Glycosylation Alterations in Multiple Sclerosis Show Increased Proinflammatory Potential.
ABSTRACT: Multiple sclerosis (MS) is an inflammatory autoimmune disorder affecting the central nervous system (CNS), with unresolved aetiology. Previous studies have implicated N-glycosylation, a highly regulated enzymatic attachment of complex sugars to targeted proteins, in MS pathogenesis. We investigated individual variation in N-glycosylation of the total plasma proteome and of IgG in MS. Both plasma protein and IgG N-glycans were chromatographically profiled and quantified in 83 MS cases and 88 age- and sex-matched controls. Comparing levels of glycosylation features between MS cases and controls revealed that core fucosylation (p = 6.96 × 10-3) and abundance of high-mannose structures (p = 1.48 × 10-2) were the most prominently altered IgG glycosylation traits. Significant changes in plasma protein N-glycome composition were observed for antennary fucosylated, tri- and tetrasialylated, tri- and tetragalactosylated, high-branched N-glycans (p-value range 1.66 × 10-2-4.28 × 10-2). Classification performance of N-glycans was examined by ROC curve analysis, resulting in an AUC of 0.852 for the total plasma N-glycome and 0.798 for IgG N-glycome prediction models. Our results indicate that multiple aspects of protein glycosylation are altered in MS, showing increased proinflammatory potential. N-glycan alterations showed substantial value in classification of the disease status, nonetheless, additional studies are warranted to explore their exact role in MS development and utility as biomarkers.
Project description:<h4>Background</h4>Primary or secondary abnormalities of glycosylation have been reported in various brain diseases. Decreased asialotransferrin to sialotransferrin ratio in cerebrospinal fluid (CSF) is a diagnostic marker of leukodystrophies related to mutations of genes encoding translation initiation factor, EIF2B. We investigated the CSF glycome of eIF2B-mutated patients and age-matched normal individuals in order to further characterize the glycosylation defect for possible use as a biomarker.<h4>Methodology/principal findings</h4>We conducted a differential N-glycan analysis using MALDI-TOF/MS of permethylated N-glycans in CSF and plasma of controls and eIF2B-mutated patients. We found in control CSF that tri-antennary/bisecting and high mannose structures were highly represented in samples obtained between 1 to 5 years of age, whereas fucosylated, sialylated structures were predominant at later age. In CSF, but not in plasma, of eIF2B-mutated patient samples, we found increased relative intensity of bi-antennary structures and decreased tri-antennary/bisecting structures in N-glycan profiles. Four of these structures appeared to be biomarker candidates of glycomic profiles of eIF2B-related disorders.<h4>Conclusion</h4>Our results suggest a dynamic development of normal CSF N-glycan profiles from high mannose type structures to complex sialylated structures that could be correlated with postnatal brain maturation. CSF N-glycome analysis shows relevant quantitative changes associated with eIF2B related disorders. This approach could be applied to other neurological disorders involving developmental gliogenesis/synaptogenesis abnormalities.
Project description:<h4>Background</h4>Chronic obstructive pulmonary disease (COPD) is a complex condition, whose diagnosis requires spirometric assessment. However, considering its heterogeneity, subjects with similar spirometric parameters do not necessarily have the same functional status. To overcome this limitation novel biomarkers for COPD have been investigated. Therefore, we aimed to explore the potential value of N-glycans as COPD biomarkers and to examine the individual variation of plasma protein and immunoglobulin G (IgG) glycosylation profiles in subjects with COPD and healthy controls.<h4>Methods</h4>Both the total plasma protein and IgG N-glycome have been profiled in the total of 137 patients with COPD and 95 matching controls from Croatia. Replication cohort consisted of 61 subjects with COPD and 148 controls recruited at another Croatian medical centre.<h4>Results</h4>Plasma protein N-glycome in COPD subjects exhibited significant decrease in low branched and conversely, an increase in more complex glycan structures (tetragalactosylated, trisialylated, tetrasialylated and antennary fucosylated glycoforms). We also observed a significant decline in plasma monogalactosylated species, and the same change replicated in IgG glycome. N-glycans also showed value in distinguishing subjects in different COPD GOLD stages, where the relative abundance of more complex glycan structures increased as the disease progressed. Glycans also showed statistically significant associations with the frequency of exacerbations and demonstrated to be affected by smoking, which is the major risk factor for COPD development.<h4>Conclusions</h4>This study showed that complexity of glycans associates with COPD, mirroring also the disease severity. Moreover, changes in N-glycome associate with exacerbation frequency and are affected by smoking. In general, this study provided new insights into plasma protein and IgG N-glycome changes occurring in COPD and pointed out potential novel markers of the disease progression and severity.
