Glycan Changes Research Articles

Abstract PR12: The glycan CA19-9 promotes pancreatitis and pancreatic cancer in mice

Abstract Glycosylation alterations are indicative of tissue inflammation and neoplasia, but whether these alterations contribute to disease pathogenesis is largely unknown. To study the role of glycan changes in pancreatic disease, we inducibly expressed human fucosyltransferase 3 and b1,3-galactosyltransferase 5 in mice, reconstituting the glycan sialyl-Lewis a, also known as carbohydrate antigen 19-9 (CA19-9). Notably, CA19-9 expression in mice resulted in rapid and severe pancreatitis with hyperactivation of epidermal growth factor receptor (EGFR) signaling. Mechanistically, CA19-9 modification of the matricellular protein fibulin-3 increased its interaction with EGFR, and blockade of fibulin-3, EGFR, or CA19-9 prevented EGFR hyperactivation in organoids. CA19-9–mediated pancreatitis was reversible and could be suppressed with CA19-9 antibodies. CA19-9 also cooperated with the KrasG12D oncogene to produce aggressive pancreatic cancer. These findings implicate CA19-9 in the etiology of pancreatitis and pancreatic cancer and nominate CA19-9 as a therapeutic target. This abstract is also being presented as Poster C16. Citation Format: Dannielle Engle, Herve Tiriac, Keith Rivera, Arnaud Pommier, Sean Whalen, Tobiloba Oni, Brinda Alagesan, Eun Lee, Melissa Yao, Matthew Lucito, Benjamin Spielman, Kenneth Yu, Robert Grutzmann, Daniela Aust, Phyllis Gimotty, Katherine Pollard, Ralph Hruban, Michael Goggins, Christian Pilarsky, Young Park, Darryl Pappin, Michael A. Hollingsworth, David Tuveson. The glycan CA19-9 promotes pancreatitis and pancreatic cancer in mice [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr PR12.

The Association Between Normal BMI With Central Adiposity And Proinflammatory Potential Immunoglobulin G N-Glycosylation.

BackgroundThe mechanism by which normal body mass index (BMI) with central adiposity (NWCA) increases the risk of the diseases has not been completely elucidated. The inflammatory role of immunoglobulin G (IgG) N-glycosylation in obesity defined by BMI or central adiposity defined by waist-to-hip ratio (WHR) was reported, respectively. We undertook this three-center cross-sectional study to determine the association between the IgG N-glycans and NWCA.MethodsThe participants were categorized into four different phenotypes: normal BMI with normal WHR (NW), normal BMI with central adiposity (NWCA), obesity with normal WHR (ONCA) and obesity with central adiposity (OCA). The IgG N-glycans were analyzed using ultra-performance liquid chromatography analysis of released glycans, and differences among groups were compared.ResultsIn total, 17 out of 24 initial IgG N-glycans were significantly different among the four groups (NW, ONCA, NWCA and OCA) (P<0.05/6*78=0.0001). The changes of IgG glycans in central obesity (12 GPs) were more than those in obesity (3 GPs). In addition, lower galactosylation and bisecting GlcNAc and higher fucosylation were associated with increased risk of NWCA.ConclusionCentral obesity was involved in more changes of IgG N-glycosylation representing stronger inflammation than obesity, which might make a greater contribution to the risk of related disorders. NWCA was associated with an increased pro-inflammatory of IgG N-glycosylation, which was accompanied by the development of central obesity and other related disorders.

Glycan Analysis as Biomarkers for Testicular Cancer.

