ABSTRACT
HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8+ T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , CD8-Positive T-Lymphocytes/immunology , Glioma/immunology , Glycosphingolipids/metabolism , Glycosyltransferases/metabolism , HLA Antigens/metabolism , Histocompatibility Antigens Class I/metabolism , Immunotherapy/methods , Antigen Presentation , Aspartic Acid Endopeptidases/genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Glioma/mortality , Glycosphingolipids/immunology , HLA Antigens/immunology , Histocompatibility Antigens Class I/immunology , Humans , Lymphocyte Activation , Signal Transduction , Survival Analysis , Tumor EscapeABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis and high mortality. Transforming growth factor-ß (TGF-ß) plays a key role in PDAC tumor progression, which is often associated with aberrant glycosylation. However, how PDAC cells respond to TGF-ß and the role of glycosylation therein is not well known. Here, we investigated the TGF-ß-mediated response and glycosylation changes in the PaTu-8955S (PaTu-S) cell line deficient in SMA-related and MAD-related protein 4 (SMAD4), a signal transducer of the TGF-ß signaling. PaTu-S cells responded to TGF-ß by upregulating SMAD2 phosphorylation and target gene expression. We found that TGF-ß induced expression of the mesenchymal marker N-cadherin but did not significantly affect epithelial marker E-cadherin expression. We also examined differences in N-glycans, O-glycans, and glycosphingolipid-linked glycans in PaTu-S cells upon TGF-ß stimulation. TGF-ß treatment primarily induced N-glycome aberrations involving elevated levels of branching, core fucosylation, and sialylation in PaTu-S cells, in agreement with TGF-ß-induced changes in the expression of glycosylation-associated genes. In addition, we observed differences in O glycosylation and glycosphingolipid glycosylation profiles after TGF-ß treatment, including lower levels of sialylated Tn antigen and neoexpression of globosides. Furthermore, the expression of transcription factor sex-determining region Y-related high-mobility group box 4 was upregulated upon TGF-ß stimulation, and its depletion blocked TGF-ß-induced N-glycomic changes. Thus, TGF-ß-induced N-glycosylation changes can occur in a sex-determining region Y-related high-mobility group box 4-dependent and SMAD4-independent manner in the pancreatic PaTu-S cancer cell line. Our results open up avenues to study the relevance of glycosylation in TGF-ß signaling in SMAD4-inactivated PDAC.
Subject(s)
Carcinoma, Pancreatic Ductal , Glycosphingolipids , Pancreatic Neoplasms , Transforming Growth Factor beta , Carcinoma, Pancreatic Ductal/metabolism , Cell Line, Tumor , Glycosphingolipids/metabolism , Humans , Pancreatic Neoplasms/metabolism , Polysaccharides , Transforming Growth Factor beta/metabolismABSTRACT
The choice for adjuvant chemotherapy in stage II colorectal cancer is controversial as many patients are cured by surgery alone and it is difficult to identify patients with high risk of recurrence of the disease. There is a need for better stratification of this group of patients. Mass spectrometry imaging could identify patients at risk. We report here the N-glycosylation signatures of the different cell populations in a group of stage II colorectal cancer tissue samples. The cancer cells, compared with normal epithelial cells, have increased levels of sialylation and high-mannose glycans, as well as decreased levels of fucosylation and highly branched N-glycans. When looking at the interface between cancer and its microenvironment, it seems that the cancer N-glycosylation signature spreads into the surrounding stroma at the invasive front of the tumor. This finding was more outspoken in patients with a worse outcome within this sample group.
Subject(s)
Colorectal Neoplasms/metabolism , Tumor Microenvironment , Aged , Aged, 80 and over , Colon/metabolism , Colorectal Neoplasms/pathology , Female , Glycomics , Glycosylation , Humans , Intestinal Mucosa/metabolism , Male , Mannose/metabolism , Middle Aged , Neoplasm Staging , Polysaccharides/metabolism , PrognosisABSTRACT
Most patients with multiple myeloma develop a severe osteolytic bone disease. The myeloma cells secrete immunoglobulins, and the presence of monoclonal immunoglobulins in the patient's sera is an important diagnostic criterion. Here, we show that immunoglobulins isolated from myeloma patients with bone disease promote osteoclast differentiation when added to human preosteoclasts in vitro, whereas immunoglobulins from patients without bone disease do not. This effect was primarily mediated by immune complexes or aggregates. The function and aggregation behavior of immunoglobulins are partly determined by differential glycosylation of the immunoglobulin-Fc part. Glycosylation analyses revealed that patients with bone disease had significantly less galactose on immunoglobulin G (IgG) compared with patients without bone disease and also less sialic acid on IgG compared with healthy persons. Importantly, we also observed a significant reduction of IgG sialylation in serum of patients upon onset of bone disease. In the 5TGM1 mouse myeloma model, we found decreased numbers of lesions and decreased CTX-1 levels, a marker for osteoclast activity, in mice treated with a sialic acid precursor, N-acetylmannosamine (ManNAc). ManNAc treatment increased IgG-Fc sialylation in the mice. Our data support that deglycosylated immunoglobulins promote bone loss in multiple myeloma and that altering IgG glycosylation may be a therapeutic strategy to reduce bone loss.
