Changes In Protein Glycosylation Research Articles

Characterization of Cell Surface Glycoproteins Using Enzymatic Treatment and Mass Spectrometry.

Almost all proteins on the cell surface are modified by glycosylation. Cell surface glycoproteins participate in various cellular pathways, such as cell adhesion, cell-cell communication, and immune response. Due to their functional importance, glycoproteins on the cell surface often serve as potential therapeutic targets. Recent advancements in mass spectrometry (MS) have facilitated the characterization of glycoproteins that are generally localized on the cell surface, secreted to the extracellular environment, or found in intracellular organelles such as the endoplasmic reticulum, Golgi apparatus, and peroxisome. However, the selective characterization of glycoproteins on the cell surface remains challenging. In this study, we applied enzymatic treatment to live cells, followed by MS-based glycoproteomics analysis, to assess changes in protein glycosylation at different treatment time points as a method to identify cell surface glycoproteins. To demonstrate this approach, a renal cell carcinoma cell line, A498, was treated with glycosidases, sialidase and PNGase F, over two treatment time intervals, 2 and 24 h. Glycoproteins were identified as cell surface glycoproteins from A498 cells when enzyme treatment altered the glycosylation of the glycoproteins. The results revealed the effectiveness of integrating enzymatic treatment with MS-based glycoproteomics for analyzing cell surface glycoproteins. Our established method has demonstrated the potential applications for assessing accessibility of therapeutic targets on the cell surface over time and supporting the development of new targeted therapies.

Cardiomyocyte Reduction of Hybrid/Complex N-Glycosylation in the Adult Causes Heart Failure With Reduced Ejection Fraction in the Absence of Cellular Remodeling.

Heart failure (HF) presents a massive burden to health care with a complex pathophysiology that results in HF with reduced left ventricle ejection fraction (EF) or HF with preserved EF. It has been shown that relatively modest changes in protein glycosylation, an essential posttranslational modification, are associated with clinical presentations of HF. We and others previously showed that such aberrant protein glycosylation in animal models can lead to HF. We develop and characterize a novel, tamoxifen-inducible, cardiomyocyte Mgat1 knockout mouse strain, achieved through deletion of Mgat1, alpha-1,3-mannosyl-glycoproten 2-beta-N-acetlyglucosaminyltransferase, which encodes N-acetylglucosaminyltransferase I. We investigate the role of hybrid/complex N-glycosylation in adult HFrEF pathogenesis at the ion channel, cardiomyocyte, tissue, and gross cardiac level. The data demonstrate successful reduction of N-acetylglucosaminyltransferase I activity and confirm that hybrid/complex N-glycans modulate gating of cardiomyocyte voltage-gated calcium channels. A longitudinal study shows that the tamoxifen-inducible, cardiomyocyte Mgat1 knockout mice present with significantly reduced systolic function by 28 days post induction that progresses into HFrEF by 8 weeks post induction, without significant ventricular dilation or hypertrophy. Further, there was minimal, if any, physiologic or pathophysiologic cardiomyocyte electromechanical remodeling or fibrosis observed before (10-21 days post induction) or after (90-130 days post induction) HFrEF development. The tamoxifen-inducible, cardiomyocyte Mgat1 knockout mouse strain created and characterized here provides a model to describe novel mechanisms and causes responsible for HFrEF onset in the adult, likely occurring primarily through tissue-level reductions in electromechanical activity in the absence of (or at least before) cardiomyocyte remodeling and fibrosis.

Liposome-encapsulated mannose-1-phosphate therapy improves global N-glycosylation in different congenital disorders of glycosylation