The U.S. Preventive Services Task Force does not recommend routine screening for testicular cancer (TC) in asymptomatic men, essentially because serological testicular cancer (TC) biomarkers are not reliable. The main reason is that two of the most important TC biomarkers, α-fetoprotein (AFP) and human chorionic gonadotropin (hCG), are not produced solely due to TC. Moreover, up to 40% of patients with TC do not have elevated serological biomarkers, which is why serial imaging with CT is the chief means of monitoring progress. On the other hand, exposure to radiation can lead to an increased risk of secondary malignancies. This review provides the first comprehensive account of the applicability of protein glycoprofiling as a promising biomarker for TC with applications in disease diagnostics, monitoring and recurrence evaluation. The review first deals with the description and classification of TC. Secondly, the limitations of current TC biomarkers such as hCG, AFP and lactate dehydrogenase are provided together with an extensive overview of the glycosylation of hCG and AFP related to TC. The final part of the review summarises the potential of glycan changes on either hCG and AFP as TC biomarkers for diagnostics and prognostics purposes, and for disease recurrence evaluation. Finally, an analysis of glycans in serum and tissues as TC biomarkers is also provided.

Omics-based Identification of Glycan Structures as Biomarkers for a Variety of Diseases.

Glycans play important roles in cell communication, protein interaction, and immunity, and structural changes in glycans are associated with the regulation of a range of biological pathways involved in disease. However, our understanding of the detailed relationships between specific diseases and glycans is very limited. In this study, we proposed an omics-based method to investigate the correlations between glycans and a wide range of human diseases. We analyzed the gene expression patterns of glycogenes (glycosyltransferases and glycosidases) for 79 different diseases. A biological pathway-based glycogene signature was constructed to identify the alteration in glycan biosynthesis and the associated glycan structures for each disease state. The degradation of N-glycan and keratan sulfate, for example, may promote the growth or metastasis of multiple types of cancer, including endometrial, gastric, and nasopharyngeal. Our results also revealed that commonalities between diseases can be interpreted using glycogene expression patterns, as well as the associated glycan structure patterns at the level of the affected pathway. The proposed method is expected to be useful for understanding the relationships between glycans, glycogenes, and disease and identifying disease-specific glycan biomarkers.

Glycoanalysis of the placental membrane glycoproteins throughout placental development

Structural changes of glycans are observed in different (patho)physiological conditions. Human placental membrane (glyco)proteins were isolated from the first and third trimester placentas of mothers at different ages. By using lectin microarray, we demonstrated that the placental membrane N-glycome contains several N-glycan groups: high mannose, asialylated and sialylated biantennary moieties, bisected, core fucosylated, fucosylated at other positions (bearing terminal and/or antennary Fuc), α2-6 and α2-3 sialylated structures. Employing MALDI-TOF MS enabled identification of over sixty different N-glycan structures in all samples, with 17 moieties exceeding the relative abundance of 2%. The major MS peaks originated from: 1) biantennary complex type N-glycan with a bisecting GlcNAc residue and 2) a core Fuc paucimannosidic and high mannose type structures M3-M9. Age of mothers and the stage of placental development affected N-glycome. The work presented in this article is the first comprehensive mass spectrometric study of the N-glycome of human placental membrane proteins. Our results may be seen as the baseline which can serve for future MALDI MS profiling of the placental membrane N-glycome in different pathophysiological conditions.

Human Influenza A Virus Hemagglutinin Glycan Evolution Follows a Temporal Pattern to a Glycan Limit.

Human antibody-based immunity to influenza A virus is limited by antigenic drift resulting from amino acid substitutions in the hemagglutinin (HA) head domain. Glycan addition can cause large antigenic changes but is limited by fitness costs to viral replication. Here, we report that glycans are added to H1 and H3 HAs at discrete 5-to-7-year intervals, until they reach a functional glycan limit, after which glycans are swapped at approximately 2-fold-longer intervals. Consistent with this pattern, 2009 pandemic H1N1 added a glycan at residue N162 over the 2015-2016 season, an addition that required two epistatic HA head mutations for complete glycosylation. These strains rapidly replaced H1N1 strains globally, by 2017 dominating H3N2 and influenza B virus strains for the season. The pattern of glycan modulation that we outline should aid efforts for tracing the epidemic potential of evolving human IAV strains.IMPORTANCE Frequent mutation of its major antibody target, the glycoprotein hemagglutinin, ensures that the influenza virus is perennially both a rapidly emerging virus and a major threat to public health. One type of mutation escapes immunity by adding a glycan onto an area of hemagglutinin that many antibodies recognize. This study revealed that these glycan changes follow a simple temporal pattern. Every 5 to 7 years, hemagglutinin adds a new glycan, up to a limit. After this limit is reached, no net additions of glycans occur. Instead, glycans are swapped or lost at longer intervals. Eventually, a pandemic replaces the terminally glycosylated hemagglutinin with a minimally glycosylated one from the animal reservoir, restarting the cycle. This pattern suggests the following: (i) some hemagglutinins are evolved for this decades-long process, which is both defined by and limited by successive glycan addition; and (ii) hemagglutinin's antibody dominance and its capacity for mutations are highly adapted features that allow influenza to outpace our antibody-based immunity.