Subject(s)
Antibodies, Monoclonal/immunology , Bone Resorption/immunology , Immunoglobulin G/immunology , Multiple Myeloma/immunology , Neoplasm Proteins/immunology , Aged , Animals , Bone Resorption/pathology , Cell Line, Tumor , Female , Humans , Male , Mice , Middle Aged , Multiple Myeloma/pathologyABSTRACT
Alterations in protein glycosylation in colorectal cancer (CRC) have been extensively studied using cell lines as models. However, little is known about their O-glycome and the differences in glycan biosynthesis in different cell types. To provide a better understanding of the variation in O-glycosylation phenotypes and their association with other molecular features, an in-depth O-glycosylation analysis of 26 different CRC cell lines was performed. The released O-glycans were analysed on porous graphitized carbon nano-liquid chromatography system coupled to a mass spectrometer via electrospray ionization (PGC-nano-LC-ESI-MS/MS) allowing isomeric separation as well as in-depth structural characterization. Associations between the observed glycan phenotypes with previously reported cell line transcriptome signatures were examined by canonical correlation analysis. Striking differences are observed between the O-glycomes of 26 CRC cell lines. Unsupervized principal component analysis reveals a separation between well-differentiated colon-like and undifferentiated cell lines. Colon-like cell lines are characterized by a prevalence of I-branched and sialyl Lewis x/a epitope carrying glycans, while most undifferentiated cell lines show absence of Lewis epitope expression resulting in dominance of truncated α2,6-core sialylated glycans. Moreover, the expression of glycan signatures associates with the expression of glycosyltransferases that are involved in their biosynthesis, providing a deeper insight into the regulation of glycan biosynthesis in different cell types. This untargeted in-depth screening of cell line O-glycomes paves the way for future studies exploring the role of glycosylation in CRC development and drug response leading to discovery of novel targets for the development of anti-cancer antibodies.
Subject(s)
Cell Differentiation , Glycomics/methods , Polysaccharides/analysis , Carbohydrate Sequence , Cell Line, Tumor , Chromatography, High Pressure Liquid , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Glycosylation , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Humans , Phenotype , Polysaccharides/metabolism , Principal Component Analysis , Tandem Mass SpectrometryABSTRACT
Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric basic structure of the nuclear egress complex (core NEC). These core NECs serve as a hexameric lattice-structured platform for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina- and membrane-rearranging functions (multicomponent NEC). Here, we report the X-ray structures of ß- and γ-herpesvirus core NECs obtained through an innovative recombinant expression strategy based on NEC-hook::NEC-groove protein fusion constructs. This approach yielded the first structure of γ-herpesviral core NEC, namely the 1.56 Å structure of Epstein-Barr virus (EBV) BFRF1-BFLF2, as well as an increased resolution 1.48 Å structure of human cytomegalovirus (HCMV) pUL50-pUL53. Detailed analysis of these structures revealed that the prominent hook segment is absolutely required for core NEC formation and contributes approximately 80% of the interaction surface of the globular domains of NEC proteins. Moreover, using HCMV::EBV hook domain swap constructs, computational prediction of the roles of individual hook residues for binding, and quantitative binding assays with synthetic peptides presenting the HCMV- and EBV-specific NEC hook sequences, we characterized the unique hook-into-groove NEC interaction at various levels. Although the overall physicochemical characteristics of the protein interfaces differ considerably in these ß- and γ-herpesvirus NECs, the binding free energy contributions of residues displayed from identical positions are similar. In summary, the results of our study reveal critical details of the molecular mechanism of herpesviral NEC interactions and highlight their potential as an antiviral drug target.
