Cystatin F, a cysteine peptidase inhibitor, is a potent modulator of NK cytotoxicity. By inhibiting granule-mediated cytotoxicity pathway, cystatin F induces formation of non-functional NK cell... Show moreCystatin F, a cysteine peptidase inhibitor, is a potent modulator of NK cytotoxicity. By inhibiting granule-mediated cytotoxicity pathway, cystatin F induces formation of non-functional NK cell stage, called split-anergy. We show that N-glycosylation determines the localization and cellular function of cystatin F. Cystatin F mostly exhibited high-mannose glycosylation in U-937 cells, both high-mannose and complex glycosylation in NK-92 and primary NKs, and predominantly complex glycosylation in super-charged NKs. Manipulating N-glycosylation with kifunensine increased high-mannose glycosylation of cystatin F and lysosome localisation, which decreased cathepsin C activity and reduced NK cytotoxicity. Mannose-6-phosphate could significantly reduce the internalization of extracellular cystatin F. By comparing NK cells with different cytotoxic potentials, we found that high-mannose cystatin F was strongly associated with lysosomes and cathepsin C in NK-92 cell line. In contrast, in highly cytotoxic super-charged NKs, cystatin F with complex glycosylation was associated with the secretory pathway and less prone to inhibit cathepsin C. Modulating glycosylation to alter cystatin F localisation could increase the cytotoxicity of NK cells, thereby enhancing their therapeutic potential for treating cancer patients. Show less
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
Deschepper, F.M.; Zoppi, R.; Pirro, M.; Hensbergen, P.J.; Dall'Olio, F.; Kotsias, M.; ... ; Videira, P.A. 2020
Metastasis is the main cause of death among colorectal cancer (CRC) patients. E-selectin and its carbohydrate ligands, including sialyl Lewis X (sLe(X)) antigen, are key players in the binding of... Show moreMetastasis is the main cause of death among colorectal cancer (CRC) patients. E-selectin and its carbohydrate ligands, including sialyl Lewis X (sLe(X)) antigen, are key players in the binding of circulating tumor cells to the endothelium, which is one of the major events leading to organ invasion. Nevertheless, the identity of the glycoprotein scaffolds presenting these glycans in CRC remains unclear. In this study, we firstly have characterized the glycoengineered cell line SW620 transfected with the fucosyltransferase 6 (FUT6) coding for the alpha 1,3-fucosyltransferase 6 (FUT6), which is the main enzyme responsible for the synthesis of sLe(X) in CRC. The SW620FUT6 cell line expressed high levels of sLe(X) antigen and E-selectin ligands. Moreover, it displayed increased migration ability. E-selectin ligand glycoproteins were isolated from the SW620FUT6 cell line, identified by mass spectrometry, and validated by flow cytometry and Western blot (WB). The most prominent E-selectin ligand we identified was the neural cell adhesion molecule L1 (L1CAM). Previous studies have shown association of L1CAM with metastasis in cancer, thus the novel role as E-selectin counter-receptor contributes to understand the molecular mechanism involving L1CAM in metastasis formation. 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
Background: The Ca2+-dependent C-type lectin receptor Macrophage Galactose-type Lectin (MGL) is highly expressed by tolerogenic dendritic cells (DC) and macrophages. MGL exhibits a high binding... Show moreBackground: The Ca2+-dependent C-type lectin receptor Macrophage Galactose-type Lectin (MGL) is highly expressed by tolerogenic dendritic cells (DC) and macrophages. MGL exhibits a high binding specificity for terminal alpha- and beta-linked GaINAc residues found in Tn, sTn and LacdiNAc antigens. These glycan epitopes are often overexpressed in colorectal cancer (CRC), and, as such, MGL can be used to discriminate tumor from the corresponding healthy tissues. Moreover, the high expression of MGL ligands is associated with poor diseasefree survival in stage III of CRC tumors. Nonetheless, the glycoproteins expressed by tumor cells that are recognized by MGL have hitherto remained elusive.Methods: Using a panel of three CRC cell lines (HCT116, HT29 and LS174T), recapitulating CRC diversity, we performed FACS staining and pull-down assays using a recombinant soluble form of MGL (and a mutant MGL as control) combined with mass spectrometry-based (glyco)proteomics.Results: HCT116 and HT29, but not LS174T, are high MGL-binding CRC cell lines. On these cells, the major cell surface binding proteins are receptors (e.g. MET, PTK7, SORL1, PTPRF) and integrins (ITGB1, ITGA3). From these proteins, several N- and/or O-glycopeptides were identified, of which some carried either a LacdiNAc or Tn epitope.Conclusions: We have identified cell surface MGL-ligands on CRC cell lines.General significance: Advances in (glyco)proteomics have led to identification of candidate key mediators of immune-evasion and tumor growth in CRC. Show less