Intact glycosphingolipidomic analysis of the cell membrane during differentiation yields extensive glycan and lipid changes

Glycosphingolipids (GSLs) are found in cellular membranes of most organisms and play important roles in cell-cell recognition, signaling, growth, and adhesion, among others. A method based on nanoflow high performance liquid chromatography-chip-quadrupole-time-of-flight mass spectrometry (nanoHPLC Chip-Q-TOF MS) was applied towards identifying and quantifying intact GSLs from a variety of samples, including cultured cell lines and animal tissue. The method provides the composition and sequence of the glycan, as well as variations in the ceramide portion of the GSL. It was used to profile the changes in the glycolipidome of Caco-2 cells as they undergo differentiation. A total of 226 unique GSLs were found among Caco-2 samples from five differentiation time-points. The method provided a comprehensive glycolipidomic profile of a cell during differentiation to yield the dynamic variation of intact GSL structures.

Abstract 2423: Hypoxia regulates tumor cell invasiveness through altered glycosylation

Abstract Glycosylation, one of the most fundamental post-translational modifications, is altered in many cancers. These alterations have been proven to impact on the progression of the tumor cell and promote survival. In literature published over the last half century, it is obvious there is a clear link between (a) chemo-resistance and hypoxia (b) hypoxia and epigenetics and more recently (c) glycosylation and epigenetics. We aim to bring these paradigms together with the remit of offering a more complete story and opening up new avenues of approach for the detection, diagnosis and treatment of breast and ovarian cancer. Ovarian and breast cancer cells were exposed to differential hypoxic conditions (0.5%, 1.0%, 2.0%) and normoxia. Firstly, we compared the methylation status of hypoxia exposed cells to the normoxic controls. A combination of hydrophilic interaction liquid chromatography (HILIC) and statistical analyses allowed comparisons of the secreted N-glycans from cells exposed to differential hypoxic conditions. Western blots assessed apoptosis, senescence, autophagy and epithelial to mesenchymal transition. The OrisTM migration assay assessed the migration of the cells pre- and post-differential hypoxic exposure. RT-qPCR was used to measure gene expression of appropriate glycosyltransferases and possible transcription factors (TFs) in these samples to determine any associations with changes seen in N-glycosylation. There were non-significant trends observed between the percentage oxygen that the cells were exposed to and resultant changes seen in branching and sialylation on secreted N-glycans from the breast and ovarian cancer cell lines. While only some of these changes could be explained by RT-qPCR data, GATA2/3 transcripts, identified in-silico as possible TFs, have been shown to significantly correlate with ST3GAL4 and MGAT5 glycosyltransferases, respectively. Hypoxia exposed cells also displayed increased migration with a greater effect seen in the 0.5% hypoxia exposed samples. The GATA2 and GATA3 transcription factors are gaining popularity in recent years, with links to cancer stage, increased invasiveness and as possible therapeutic targets. Our recent data shows a strong correlation between GATA2 and GATA3 and the levels of glycosyltransferases involved in branching and sialyation. These glycan changes are known to be strongly involved in cancer cell survival and metastases. Here for the first time we may have a possible mechanism of action linking GATA2 and 3 and invasiveness of breast and ovarian cancer cells. Citation Format: Gordon Greville, Esther Llop, Rosa Peracaula Miró, Amanda McCann, Pauline M. Rudd, Radka Saldova. Hypoxia regulates tumor cell invasiveness through altered glycosylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2423.