Phosphomannomutase 2 (PMM2) converts mannose-6-phospahate to mannose-1-phosphate; the substrate for GDP-mannose, a building block of the glycosylation biosynthetic pathway. Pathogenic variants in the PMM2 gene have been shown to be associated with protein hypoglycosylation causing PMM2-congenital disorder of glycosylation (PMM2-CDG). While mannose supplementation improves glycosylation in vitro, but not in vivo, we hypothesized that liposomal delivery of mannose-1-phosphate could increase the stability and delivery of the activated sugar to enter the targeted compartments of cells. Thus, we studied the effect of liposome-encapsulated mannose-1-P (GLM101) on global protein glycosylation and on the cellular proteome in skin fibroblasts from individuals with PMM2-CDG, as well as in individuals with two N-glycosylation defects early in the pathway, namely ALG2-CDG and ALG11-CDG. We leveraged multiplexed proteomics and N-glycoproteomics in fibroblasts derived from different individuals with various pathogenic variants in PMM2, ALG2 and ALG11 genes. Proteomics data revealed a moderate but significant change in the abundance of some of the proteins in all CDG fibroblasts upon GLM101 treatment. On the other hand, N-glycoproteomics revealed the GLM101 treatment enhanced the expression levels of several high-mannose and complex/hybrid glycopeptides from numerous cellular proteins in individuals with defects in PMM2 and ALG2 genes. Both PMM2-CDG and ALG2-CDG exhibited several-fold increase in glycopeptides bearing Man6 and higher glycans and a decrease in Man5 and smaller glycan moieties, suggesting that GLM101 helps in the formation of mature glycoforms. These changes in protein glycosylation were observed in all individuals irrespective of their genetic variants. ALG11-CDG fibroblasts also showed increase in high mannose glycopeptides upon treatment; however, the improvement was not as dramatic as the other two CDG. Overall, our findings suggest that treatment with GLM101 overcomes the genetic block in the glycosylation pathway and can be used as a potential therapy for CDG with enzymatic defects in early steps in protein N-glycosylation.

Induction of acrosome reaction by 4-Br-A23187 alters the glycoproteomic profile of boar spermatozoa

Protein glycosylation is a post-translational modification involved in wide range of biological processes. In mammalian spermatozoa this modification has been identified in numerous proteins, and membrane glycoproteins are involved in the fertilization process. The objective of the present study was to identify changes in protein glycosylation after acrosome reaction (AR) induction using the 4-Br-A23187 ionophore. Our results showed that treatment with 10 μM of 4-Br-A23187 for 20 min significantly increased the percentage of live acrosome-reacted spermatozoa compared to the control (69.8 ± 0.8 vs. 6.4 ± 0.5; mean % ± SEM, respectively). Also, we observed an increase in 32 kDa tyrosine-phosphorylated protein (p32) and a decrease in serine/threonine phosphorylation of the protein kinase A substrates (phospho-PKA-substrates) after ionophore treatment. Furthermore, changes in glycosylated proteins following AR induction were analyzed using different HRP-conjugated lectins (GNA, DSA, and SNA), revealing changes in mannose and sialic acid residues. Proteomic analysis of isolated proteins using GNA lectin revealed that 50 proteins exhibited significantly different abundance (q-value < 0.01). Subsequent analysis using Uniprot database identified 39 downregulated and 11 upregulated proteins in the presence of 4-Br-A23187. Notably, six of these proteins were classified as transmembrane proteins, namely LRRC37A/B like protein 1 C-terminal domain-containing protein, Membrane metalloendopeptidase like 1, VWFA domain-containing protein, Syndecan, Membrane spanning 4-domains A14 and Serine protease 54. This study shows a novel protocol to induce acrosome reaction in boar spermatozoa and identifies new transmembrane proteins containing mannose residues. Further work is needed to elucidate the role of these proteins in sperm-oocyte fusion.

Protein glycosylation in lung cancer from a mass spectrometry perspective.

Lung cancer is a severe disease for which better diagnostic and therapeutic approaches are urgently needed. Increasing evidence implies that aberrant protein glycosylation plays a crucial role in the pathogenesis and progression of lung cancer. Differences in glycosylation patterns have been previously observed between healthy and cancerous samples as well as between different lung cancer subtypes, which suggests untapped diagnostic potential. In addition, understanding the changes mediated by glycosylation may shed light on possible novel therapeutic targets and personalized treatment strategies for lung cancer patients. Mass spectrometry based glycomics and glycoproteomics have emerged as powerful tools for in-depth characterization of changes in protein glycosylation, providing valuable insights into the molecular basis of lung cancer. This paper reviews the literature on the analysis of protein glycosylation in lung cancer using mass spectrometry, which is dominated by manuscripts published over the past 5 years. Studies analyzing N-glycosylation, O-glycosylation, and glycosaminoglycan patterns in tissue, serum, plasma, and rare biological samples of lung cancer patients are highlighted. The current knowledge on the potential utility of glycan and glycoprotein biomarkers is also discussed.

