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.
Highlights
Glycosylation is one of the most frequent post-translational modifications on proteins and lipids [1]
One of the most common process is represented by the dysregulation of the initial steps of O-glycan biosynthesis mediated by polypeptide α-N-acetylgalactosaminyl-transferases that transfer UDP-GalNAc to Ser/Thr residues in a polypeptide, forming Tn antigen (GalNAcα1-Ser/Thr) [6,8]
We studied theproteins of Colorectal cancer (CRC) cell lines using a combination of lectin staining, MGL pull-downs in the absence and presence of PNGase F treatment and overall comparative quantitative proteomics using Tandem Mass Tag (TMT) labeling
Summary
Glycosylation is one of the most frequent post-translational modifications on proteins and lipids [1]. Humans have 20 genes encoding ppGalNAcTs, and an overexpression, a higher activity and/or a modified subcellular location of these transferases can lead to higher Tn antigen expression, frequently decorating mucins on epithelial tumors [9]. The expression of Tn and STn can be enhanced by defects in the activity of T-synthase or mutations in its chaperone (Cosmc), which together mediate the synthesis of core 1 O-glycans (T antigen, Galβ1,3-GalNAcα1-O-Ser/Thr) [10]. Enhanced expression of sialyltransferases modifies the latter antigens into the sialylated species SLex, and SLey. Higher activity of the mannoside acetyl-glucosaminyltransferase 5 (MGAT5) increases N-glycan branching, while dysregulation of the β4-N-acetylgalactosaminyltransferase 3 (B4GALNT3) may lead to the expression of terminal LacdiNAc (GalNAcβ1,4GlcNAcβ1-) on both N-linked [11] and O-linked glycans [12]
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