Co-evolution of HIV Envelope and Apex-Targeting Neutralizing Antibody Lineage Provides Benchmarks for Vaccine Design

SummaryBroadly neutralizing antibodies (bnAbs) targeting the HIV envelope glycoprotein (Env) typically take years to develop. Longitudinal analyses of both neutralizing antibody lineages and viruses at serial time points during infection provide a basis for understanding the co-evolutionary contest between HIV and the humoral immune system. Here, we describe the structural characterization of an apex-targeting antibody lineage and autologous clade A viral Env from a donor in the Protocol C cohort. Comparison of Ab-Env complexes at early and late time points reveals that, within the antibody lineage, the CDRH3 loop rigidifies, the bnAb angle of approach steepens, and surface charges are mutated to accommodate glycan changes. Additionally, we observed differences in site-specific glycosylation between soluble and full-length Env constructs, which may be important for tuning optimal immunogenicity in soluble Env trimers. These studies therefore provide important guideposts for design of immunogens that prime and mature nAb responses to the Env V2-apex.

Glycosylation on proteins of the intestine and perimicrovillar membrane of Triatoma (Meccus) pallidipennis, under different feeding conditions.

Trypanosoma cruzi, the causative agent of Chagas disease, interacts with molecules in the midgut of its insect vector to multiply and reach the infective stage. Many studies suggest that the parasite binds to midgut-specific glycans. We identified several glycoproteins expressed in the intestine and perimicrovillar membrane (PMM) of Triatoma (Meccus) pallidipennis under different feeding conditions. In order to assess changes in protein-linked glycans, we performed lectin and immunoblot analyses on glycoprotein extracts from these intestinal tissues using well-characterized lectins, and an antibody, which collectively recognize a wide range of different glycans epitopes. We observed that the amount and composition of proteins and glycoproteins associated with different glycans structures changed over time in the intestines and PMM under different physiological conditions. PMM extracts contained a wide variety of glycoproteins with different sugar residues, including abundant high-mannose and complex sialylated glycans. We propose that these molecules could be involved in the process of parasite-vector interactions.

Alterations of the Human Skin N- and O-Glycome in Basal Cell Carcinoma and Squamous Cell Carcinoma.

The glycome of one of the largest and most exposed human organs, the skin, as well as glycan changes associated with non-melanoma skin cancers have not been studied in detail to date. Skin cancers such as basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are among the most frequent types of cancers with rising incidence rates in the aging population. We investigated the healthy human skin N- and O-glycome and its changes associated with BCC and SCC. Matched patient samples were obtained from frozen biopsy and formalin-fixed paraffin-embedded tissue samples for glycomics analyses using two complementary glycomics approaches: porous graphitized carbon nano-liquid chromatography electro spray ionization tandem mass spectrometry and capillary gel electrophoresis with laser induced fluorescence detection. The human skin N-glycome is dominated by complex type N-glycans that exhibit almost similar levels of α2-3 and α2-6 sialylation. Fucose is attached exclusively to the N-glycan core. Core 1 and core 2 type O-glycans carried up to three sialic acid residues. An increase of oligomannose type N-glycans and core 2 type O-glycans was observed in BCC and SCC, while α2-3 sialylation levels were decreased in SCC but not in BCC. Furthermore, glycopeptide analyses provided insights into the glycoprotein candidates possibly associated with the observed N-glycan changes, with glycoproteins associated with binding events being the most frequently identified class.

Drug resistance related to aberrant glycosylation in colorectal cancer.

Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths in the world. Drug resistance of tumour cells remains the main challenge toward curative treatments efficiency. Several epidemiologic studies link emergence and recurrence of this cancer to metabolic disorders. Glycosylation that modifies more than 80% of human proteins is one of the most widepread nutrient-sensitive post-translational modifications. Aberrant glycosylation participates in the development and progression of cancer. Thus, some of these glycan changes like carbohydrate antigen CA 19-9 (sialyl Lewis a, sLea) or those found on carcinoembryonic antigen (CEA) are already used as clinical biomarkers to detect and monitor CRC. The current review highlights emerging evidences accumulated mainly during the last decade that establish the role played by altered glycosylations in CRC drug resistance mechanisms that induce resistance to apoptosis and activation of signaling pathways, alter drug absorption and metabolism, and led to stemness acquisition. Knowledge in this field of investigation could aid to the development of better therapeutic approaches with new predictive biomarkers and targets tied in with adapted diet.

Simulated Dynamics of Glycans on Ligand-Binding Domain of NMDA Receptors Reveals Strong Dynamic Coupling between Glycans and Protein Core.

N-Methyl-d-aspartate (NMDA) receptors, key neuronal receptors playing the central role in learning and memory, are heavily glycosylated in vivo. Astonishingly little is known about the structure, dynamics, and physiological relevance of glycans attached to them. We recently demonstrated that certain glycans on the ligand binding domain (LBD) of NMDA receptors (NMDARs) can serve as intramolecular potentiators, changing EC50 of NMDAR coagonists. In this work, we use molecular dynamics trajectories, in aggregate 86.5 μs long, of the glycosylated LBD of the GluN1 subunit of the NMDAR to investigate the behavior of glycans on NMDARs. Though all glycans in our simulations were structurally the same (Man5), the dynamics of glycans at different locations on NMDARs was surprisingly different. The slowest-time scale motions that we detected in various glycans in some cases corresponded to a flipping of parts of glycans relative to each other, while in other cases they reduced to a head-to-tail bending of a glycan. We predict that time scales of conformational changes in glycans on the GluN1 LBD of NMDARs range from nanoseconds to at least hundreds of microseconds. Some of the conformational changes in the glycans correlate with the physiologically important clamshell-like opening and closing of the GluN1 LBD domain. Thus, glycans are an integral part of NMDARs, and computational models of NMDARs should include glycans to faithfully represent the structure and the dynamics of these receptors.

Lectins: an effective tool for screening of potential cancer biomarkers.

In recent years, the use of lectins for screening of potential biomarkers has gained increased importance in cancer research, given the development in glycobiology that highlights altered structural changes of glycans in cancer associated processes. Lectins, having the properties of recognizing specific carbohydrate moieties of glycoconjugates, have become an effective tool for detection of new cancer biomarkers in complex bodily fluids and tissues. The specificity of lectins provides an added advantage of selecting peptides that are differently glycosylated and aberrantly expressed in cancer patients, many of which are not possibly detected using conventional methods because of their low abundance in bodily fluids. When coupled with mass spectrometry, research utilizing lectins, which are mainly from plants and fungi, has led to identification of numerous potential cancer biomarkers that may be used in the future. This article reviews lectin-based methods that are commonly adopted in cancer biomarker discovery research.

Profiling of glycan alterations in regrowing limb tissues of Cynops orientalis.

Glycans are known to play important roles in molecular recognition, cell-cell adhesion, molecular trafficking, receptor activation, and signal transduction during development and regeneration. Despite numerous investigations of regenerating salamander limbs, global analysis of the precise variation of glycans during the limb regeneration process has received little attention. Here, we have used lectin microarrays and lectin histochemistry to analyze the alterations and distribution of glycans during the early stages leading to blastema formation during Cynops orientalis limb regeneration in response to limb amputation. Compared with the control group, analysis at several time points (3, 7, and 14 days postamputation) using microarrays containing 37 lectins showed that limb tissues expressed significantly different complements of glycans recognized by 9 different lectins. Postamputation limb tissues showed higher expression of two glycan structures recognized by the lectins STL and LTL and lower expression of seven glycan structures recognized by PHA-E, MAL-I, SNA, UEA-I, PHA-E + L, VVA, and GNA. We also observed significant changes in glycans specifically at 7 days postamputation, including higher binding capacity by WFA, GSL-I, and NPA and lower binding capacity by PNA, HHL, ConA, LCA, GSL-II, and PWM. Next, we validated our lectin microarray data using lectin histochemistry in limb tissues. Glycans recognized by STL and GNA showed similar changes in signal intensity to those found in the lectin microarrays, with STL staining in the cytoplasm and GNA in the cytoplasm and nucleus. Our findings are the first report of significant postamputation changes in glycans in limb tissues and suggest that those glycans perform potentially important functions during the early stages of C. orientalis limb regeneration.