Enrichment methods of N-linked glycopeptides from human serum or plasma: A mini-review

Human diseases often correlate with changes in protein glycosylation, which can be observed in serum or plasma samples. N-glycosylation, the most common form, can provide potential biomarkers for disease prognosis and diagnosis. However, glycoproteins constitute a relatively small proportion of the total proteins in human serum and plasma compared to the non-glycosylated protein albumin, which constitutes the majority. The detection of microheterogeneity and low glycan abundance presents a challenge. Mass spectrometry facilitates glycoproteomics research, yet it faces challenges due to interference from abundant plasma proteins. Therefore, methods have emerged to enrich N-glycans and N-linked glycopeptides using glycan affinity, chemical properties, stationary phase chemical coupling, bioorthogonal techniques, and other alternatives. This review focuses on N-glycans and N-glycopeptides enrichment in human serum or plasma, emphasizing methods and applications. Although not exhaustive, it aims to elucidate principles and showcase the utility and limitations of glycoproteome characterization.

C1GalT1 expression reciprocally controls tumour cell-cell and tumour-macrophage interactions mediated by galectin-3 and MGL with double impact on cancer development and progression

Although most cell membrane proteins are modified by glycosylation, our understanding of the role and actions of protein glycosylation is still very limited. β1,3galactosyltransferase (C1GalT1) is a key glycosyltransferase that controls the biosynthesis of the Core 1 structure of O-linked mucin type glycans and is overexpressed by many common types of epithelial cancers. This study reports that suppression of C1GalT1 expression in human colon cancer cells caused substantial changes of protein glycosylation of cell membrane proteins, many of which were ligands of the galactoside-binding galectin-3 and the macrophage galactose-type lectin (MGL). This led to significant reduction of cancer cell proliferation, adhesion, migration and the ability of tumour cells to form colonies. Crucially, C1GalT1 suppression significantly reduced galectin-3-mediated tumour cell-cell interaction and galectin-3-promoted tumour cell activities. In the meantime, C1GalT1 suppression substantially increased MGL-mediated macrophage-tumour cell interaction and macrophage-tumour cell phagocytosis and cytokine secretion. C1GalT1-expressing cancer cells implanted in chick embryos resulted in the formation of significantly bigger tumours than C1GalT1-suppressed cells and the presence of galectin-3 increased tumour growth of C1GalT1-expressing but not C1GalT1-suppressed cells. More MGL-expressing macrophages and dendritic cells were seen to be attracted to the tumour microenvironment in ME C1galt1-/-/Erb mice than in C1galt1f/f /Erb mice. These results indicate that expression of C1GalT1 by tumour cells reciprocally controls tumour cell-cell and tumour-macrophage interactions mediated by galectin-3 and MGL with double impact on cancer development and progression. C1GalT1 overexpression in epithelial cancers therefore may represent a fundamental mechanism in cancer promotion and in reduction of immune response/surveillance in cancer progression.

Increased expression of the pathological O-glycosylated form of oncofetal fibronectin in the multidrug resistance phenotype of cancer cells