Subject(s)
Betaherpesvirinae/metabolism , Gammaherpesvirinae/metabolism , Viral Proteins/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Cytomegalovirus/metabolism , HeLa Cells , Herpesvirus 4, Human/metabolism , Humans , Peptides/chemistry , Peptides/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Surface Plasmon Resonance , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Human C1-inhibitor (C1-Inh) is a serine protease inhibitor and the major regulator of the contact activation pathway as well as the classical and lectin complement pathways. It is known to be a highly glycosylated plasma glycoprotein. However, both the structural features and biological role of C1-Inh glycosylation are largely unknown. Here, we performed for the first time an in-depth site-specific N- and O-glycosylation analysis of C1-Inh combining various mass spectrometric approaches, including C18-porous graphitized carbon (PGC)-LC-ESI-QTOF-MS/MS applying stepping-energy collision-induced dissociation (CID) and electron-transfer dissociation (ETD). Various proteases were applied, partly in combination with PNGase F and exoglycosidase treatment, in order to analyze the (glyco)peptides. The analysis revealed an extensively O-glycosylated N-terminal region. Five novel and five known O-glycosylation sites were identified, carrying mainly core1-type O-glycans. In addition, we detected a heavily O-glycosylated portion spanning from Thr82-Ser121 with up to 16 O-glycans attached. Likewise, all known six N-glycosylation sites were covered and confirmed by this site-specific glycosylation analysis. The glycoforms were in accordance with results on released N-glycans by MALDI-TOF/TOF-MS/MS. The comprehensive characterization of C1-Inh glycosylation described in this study will form the basis for further functional studies on the role of these glycan modifications.
Subject(s)
Complement C1 Inhibitor Protein/metabolism , Glycosylation , Humans , Polysaccharides/metabolism , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass SpectrometryABSTRACT
Cancer is a major cause of death in both developing and developed countries. Early detection and efficient therapy can greatly enhance survival. Aberrant glycosylation has been recognized to be one of the hallmarks of cancer as glycans participate in many cancer-associated events. Cancer-associated glycosylation changes often involve sialic acids which play important roles in cell-cell interaction, recognition and immunological response. This review aims at giving a comprehensive overview of the literature on changes of sialylation in serum of cancer patients. Furthermore, the methods available to measure serum and plasma sialic acids as well as possible underlying biochemical mechanisms involved in the serum sialylation changes are surveyed. In general, total serum sialylation levels appear to be increased with various malignancies and show a potential for clinical applications, especially for disease monitoring and prognosis. In addition to overall sialic acid levels and the amount of sialic acid per total protein, glycoprofiling of specific cancer-associated glycoproteins, acute phase proteins and immunoglobulins in serum as well as the measurements of sialylation-related enzymes such as sialidases and sialyltransferases have been reported for early detection of cancer, assessing cancer progression and improving prognosis of cancer patients. Moreover, sialic-acid containing glycan antigens such as CA19-9, sialyl Lewis X and sialyl Tn on serum proteins have also displayed their value in cancer diagnosis and management whereby increased levels of these factors positively correlated with metastasis or poor prognosis.
Subject(s)
Biomarkers, Tumor/blood , Neoplasms/blood , Protein Processing, Post-Translational , Sialic Acids/metabolism , Humans , Sialic Acids/bloodABSTRACT
Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation.
Subject(s)
Colorectal Neoplasms/metabolism , Glycomics/methods , Homeodomain Proteins/metabolism , Microfilament Proteins/metabolism , Polysaccharides/metabolism , Caco-2 Cells , Cell Differentiation/genetics , Cell Line, Tumor , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Fucose/metabolism , Gene Expression Regulation, Neoplastic , Glycosylation , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , HCT116 Cells , HT29 Cells , Homeodomain Proteins/genetics , Humans , Microfilament Proteins/genetics , Principal Component Analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass SpectrometryABSTRACT
The human acute monocytic leukemia cell line THP-1 is widely used as an in vitro phagocytic cell model because it exhibits several immune properties similar to native monocyte-derived macrophages. In this study, we investigated the alteration of N- and O-linked glycans as well as glycosphingolipids, during THP-1 differentiation, combining mass spectrometry, flow cytometry, and quantitative real-time PCR. Mass spectrometry revealed that macrophage differentiation led to a marked upregulation of expression of GM3 ganglioside as well as an increase in complex-type structures, particularly triantennary glycans, occurring at the expense of high-mannose N-glycans. Moreover, we observed a slight decrease in the proportion of multifucosylated N-glycans and α2,6-sialylation. The uncovered changes in glycosylation correlated with variations of gene expression of relevant glycosyltransferases and glycosidases including sialyltransferases, ß-N-acetylglucosaminyltransferases, fucosyltransferases, and neuraminidase. Furthermore, using flow cytometry and antibodies directed against glycan structures, we confirmed that the alteration of glycosylation occurs at the cell surface of THP-1 macrophage-like cells. Altogether, we established that macrophagic maturation of THP-1 induces dramatic modifications of the surface glycosylation pattern that may result in differential interaction of monocytic and macrophagic THP-1 with immune or bacterial lectins.