Assessment of tumor characteristics based on glycoform analysis of membrane-tethered MUC1

Clinical tissue specimens are useful for pathological diagnosis, which is, in some cases, supported by visualization of biomolecule localization. In general, diagnostic specificity in molecular pathology is increased by the acquisition of a probe to distinguish the modification of isomers. Although glycosylation is one of the candidate modifications in a protein, comparative glycan analysis of disease-associated proteins derived from a single tissue section is still challenging because of the lack of analytical sensitivity. Here we demonstrate a possible method for differential glycoform analysis of an endogenous tumor-associated glycoprotein MUC1 by an antibody-overlay lectin microarray. Tissue sections (5 μm thick) of patients with cholangiocarcinoma (CCA; n=21) and pancreatic ductal adenocarcinoma (PDAC; n=50) were stained with an anti-MUC1 antibody MY.1E12 that was established as a monoclonal antibody recognizing an MUC1 glycosylation isoform with a sialyl-core 1 structure (NeuAcα2-3galactosyl β1-3-N-acetylgalactosamine). MY.1E12-positive tissue areas (2.5 mm2) were selectively dissected with a laser capture microdissection procedure. The membrane MUC1 was enriched by immunoprecipitation with MY.1E12 and subjected to lectin microarray analysis. Even though the reactivities of MY.1E12 between CCA and PDAC were similar, the lectin-binding patterns varied. We found Maackia amurensis leukoagglutinin and pokeweed lectin distinguished MY.1E12-reactive MUC1 of CCA from that of PDAC. Moreover, MUC1 with M. amurensis hemagglutinin (MAH) reactivity potentially reflected the degree of malignancy. These results were confirmed with MAH-MY.1E12 double fluorescent immunostaining. These glycan changes on MUC1 were detected with high sensitivity owing to the cluster effect of immobilized lectins on a tandem repeat peptide antigen covered with highly dense glycosylation such as mucin. Our approach provides the information to investigate novel glycodynamics in biology, for example, glycoalteration, as well as diseases related to not only MUC1 but also other membrane proteins.

Draft Genome Sequences of Salmonella enterica Serovar Typhimurium LT2 with Deleted Chitinases That Are Emerging Virulence Factors.

ABSTRACTChitinases are glycosyl hydrolases that catalyze the hydrolysis of the β-1,4 linkages in complex carbohydrates and those that contain GlcNAc. These enzymes are considered emerging virulence factors during infection because the host glycan changes. This is the release of four single chitinase deletion mutants in Salmonella enterica serovar Typhimurium LT2.

LC-MS/MS isomeric profiling of permethylated N-glycans derived from serum haptoglobin of hepatocellular carcinoma (HCC) and cirrhotic patients.