Changes in protein glycosylation are a hallmark of transformed cells and modulate numerous phenomena associated with cancer progression, such as the acquisition of multidrug resistance (MDR) phenotype. Different families of glycosyltransferases and their products have already been described as possible modulators of the MDR phenotype. Among the glycosyltransferases intensively studied in cancer research, UDP-N-acetyl-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase-6 (pp-GalNAc-T6), which is widely expressed in many organs and tissues, stands out. Its influence in several events associated with kidney, oral, pancreatic, renal, lung, gastric and breast cancer progression has already been described. However, its participation in the MDR phenotype has never been studied. Here, we demonstrate that human breast adenocarcinoma MCF-7 MDR cell lines, generated by chronic exposure to doxorubicin, in addition to exhibiting increased expression of proteins belonging to the ABC superfamily (ABCC1 and ABCG2), and anti-apoptotic proteins (Blcl-2 and Bcl-xL), also present high expression of pp-GalNAc-T6, the enzyme currently proposed as the main responsible for the biosynthesis of oncofetal fibronectin (onf-FN), a major extracellular matrix component expressed by cancer cells and embryonic tissues, but absent in healthy cells. Our results show that onf-FN, which is generated by the addition of a GalNAc unit at a specific threonine residue inside the type III homology connective segment (IIICS) domain of FN, is strongly upregulated during the acquisition of the MDR phenotype. Also, the silencing of pp-GalNAc-T6, not only compromises the expression of the oncofetal glycoprotein, but also made the MDR cells more sensitive to all anticancer drugs tested, partially reversing the MDR phenotype. Taken together, our results demonstrate for the first time the upregulation of the O-glycosylated oncofetal fibronectin, as well as the direct participation of pp-GalNAc-T6 during the acquisition of a MDR phenotype in a breast cancer model, giving credence to the hypothesis that in transformed cells, glycosyltransferases and/or their products, such as unusual extracellular matrix glycoproteins can be used as potential therapeutic targets for the treatment of cancer.

Twin research shows glycan changes are more susceptible to environmental factors than their carrier glycoproteins.

Changes in protein glycosylation are clinically used as biomarkers. In the present study, we employed a twin cohort to investigate the contributions of genetic and environmental factors to glycan modifications of glycoproteins. Mac-2 binding protein (Mac-2bp), haptoglobin (Hp), and their glycosylated forms are liver fibrosis and cancer biomarkers. Sera from 107 twin pairs without clinical information were used as a training cohort for the Mac-2bp and Mac-2bp glycosylation isomer (M2BPGi) assay. As a validation cohort, 22 twin pairs were enrolled in the study. For each twin pair, one twin was diagnosed with liver or pancreatic disease. For the training cohort, the correlation ratios of serum Mac-2bp and M2BPGi levels in twin sera with random sequences were 0.30 and 0.018, respectively. The correlation ratios between twin pairs in the validation cohort for serum Mac-2bp and M2BPGi levels were 0.75 and 0.35, respectively. In contrast, correlation ratios of serum Hp and fucosylated haptoglobin (Fuc-Hp) levels between twin sera with liver and pancreatic disease were 0.49 and 0.16, respectively. Although serum protein levels of glycoproteins are susceptible to genetic factors, characteristic glycan changes of these glycoproteins are more susceptible to environmental factors, including liver and pancreatic disease.

Aberrant N-glycosylation in cancer: MGAT5 and β1,6-GlcNAc branched N-glycans as critical regulators of tumor development and progression.

Changes in protein glycosylation are widely observed in tumor cells. N-glycan branching through adding β1,6-linked N-acetylglucosamine (β1,6-GlcNAc) to an α1,6-linked mannose, which is catalyzed by the N-acetylglucosaminyltransferase V (MGAT5 or GnT-V), is one of the most frequently observed tumor-associated glycan structure formed. Increased levels of this branching structure play a pro-tumoral role in various ways, for example, through the stabilization of growth factor receptors, the destabilization of intercellular adhesion, or the acquisition of a migratory phenotype. In this review, we provide an updated and comprehensive summary of the physiological and pathophysiological roles of MGAT5 and β1,6-GlcNAc branched N-glycans, including their regulatory mechanisms. Specific emphasis is given to the role of MGAT5 and β1,6-GlcNAc branched N-glycans in cellular mechanisms that contribute to the development and progression of solid tumors. We also provide insight into possible future clinical implications, such as the use of MGAT5 as a prognostic biomarker.

Quantitative analysis of fucosylated glycoproteins by immobilized lectin-affinity fluorescent labeling.