Subject(s)
Cell Differentiation/immunology , Glycosphingolipids/chemistry , Macrophages/chemistry , Monocytes/chemistry , Polysaccharides/chemistry , Carbohydrate Conformation , Carbohydrate Sequence , Cell Line , Fucosyltransferases/genetics , Fucosyltransferases/immunology , G(M3) Ganglioside/chemistry , G(M3) Ganglioside/immunology , Gene Expression Regulation , Glycosphingolipids/immunology , Glycosylation , Glycosyltransferases/genetics , Glycosyltransferases/immunology , Humans , Macrophages/cytology , Macrophages/immunology , Mannose/chemistry , Mannose/immunology , Monocytes/cytology , Monocytes/immunology , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/immunology , Neuraminidase/genetics , Neuraminidase/immunology , Polysaccharides/immunology , Sialic Acids/chemistry , Sialic Acids/immunology , Sialyltransferases/genetics , Sialyltransferases/immunologyABSTRACT
Fetal or neonatal alloimmune thrombocytopenia (FNAIT) is a potentially life-threatening disease where fetal platelets are destroyed by maternal anti-platelet IgG alloantibodies. The clinical outcome varies from asymptomatic, to petechiae or intracranial haemorrhage, but no marker has shown reliable correlation with severity, making screening for FNAIT impractical and highly inefficient. We recently found IgG Fc-glycosylation towards platelet and red blood cell antigens to be skewed towards decreased fucosylation, increased galactosylation and sialylation. The lowered core-fucosylation increases the affinity of the pathogenic antibodies to FcγRIIIa and FcγRIIIb, and hence platelet destruction. Here we analysed the N-linked glycans of human platelet antigen (HPA)-1a specific IgG1 with mass spectrometry in large series of FNAIT cases (n = 166) including longitudinal samples (n = 26). Besides a significant decrease in Fc-fucosylation after the first pregnancy (P = 0·0124), Fc-glycosylation levels remained stable during and after pregnancy and in subsequent pregnancies. Multiple logistic regression analysis identified anti-HPA-1a -fucosylation (P = 0·006) combined with galactosylation (P = 0·021) and antibody level (P = 0·038) correlated with bleeding severity, making these parameters a feasible marker in screening for severe cases of FNAIT.
Subject(s)
Blood Platelets/immunology , Glycosylation , Immunoglobulin G/analysis , Isoantibodies/chemistry , Thrombocytopenia, Neonatal Alloimmune/immunology , Antibodies, Anti-Idiotypic/chemistry , Antigens, Human Platelet/immunology , Female , Fucose/chemistry , Galactose/chemistry , Hemorrhage/immunology , Humans , Integrin beta3 , Isoantibodies/blood , Mass Spectrometry , N-Acetylneuraminic Acid/chemistry , Predictive Value of Tests , Pregnancy , Severity of Illness IndexABSTRACT
On-tissue digestion matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to record spatially correlated molecular information from formalin-fixed, paraffin-embedded (FFPE) tissue sections. In this work, we present the in situ multimodal analysis of N-linked glycans and proteins from the same FFPE tissue section. The robustness and applicability of the method are demonstrated for several tumors, including epithelial and mesenchymal tumor types. Major analytical aspects, such as lateral diffusion of the analyte molecules and differences in measurement sensitivity due to the additional sample preparation methods, have been investigated for both N-glycans and proteolytic peptides. By combining the MSI approach with extract analysis, we were also able to assess which mass spectral peaks generated by MALDI-MSI could be assigned to unique N-glycan and peptide identities.