Maturation of human Dendritic Cells (DCs) is characterized by increased expression of antigen presentation molecules, and overall decreased levels of sialic acid at cell surface. Here, we aimed to... Show moreMaturation of human Dendritic Cells (DCs) is characterized by increased expression of antigen presentation molecules, and overall decreased levels of sialic acid at cell surface. Here, we aimed to identify sialylated proteins at DC surface and comprehend their role and modulation. Mass spectrometry analysis of DC's proteins, pulled down by a sialic acid binding lectin, identified molecules of the major human histocompatibility complex class I (MHC-I), known as human leucocyte antigen (HLA). After desialylation, DCs showed significantly higher reactivity with antibodies specific for properly folded MHC-I-beta 2-microglobulin complex and for beta 2-microglobulin but showed significant lower reactivity with an antibody specific for free MHC-I heavy chain. Similar results for antibody reactivities were observed for TAP2-deficient lymphoblastoid T2 cells, which express HLA-A*02:01. Using fluorescent peptide specifically fitting the groove of HLA-A*02:01, instead of antibody staining, also showed higher peptide binding on desialylated cells, confirming higher surface expression of MHC-I complex. A decay assay showed that desialylation doubled the half-life of MHC-I molecules at cell surface in both DCs and T2 cells. The biological impact of DC ' s desialylation was evaluated in co-cultures with autologous T cells, showing higher number and earlier immunological synapses, and consequent significantly increased production of IFN-gamma by T cells. In summary, sialic acid content modulates the expression and stability of complex MHC-I, which may account for the improved DC-T synapses. Show less
Kotsias, M.; Blanas, A.; Vliet, S.J. van; Pirro, M.; Spencer, D.I.R.; Kozak, R.P. 2019
The study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes.... Show moreThe study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes. Monitoring glycosylation in diagnosis, prognosis, as well as bio-pharmaceutical development and quality control are important research areas. A number of techniques for the analysis of protein N-glycosylation are currently available. Here we examine three methodologies routinely used for the release of N-glycans, in the effort to establish and standardize glycoproteomics technologies for quantitative glycan analysis from cultured cell lines. N-glycans from human gamma immunoglobulins (IgG), plasma and a pool of four cancer cell lines were released following three approaches and the performance of each method was evaluated. Show less
Pirro, M.; Schoof, E.; Vliet, S.J. van; Rombouts, Y.; Stella, A.; Ru, A. de; ... ; Hensbergen, P.J. 2019
C-type lectins are a diverse group of proteins involved in many human physiological and pathological processes. Most C-type lectins are glycan-binding proteins, some of which are pivotal for innate... Show moreC-type lectins are a diverse group of proteins involved in many human physiological and pathological processes. Most C-type lectins are glycan-binding proteins, some of which are pivotal for innate immune responses against pathogens. Other C-type lectins, such as the macrophage galactose-type lectin (MGL), have been shown to induce immunosuppressive responses upon the recognition of aberrant glycosylation on cancer cells. MGL is known to recognize terminal N-acetylgalactosamine (GalNAc), such as the Tn antigen, which is commonly found on malignant cells. Even though this glycan specificity of MGL is well described, there is a lack of understanding of the actual glycoproteins that bind MGL. We present a glycoproteomic workflow for the identification of MGL-binding proteins, which we applied to study MGL ligands on the human Jurkat leukemia cell line. In addition to the known MGL ligands and Tn antigen-carrying proteins CD43 and CD45 on these cells, we have identified a set of novel cell-surface ligands for MGL. Importantly, for several of these, O-glycosylation has hitherto not been described. Altogether, our data provide new insight into the identification and structure of novel MGL ligands that presumably act as modulatory molecules in cancer immune responses. Show less
Kotsias, M.; Blanas, A.; Vliet, S.J. van; Pirro, M.; Spencer, D.I.R.; Kozak, R.P. 2019
The study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes.... Show moreThe study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes. Monitoring glycosylation in diagnosis, prognosis, as well as biopharmaceutical development and quality control are important research areas. A number of techniques for the analysis of protein N-glycosylation are currently available. Here we examine three methodologies routinely used for the release of N-glycans, in the effort to establish and standardize glycoproteomics technologies for quantitative glycan analysis from cultured cell lines. N-glycans from human gamma immunoglobulins (IgG), plasma and a pool of four cancer cell lines were released following three approaches and the performance of each method was evaluated. Show less