Human biofluids are often used to discover disease-specific glycosylation, since abnormal changes in protein glycosylation can discern physiopathological states. Highly glycosylated proteins in biofluids make it possible to identify disease signatures. Glycoproteomic studies on saliva glycoproteins showed that fucosylation was significantly increased during tumorigenesis and that glycoproteins became hyperfucosylated in lung metastases, and tumor stage is associated with fucosylation. Quantification of salivary fucosylation can be achieved by mass spectrometric analysis of fucosylated glycoproteins or fucosylated glycans; however, the use of mass spectrometry is non-trivial for clinical practice. Here, we developed a high-throughput quantitative method, lectin-affinity fluorescent labeling quantification (LAFLQ), to quantify fucosylated glycoproteins without relying on mass spectrometry. Lectins with a specific affinity for fucoses are immobilized on the resin and effectively capture fluorescently labeled fucosylated glycoproteins, which are further quantitatively characterized by fluorescence detection in a 96-well plate. Our results demonstrated that serum IgG can be accurately quantified by lectin and fluorescence detection. Quantification in saliva showed significantly higher fucosylation in lung cancer patients compared to healthy controls or other non-cancer diseases, suggesting that this method has the potential to quantify stage-related fucosylation in lung cancer saliva.

An Integrated Proteomic and Glycoproteomic Investigation Reveals Alterations in the N-Glycoproteomic Network Induced by 2-Deoxy-D-Glucose in Colorectal Cancer Cells.

As a well-known glycolysis inhibitor for anticancer treatment, 2-Deoxy-D-glucose (2DG) inhibits the growth and survival of cancer cells by interfering with the ATP produced by the metabolism of D-glucose. In addition, 2DG inhibits protein glycosylation in vivo by competing with D-mannose, leading to endoplasmic reticulum (ER) stress and unfolded protein responses in cancer cells. However, the molecular details underlying the impact of 2DG on protein glycosylation remain largely elusive. With an integrated approach to glycoproteomics and proteomics, we characterized the 2DG-induced alterations in N-glycosylation, as well as the cascading impacts on the whole proteome using the HT29 colorectal cancer cell line as a model system. More than 1700 site-specific glycoforms, represented by unique intact glycopeptides (IGPs), were identified. The treatment of 2DG had a broad effect on the N-glycoproteome, especially the high-mannose types. The glycosite occupancy of the high-mannose N-glycans decreased the most compared with the sialic acid and fucose-containing N-glycans. Many of the proteins with down-regulated high-mannose were implicated in functional networks related to response to topologically incorrect protein, integrin-mediated signaling, lysosomal transport, protein hydroxylation, vacuole, and protein N-glycosylation. The treatment of 2DG also functionally disrupted the global cellular proteome, evidenced by significant up-regulation of the proteins implicated in protein folding, endoplasmic reticulum, mitochondrial function, cellular respiration, oxidative phosphorylation, and translational termination. Taken together, these findings reveal the complex changes in protein glycosylation and expression underlying the various effects of 2DG on cancer cells, and may provide insightful clues to inform therapeutic development targeting protein glycosylation.

Protein glycosylation changes during systemic acquired resistance in Arabidopsis thaliana

N-glycosylation, an important post-translational modification of proteins in all eukaryotes, has been clearly shown to be involved in numerous diseases in mammalian systems. In contrast, little is known regarding the role of protein N-glycosylation in plant defensive responses to pathogen infection. We identified, for the first time, glycoproteins related to systemic acquired resistance (SAR) in an Arabidopsis thaliana model, using a glycoproteomics platform based on high-resolution mass spectrometry. 407 glycosylation sites corresponding to 378 glycopeptides and 273 unique glycoproteins were identified. 65 significantly changed glycoproteins with 80 N-glycosylation sites were detected in systemic leaves of SAR-induced plants, including numerous GDSL-like lipases, thioglucoside glucohydrolases, kinases, and glycosidases. Functional enrichment analysis revealed that significantly changed glycoproteins were involved mainly in N-glycan biosynthesis and degradation, phenylpropanoid biosynthesis, cutin and wax biosynthesis, and plant-pathogen interactions. Comparative analysis of glycoproteomics and proteomics data indicated that glycoproteomics analysis is an efficient method for screening proteins associated with SAR. The present findings clarify glycosylation status and sites of A. thaliana proteins, and will facilitate further research on roles of glycoproteins in SAR induction.