Subject(s)
Peptides/analysis , Polysaccharides/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Antigens/metabolism , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Glycoproteins/metabolism , Humans , Leiomyosarcoma/metabolism , Leiomyosarcoma/pathology , Liposarcoma, Myxoid/metabolism , Liposarcoma, Myxoid/pathology , Paraffin Embedding , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism , Peptides/metabolism , Polysaccharides/metabolismABSTRACT
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging is a rapidly evolving field in which mass spectrometry techniques are applied directly on tissues to characterize the spatial distribution of various molecules such as lipids, protein/peptides, and recently also N-glycans. Glycans are involved in many biological processes and several glycan changes have been associated with different kinds of cancer, making them an interesting target group to study. An important analytical challenge for the study of glycans by MALDI mass spectrometry is the labile character of sialic acid groups which are prone to in-source/postsource decay, thereby biasing the recorded glycan profile. We therefore developed a linkage-specific sialic acid derivatization by dimethylamidation and subsequent amidation and transferred this onto formalin-fixed paraffin-embedded (FFPE) tissues for MALDI imaging of N-glycans. Our results show (i) the successful stabilization of sialic acids in a linkage specific manner, thereby not only increasing the detection range, but also adding biological meaning, (ii) that no noticeable lateral diffusion is induced during to sample preparation, (iii) the potential of mass spectrometry imaging to spatially characterize the N-glycan expression within heterogeneous tissues.
Subject(s)
Formaldehyde/chemistry , Paraffin Embedding , Polysaccharides/chemistry , Sialic Acids/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Carbohydrate ConformationABSTRACT
Glycosylation is a post-translational modification of key importance with heterogeneous structural characteristics. Previously, we have developed a robust, high-throughput MALDI-TOF-MS method for the comprehensive profiling of human plasma N-glycans. In this approach, sialic acid residues are derivatized with linkage-specificity, namely the ethylation of α2,6-linked sialic acid residues with parallel lactone formation of α2,3-linked sialic acids. In the current study, this procedure was used as a starting point for the automation of all steps on a liquid-handling robot system. This resulted in a time-efficient and fully standardized procedure with throughput times of 2.5 h for a first set of 96 samples and approximately 1 h extra for each additional sample plate. The mass analysis of the thus-obtained glycans was highly reproducible in terms of relative quantification, with improved interday repeatability as compared to that of manual processing.
Subject(s)
Automation/methods , Glycoproteins/blood , Glycoproteins/chemistry , High-Throughput Screening Assays/methods , N-Acetylneuraminic Acid/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Esterification , Glycoproteins/metabolism , Glycosylation , N-Acetylneuraminic Acid/chemistry , Reproducibility of ResultsABSTRACT
Cancer is a leading cause of death and alterations of glycosylation are characteristic features of malignant cells. Colorectal cancer is one of the most common cancers and its exact causes and biology are not yet well understood. Here, we compared glycosylation profiles of colorectal tumor tissues and corresponding control tissues of 13 colorectal cancer patients to contribute to the understanding of this cancer. Using MALDI-TOF(/TOF)-MS and 2-dimensional LC-MS/MS we characterized enzymatically released and 2-aminobenzoic acid labeled glycans from glycosphingolipids. Multivariate data analysis revealed significant differences between tumor and corresponding control tissues. Main discriminators were obtained, which represent the overall alteration in glycosylation of glycosphingolipids during colorectal cancer progression, and these were found to be characterized by (1) increased fucosylation, (2) decreased acetylation, (3) decreased sulfation, (4) reduced expression of globo-type glycans, as well as (5) disialyl gangliosides. The findings of our current research confirm former reports, and in addition expand the knowledge of glycosphingolipid glycosylation in colorectal cancer by revealing new glycans with discriminative power and characteristic, cancer-associated glycosylation alterations. The obtained discriminating glycans can contribute to progress the discovery of biomarkers to improve diagnostics and patient treatment.