Early stage detection and cancer treatment demand the identification of reliable biomarkers. Over the past decades, efforts have been devoted to assess the variation of glycosylation level as well as the glycan structures of proteins in blood or serum, associated with the development and/or progression of several cancers, including liver. Herein, an LC-MS/MS-based analysis was conducted to define the glycosylation patterns of haptoglobin glycoprotein derived from sera collected from cirrhotic and hepatocellular carcinoma (HCC) patients. The haptoglobin samples were extracted from serum using an antibody-immobilized column prior to the release of N-glycans. A comparison of non-isomeric and isomeric permethylated glycan forms was achieved using C18 and porous graphitic carbon (PGC) columns, respectively. In the case of C18-LC-MS/MS analysis, 25 glycan structures were identified of which 10 sialylated structures were found to be statistically significant between the two cohorts. Also, 8 out of 34 glycan structures identified by PGC-LC-MS/MS were found to be statistically significant, suggesting that isomeric distributions of a particular glycan composition were different in abundances between the two cohorts. The glycan isoform patterns distinguished early stage HCC from cirrhotic patients. Both retention times and tandem mass spectra were utilized to determine the specific isomeric glycan structures. All of the glycan isomers, which were statistically significant, were either branch fucosylated or composed of α-2,6 linked sialic acid moieties. The result of this study demonstrates the potential importance of isomeric separation for defining disease prompted aberrant glycan changes. The levels of several glycan isoforms effectively distinguished early stage HCC from cirrhosis.

Abstract 5544: New technologies to probe the systems glycobiology of cancer

Abstract A robotic 384 well based platform was developed to release and label the human serum N-glycome. Improved separation of the glycan pool, based on hydrophilic interaction UPLC chromatography combined with mass spectrometry (Waters Corporation) and computer assisted data interpretation (NIBRT Glycobase) enabled us to build a database after assigning the detailed structures of more than 170 N-glycans. The aim of the project was to use these technologies and databases to link glycosylation changes in individual patients’ serum with features of cancer and with changes in a range of other –omics data acquired from the same patients. Alterations in glycosylation in various breast cancers were mapped to changes in the serum glycomes and aligned with genetic, transcriptomic and proteomic data. Pathway analysis showed strong associations between these glycan changes and the –omics data. This revealed that many of the glycan changes are directly associated with pathways involved in cancer metastasis. Our next aim was to demonstrate the feasibility of collecting personalised data from individual patients from each of the –omics analyses to build up a dataset of the changing glycosylation over time. This opens up the possibility of linking these data to explore pathways of disease and, in particular, nodal points where the patient can no longer compensate for the effects of an altered pathway that is leading to disease. To this end, single glycoproteins from 8 controls and 26 ovarian cancer patients were sequentially purified from 5ul of serum on affinity plates and the released glycans were analysed. Rather than the conventional way of looking at markers which compare a patients’ data with averages, the control can now be the blood of the patient themselves taken at an earlier time point. The automation, sensitivity, quantitation and resolution of the new technology platforms coupled with dedicated software open up new possibilities for precision medicine and early intervention based on the biochemical profile of the patient.

Enterocyte glycosylation is responsive to changes in extracellular conditions: implications for membrane functions.

Epithelial cells in the lining of the intestines play critical roles in maintaining homeostasis while challenged by dynamic and sudden changes in luminal contents. Given the high density of glycosylation that encompasses their extracellular surface, environmental changes may lead to extensive reorganization of membrane-associated glycans. However, neither the molecular details nor the consequences of conditional glycan changes are well understood. Here we assessed the sensitivity of Caco-2 and HT-29 membrane N-glycosylation to variations in (i) dietary elements, (ii) microbial fermentation products and (iii) cell culture parameters relevant to intestinal epithelial cell growth and survival. Based on global LC-MS glycomic and statistical analyses, the resulting glycan expression changes were systematic, dependent upon the conditions of each controlled environment. Exposure to short chain fatty acids produced significant increases in fucosylation while further acidification promoted hypersialylation. Notably, among all conditions, increases of high mannose type glycans were identified as a major response when extracellular fructose, galactose and glutamine were independently elevated. To examine the functional consequences of this discrete shift in the displayed glycome, we applied a chemical inhibitor of the glycan processing mannosidase, globally intensifying high mannose expression. The data reveal that upregulation of high mannose glycosylation has detrimental effects on basic intestinal epithelium functions by altering permeability, host-microbe associations and membrane protein activities.