High Diversity of Glycosphingolipid Glycans of Colorectal Cancer Cell Lines Reflects the Cellular Differentiation Phenotype.

Colorectal cancer (CRC)–associated changes of protein glycosylation have been widely studied. In contrast, the expression of glycosphingolipid (GSL) patterns in CRC has, hitherto, remained largely unexplored. Even though GSLs are major carriers of cell surface carbohydrates, they are understudied due to their complexity and analytical challenges. In this study, we provide an in-depth analysis of GSL glycans of 22 CRC cell lines using porous graphitized carbon nano–liquid chromatography coupled with electrospray ionization–mass spectrometry. Our data revealed that the GSL expression varies among different cell line classifications, with undifferentiated cell lines showing high expression of blood group A, B, and H antigens while for colon-like cell lines the most prominent GSL glycans contained (sialyl)-LewisA/X and LewisB/Y antigens. Moreover, the GSL expression correlated with relevant glycosyltransferases that are involved in their biosynthesis as well as with transcription factors (TFs) implicated in colon differentiation. Additionally, correlations between certain glycosyltransferases and TFs at mRNA expression level were found, such as FUT3, which correlated with CDX1, ETS2, HNF1A, HNF4A, MECOM, and MYB. These TFs are upregulated in colon-like cell lines pointing to their potential role in regulating fucosylation during colon differentiation. In conclusion, our study reveals novel layers of potential GSL glycans regulation relevant for future research in colon differentiation and CRC.

Role of ammonia for brain abnormal protein glycosylation during the development of hepatitis B virus-related liver diseases

BackgroundAmmonia is the most typical neurotoxin in hepatic encephalopathy (HE), but the underlying pathophysiology between ammonia and aberrant glycosylation in HE remains unknown.ResultsHere, we used HBV transgenic mice and astrocytes to present a systems-based study of glycosylation changes and corresponding enzymes associated with the key factors of ammonia in HE. We surveyed protein glycosylation changes associated with the brain of HBV transgenic mice by lectin microarrays. Upregulation of Galβ1-3GalNAc mediated by core 1 β1,3-galactosyltransferase (C1GALT1) was identified as a result of ammonia stimulation. Using in vitro assays, we validated that upregulation of C1GALT1 is a driver of deregulates calcium (Ca2+) homeostasis by overexpression of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in astrocytes.ConclusionsWe demonstrated that silencing C1GALT1 could depress the IP3R1 expression, an effective strategy to inhibit the ammonia-induced upregulation of Ca2+ activity, thereby C1GALT1 and IP3R1 may serve as therapeutic targets in hyperammonemia of HE.

Elevated N-Glycosylation Contributes to the Cisplatin Resistance of Non-Small Cell Lung Cancer Cells Revealed by Membrane Proteomic and Glycoproteomic Analysis.

Chemoresistance is the major restriction on the clinical use of cisplatin. Aberrant changes in protein glycosylation are closely associated with drug resistance. Comprehensive study on the role of protein glycosylation in the development of cisplatin resistance would contribute to precise elucidation of the complicated mechanism of resistance. However, comprehensive characterization of glycosylated proteins remains a big challenge. In this work, we integrated proteomic and N-glycoproteomic workflow to comprehensively characterize the cisplatin resistance-related membrane proteins. Using this method, we found that proteins implicated in cell adhesion, migration, response to drug, and signal transduction were significantly altered in both protein abundance and glycosylation level during the development of cisplatin resistance in the non-small cell lung cancer cell line. Accordingly, the ability of cell migration and invasion was markedly increased in cisplatin-resistant cells, hence intensifying their malignancy. In contrast, the intracellular cisplatin accumulation was significantly reduced in the resistant cells concomitant with the down-regulation of drug uptake channel protein, LRRC8A, and over-expression of drug efflux pump proteins, MRP1 and MRP4. Moreover, the global glycosylation was elevated in the cisplatin-resistant cells. Consequently, inhibition of N-glycosylation reduced cell resistance to cisplatin, whereas promoting the high-mannose or sialylated type of glycosylation enhanced the resistance, suggesting that critical glycosylation type contributes to cisplatin resistance. These results demonstrate the high efficiency of the integrated proteomic and N-glycoproteomic workflow in discovering drug resistance-related targets, and provide new insights into the mechanism of cisplatin resistance.