Subject(s)
Colorectal Neoplasms/metabolism , Glycosphingolipids/metabolism , Adult , Aged , Biomarkers, Tumor/chemistry , Biomarkers, Tumor/metabolism , Chromatography, High Pressure Liquid , Disease Progression , Female , Glycosphingolipids/chemistry , Glycosylation , Humans , Male , Metabolome , Middle Aged , Molecular Structure , Multivariate Analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass SpectrometryABSTRACT
BACKGROUND: Respiratory therapists (RTs) work alongside allied health staff, nurses, and physicians during stressful and traumatic events that can be associated with emotional and physiological implications known as second victim (SV) experiences (SVEs). This study aimed to evaluate SVEs of RTs, including both positive and negative implications. METHODS: RTs within a large academic health care organization across Minnesota, Wisconsin, Florida, and Arizona were asked to participate in an anonymous survey that included the validated Second Victim Experience and Support Tool-Revised to assess SVEs as well as desired support services. RESULTS: Of the RTs invited to participate, 30.8% (171/555) completed the survey. Of the 171 survey respondents, 91.2% (156) reported that they had been part of a stressful or traumatic work-related event as an RT, student, or department support staff member. Emotional or physiologic implications experienced by respondents as SVs included anxiety 39.1% (61/156), reliving of the event 36.5% (57/156), sleeplessness 32.1% (50/156), and guilt 28.2% (44/156). Following a stressful clinical event, 14.8% (22/149) experienced psychological distress, 14.2% (21/148) experienced physical distress, 17.7% (26/147) indicated lack of institutional support, and 15.6% (23/147) indicated turnover intentions. Enhanced resilience and growth were reported by 9.5% (14/147). Clinical and non-clinical events were reported as possible triggers for SVEs. Nearly half of respondents 49.4% (77/156) indicated feeling like an SV due to events related to COVID-19. Peer support was the highest ranked form of desired support following an SVE by 57.7% (90/156). CONCLUSIONS: RTs are involved in stressful or traumatic clinical events, resulting in psychological/physical distress and turnover intentions. The COVID-19 pandemic has had a significant impact on RTs' SVEs, highlighting the importance of addressing the SV phenomenon among this population.
Subject(s)
COVID-19 , Physicians , Humans , Pandemics , COVID-19/epidemiology , Allied Health Personnel , Anxiety , Surveys and QuestionnairesABSTRACT
Changes in glycosylation signatures of cells have been associated with pathological processes in cancer as well as infectious and autoimmune diseases. The current protocols for comprehensive analysis of N-glycomics and O-glycomics derived from cells and tissues often require a large amount of biological material. They also only allow the processing of very limited numbers of samples at a time. Here we established a workflow for sequential release of N-glycans and O-glycans based on PVDF membrane immobilization in 96-well format from 5 × 105 cells. Released glycans are reduced, desalted, purified, and reconstituted, all in 96-well format plates, without additional staining or derivatization. Glycans are then analyzed with porous graphitized carbon nano-liquid chromatography coupled to tandem mass spectrometry using negative-mode electrospray ionization, enabling the chromatographic resolution and structural elucidation of glycan species including many compositional isomers. The approach was demonstrated using glycoprotein standards and further applied to analyze the glycosylation of the murine mammary gland NMuMG cell line. The developed protocol allows the analysis of N- and O-glycans from relatively large numbers of samples in a less time consuming way with high repeatability. Inter- and intraday repeatability of the fetuin N-glycan analysis showed two median intraday coefficients of variations (CVs) of 7.6% and 8.0%, and a median interday CV of 9.8%. Median CVs of 7.9% and 8.7% for the main peaks of N- and O-glycans released from the NMuMG cell line indicate a very good repeatability. The method is applicable to purified glycoproteins as well as to biofluids and cell- or tissue-based samples.
Subject(s)
Chromatography, Liquid , Glycomics/methods , Graphite/chemistry , Polysaccharides/chemistry , Tandem Mass Spectrometry , Animals , Data Analysis , Glycoproteins/chemistry , Glycoproteins/metabolism , Glycosylation , Humans , Mice , Polysaccharides/metabolism , Porosity , WorkflowABSTRACT
The caudal-related homeobox protein 1 (CDX1) is a transcription factor, which is important in the development, differentiation, and homeostasis of the gut. Although the involvement of CDX genes in the regulation of the expression levels of a few glycosyltransferases has been shown, associations between glycosylation phenotypes and CDX1 mRNA expression have hitherto not been well studied. Triggered by our previous study, we here characterized the N-glycomic phenotype of 16 colon cancer cell lines, selected for their differential CDX1 mRNA expression levels. We found that high CDX1 mRNA expression associated with a higher degree of multi-fucosylation on N-glycans, which is in line with our previous results and was supported by up-regulated gene expression of fucosyltransferases involved in antenna fucosylation. Interestingly, hepatocyte nuclear factors (HNF)4A and HNF1A were, among others, positively associated with high CDX1 mRNA expression and have been previously proven to regulate antenna fucosylation. Besides fucosylation, we found that high CDX1 mRNA expression in cancer cell lines also associated with low levels of sialylation and galactosylation and high levels of bisection on N-glycans. Altogether, our data highlight a possible role of CDX1 in altering the N-glycosylation of colorectal cancer cells, which is a hallmark of tumor development.