic peptides from three different CRC cell lines (9-plex TMT, three cell lines in triplicate) acquired on an Orbitrap Exploris, prompted by the notion that the simultaneous quantification of the glycopeptide would be an additional asset. Moreover, we have an interest in these cell lines because we recently used them as a model system to study differential binding of a human lectin [22,23]. From the data, we selected MS/MS spectra with the fragment ion at m/z 204.087 and identified Nand O-glycopeptides using Byonic, accepting glycopeptide matches with a score above 250.…”

The myeloid cell biomarker EMR1 is ectopically expressed in colon cancer

“…An interesting possibility to explain these results is that soluble CD206 has bound to carbohydrate structures at the tumor cell surface. It is well established, that the glycosylation pattern of surface glycoproteins and glycolipids are changed in epithelial tumors compared their normal counterparts, so called "tumor-associated carbohydrate antigens" [34].…”

Glycoproteins Presenting Galactose and N-Acetylgalactosamine in Human Seminal Plasma as Potential Players Involved in Immune Modulation in the Fertilization Process

“…Galectins recognize β-galactosides and great interest on their impact on tumor progression has arised in the recent years [ 37 , 42 ]. Macrophage galactose binding lectin (MGL), present on the surface of macrophages and the other cells of immune system, is specific for GalNAc and involved in regulation of the immune response [ 53 , 54 , 55 , 56 , 57 , 58 ].…”