Protein Mannosylation as a Diagnostic and Prognostic Biomarker of Lupus Nephritis: An Unusual Glycan Neoepitope in Systemic Lupus Erythematosus

Changes in protein glycosylation are a hallmark of immune-mediated diseases. Glycans are master regulators of the inflammatory response being important molecules for the discrimination between "self"/"non-self". We here explored whether lupus nephritis (LN) exhibit an altered cellular glycosylation aiming to identify a unique glycosignature that characterizes LN pathogenesis. A comprehensive tissue glycomics characterization was performed in a cohort of human biopsy-proven LN clinical samples from SLE patients. A combination of advanced tissue mass spectrometry imaging (MSI); in situ glyco-characterization and ex vivo glycophenotyping of human kidney biopsies were performed to structurally map the N-glycans repertoire in LN samples. LN revealed a unique glycan signature characterized by an increased abundance and spatial distribution of unusual mannose-enriched glycans that are typically found in lower microorganisms; this glycosignature was specific of LN as it was not observed in other kidney diseases. Exposure of mannosylated glycans in LN was found to occur at the cell surface of kidney cells promoting an increased recognition by specific glycans-recognizing receptors, expressed by immune cells. This abnormal glyco-signature of LN was demonstrated to be due to a deficient complex N-glycosylation and a proficient O-mannosylation pathway. Moreover, levels of mannosylation detected in kidney biopsies from LN patients at diagnosis were demonstrated to predict the development of chronic kidney disease (CKD) with 93% of specificity. Cellular mannosylation is a marker of LN, predicting the development of CKD, and thus constituting a potential glycobiomarker to be included in the diagnostic and prognostic algorithm of LN.

Abstract 272: Glycomics: Protein glycosylation changes in the pathogenesis of head and neck cancer

Abstract Background: Glycosylation is the most common post-translational modification of proteins, and glycosylation changes at cell surfaces are frequently associated with malignant epithelia. These affect many functions including proliferation, motility and invasiveness, increasing the propensity to metastasise. Many head &amp; neck squamous cell carcinomata (HNSCC), especially those originating in the lymphoid mucosa of the oropharynx, are driven by so-called high risk genotypes of Human Papillomavirus (HPV). Five-year survival remains poor, averaging around 50% globally: this is partly related to late diagnosis. Specific protein glycosylation signatures on malignant keratinocytes have promise as diagnostic and prognostic markers and as therapeutic targets. Here we begin creation of a glyco-fingerprint library mapping the glyco-space of HNSCC. Materials and methods: Six established HNSCC cell lines were used to capture the qualitative and semi-quantitative profile of the N- and O-glycome using the well-established porous graphitized carbon (PGC) glycomics approach. Furthermore, we attempted to evaluate the effect sialylation has on HNSCC cell migration by enzymatically removing sialic acid residues from cell surface glycoconjugates. Therefore, we used the enzyme neuraminidase to remove sialic acids from HNSCC cells and measured cell proliferation rates by using incucyte. Results: In Human Papillomavirus (HPV) negative HNSCC cell lines, 37-41% of cells carried oligomannoses on their surfaces compared to 45% in HPV+ cell lines. Sialylated N-glycan were the most abundant type of N-glycan, contributing to &amp;gt;40% of the N-glycome in the HPV negative cell lines: but just 32-38% in HPV+ cell lines; di- and tri-sialylated forms were most frequent. The majority of complex type N-glycans (33%) carried core-fucose residues with just minor levels (&amp;lt;3%) of Lewis-type fucosylation identified. We also observed unusual types of oligomannose N-glycans carrying core-fucose residues. Enzymatic sialic acid removal resulted in 40% reduction in cell doubling numbers. Conclusion: This first systematic mapping reveals diverse glycosylation of head and neck cancer cell lines with high levels of core fucosylation and sialylation. Differences between HPV positive and negative cells may partly explain the comparatively good prognosis of HPV-driven HNC. This mapping forms the basis of histopathological profiling of real tumours which might have prognostic potential and suggest targets for immunotherapy. Removal of sialic acids resulted in decreased cell proliferation in vitro, indicating the fundamental role of protein glycosylation in the pathogenesis of HNC. Citation Format: Mohammad Rasheduzzaman, Xi Zhang, Riccardo Dolcetti, Liz Kenny, Newell W. Johnson, Daniel Kolarich, Chamindie Punyadeera. Glycomics: Protein glycosylation changes in the pathogenesis of head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 272.

