The interaction between tumor and immune cells in the microenvironment plays a key role in oncogenesis. This recognition can be mediated by changes in glycosylation on tumor cells which are sensed... Show moreThe interaction between tumor and immune cells in the microenvironment plays a key role in oncogenesis. This recognition can be mediated by changes in glycosylation on tumor cells which are sensed by lectins, such as the Macrophage Galactose-type Lectin (MGL), expressed on immune cells, resulting in immunosuppressive responses. MGL binding to the Tn-epitope on MUC1 triggers DCs to stimulate T-regulatory responses and tolerance against the tumor, while suppressing T-effector cells responsible for tumor eradication. This undesirable consequence in cancer led to interest in the characterization of targets of MGL. This thesis dealt with the analysis of glycoproteins binding to MGL expressed by different tumor cell models. For this purpose, a robust method to enrich MGL binding proteins, in combination with their mass spectrometry-based identification was established. Because of the high level of the Tn-antigen, due to a Cosmc mutation, the Jurkat cell line was initially used for this purpose (Chapter 2) but a slightly adjusted method was later applied to high- and low-MGL binding CRC cell lines as well (Chapter 3 and 5). These analyses led to the identification of hitherto unknown MGL binders. In Chapter 4, we focused on to the contribution of N-glycan MGL binding glycotopes in CRC cell lines, most probably corresponding to LacdiNAc structures, by implementing overall N-glycan release in our workflows. With this, we were able to show that N-glycoproteins represent a hitherto underestimated group of MGL binding protein in these cell lines. Also several secreted proteins from the CRC cell lines could bind to MGL (Chapter 5), indicating that the interaction with immune cells can also be mediated by this group of proteins. The results from previously published transcriptomics and N-/O-glycomic analyses could not explain the different expression of MGL binding proteins on the CRC cell lines used. For this reason, in Chapter 4, we extended our research with full comparative quantitative proteomics analyses, in an attempt to explain the differences in MGL binding, for example by different levels of MGL binding proteins or proteins involved in glycosylation pathways. Additionally, in Chapter 6, we used such a quantitative proteomics dataset also to test the suitability of a previously suggested mass spectrometry-based method to discriminate O-GalNAc (Tn) versus O-GlcNAc, which led to the first site-specific identification of O-glycosylation of both intracellular and secreted anterior gradient protein 2 (AGR2). Show less
Developments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions... Show moreDevelopments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions in glycopeptide fragmentation spectra had been used to assign different sets of glycopeptides. In particular, this was helpful to discriminate between O-GalNAc and O-GlcNAc. Here, we thought to investigate how such information can be used to examine quantitative proteomics data. For this purpose, we used tandem mass tag (TMT)-labeled samples from total cell lysates and secreted proteins from three different colorectal cancer cell lines. Following automated glycopeptide assignment (Byonic) and evaluation of the presence and relative intensity of oxonium ions, we observed that, in particular, the ratio of the ions at m/z 144.066 and 138.055, respectively, could be used to discriminate between O-GlcNAcylated and O-GalNAcylated peptides, with concomitant relative quantification between the different cell lines. Among the O-GalNAcylated proteins, we also observed anterior gradient protein 2 (AGR2), a protein which glycosylation site and status was hitherto not well documented. Using a combination of multiple fragmentation methods, we then not only assigned the site of modification, but also showed different glycosylation between intracellular (ER-resident) and secreted AGR2. Overall, our study shows the potential of broad application of the use of the relative intensities of oxonium ions for the confident assignment of glycopeptides, even in complex proteomics datasets. Show less
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... Show moreColorectal 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 ofO-linked Tn and STn glycans on most CRC glycoproteins recognized by MGL. We therefore investigated here the impact ofN-linked andO-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 thatN-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. Show less