Large-scale enrichment and identification of human urinary N-glycoproteins/N-glycopeptides

蛋白质的N-糖基化是真核细胞中一种重要的翻译后修饰,N-糖基化修饰在调控细胞黏附、迁移、信号转导及细胞凋亡等方面扮演着关键角色。蛋白质糖基化修饰的异常变化与多种重要疾病的发生相关。尿液具有蛋白质组复杂程度低和非入侵性等特点,适合大量及连续多时间点采样研究。但由于个体差异和生理条件的影响,尿蛋白丰度的生理波动较大。目前缺乏对健康人群尿液N-糖蛋白的个体差异和生理波动的专门性研究,以及生理丰度范围的构建,难以将个体差异、正常生理波动和疾病导致的变化进行有效区分,对疾病标志物研究提出很大挑战。本研究以亲水相互作用色谱法(HILIC)为基础,对该富集方法中活化、清洗与洗脱过程进行优化,其中主要对HILIC填料粒径和富集缓冲体系进行优化,并考察了不同实验条件下N-糖肽富集的鉴定数量、选择性与稳定性,发现当HILIC填料粒径为5 μm,在三氟乙酸富集体系下有更高的N-糖蛋白/N-糖肽鉴定水平。在此基础上,对20例健康男性志愿者和20例健康女性志愿者的尿液N-糖蛋白/N-糖肽进行了富集和定性、定量及功能分析。从40例尿液样本中共鉴定到1016个N-糖蛋白、2192条N-糖肽。采用非标定量策略对尿液N-糖肽的生理丰度波动范围进行了考察,尿液N-糖肽的丰度跨度约5个数量级。在此之后探索了健康人群尿蛋白N-糖基化水平的性别差异,筛选出性别相关的差异N-糖蛋白后进行了功能分析。统计学分析显示在尿液样本中性别可能是产生个体差异的重要因素。该工作为基于尿液糖蛋白质组学的功能与机制研究和临床生物标志物筛选提供了有力支撑。

Glycosylation in Autoimmune Diseases.

Autoimmune diseases are accompanied by changes in protein glycosylation, in both the immune system and target tissues. The best-studied alteration in autoimmunity is agalactosylation of immunoglobulin G (IgG), characterized primarily in rheumatoid arthritis (RA), and then detected also in systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), and multiple sclerosis (MS). The rebuilding of IgG N-glycans in RA correlates with the relapses and remissions of the disease, is associated with physiological states such as pregnancy but also depends on applied anti-inflammatory therapy. In turn, a decreased core fucosylation of the whole pool of IgG N-glycans is a serum glycomarker in autoimmune thyroid diseases (AITD) encompassing Hashimoto's thyroiditis (HT) and Grave's disease (GD). However, fucosylation of anti-thyroglobulin IgG (an immunological marker of HT) was elevated in HT serum. Core fucosylation of IgG oligosaccharides was also lowered in MS and SLE. In AITD and IBD, chronic inflammation T lymphocytes showed the reduced expression of MGAT5 gene encoding β1,6-N-acetylglucosaminyltransferase V (GnT-V) responsible for β1,6-branching of N-glycans, which is important for T cell receptor activation. Structural changes of glycans have a profound effect on the pro-inflammatory activity of immune cells and serum immune proteins, including IgG in autoimmunity.