Project description:A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methodology has been developed to differentiate core- and antennary-fucosylated glycosylation of glycopeptides. Both the glycosylation sites (heterogeneity) and multiple possible glycan occupancy at each site (microheterogeneity) can be resolved via intact glycopeptide analysis. The serum glycoprotein alpha-1-antitrypsin (A1AT) which contains both core- and antennary-fucosylated glycosites was used in this study. Sialidase was used to remove the sialic acids in order to simplify the glycosylation microheterogeneity and to enhance the MS signal of glycopeptides with similar glycan structures. ?1-3,4 galactosidase was used to differentiate core- and antennary-fucosylation. In-source dissociation was found to severely affect the identification and quantification of glycopeptides with low abundance glycan modification. The settings of the mass spectrometer were therefore optimized to minimize the in-source dissociation. A three-step mass spectrometry fragmentation strategy was used for glycopeptide identification, facilitated by pGlyco software annotation and manual checking. The collision energy used for initial glycopeptide fragmentation was found to be crucial for improved detection of oxonium ions and better selection of Y1 ion (peptide+GlcNAc). Structural assignments revealed that all three glycosylation sites of A1AT glycopeptides contain complex N-glycan structures: site Asn70 contains biantennary glycans without fucosylation; site Asn107 contains bi-, tri- and tetra-antennary glycans with both core- and antennary-fucosylation; site Asn271 contains bi- and tri-antennary glycans with both core- and antennary-fucosylation. The relative intensity of core- and antennary-fucosylation on Asn107 was similar to that of the A1AT protein indicating that the glycosylation level of Asn107 is much larger than the other two sites.
Project description:Changes in the abundance of antennary fucosylated glycans in human total plasma N-glycome (TPNG) have been associated with several diseases ranging from diabetes to various forms of cancer. However, it is challenging to address this important part of the human glycome. Most commonly, time-consuming chromatographic separations are performed to differentially quantify core and antenna fucosylation. Obtaining sufficient resolution for larger, more complex glycans can be challenging. We introduce a matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) assay for the relative quantitation of antennary fucosylation in TPNG. N-linked glycans are released from plasma by PNGase F and further treated with a core fucosidase before performing a linkage-informative sialic acid derivatization. The core fucosylated glycans are thus depleted while the remaining antennary fucosylated glycans are quantitated. Simultaneous quantitation of ?2,3-linked sialic acids and antennary fucosylation allows an estimation of the sialyl-Lewis x motif. The approach is feasible using either ultrahigh-resolution Fourier-transform ion cyclotron resonance mass spectrometry or time-of-flight mass spectrometry. The assay was used to investigate changes of antennary fucosylation as clinically relevant marker in 14 colorectal cancer patients. In accordance with a previous report, we found elevated levels of antennary fucosylation pre-surgery which decreased after tumor resection. The assay has the potential for revealing antennary fucosylation signatures in various conditions including diabetes and different types of cancer.
Project description:Protein glycosylation is a ubiquitous modification that affects the structure and function of proteins. Our recent genome wide association study identified transcription factor HNF1A as an important regulator of plasma protein glycosylation. To evaluate the potential impact of epigenetic regulation of HNF1A on protein glycosylation we analyzed CpG methylation in 810 individuals. The association between methylation of four CpG sites and the composition of plasma and IgG glycomes was analyzed. Several statistically significant associations were observed between HNF1A methylation and plasma glycans, while there were no significant associations with IgG glycans. The most consistent association with HNF1A methylation was observed with the increase in the proportion of highly branched glycans in the plasma N-glycome. The hypothesis that inactivation of HNF1A promotes branching of glycans was supported by the analysis of plasma N-glycomes in 61 patients with inactivating mutations in HNF1A, where the increase in plasma glycan branching was also observed. This study represents the first demonstration of epigenetic regulation of plasma glycome composition, suggesting a potential mechanism by which epigenetic deregulation of the glycome may contribute to disease development.
Project description:The N-linked glycoprofile of bovine whey is the combined result of individual protein glycoprofiles. In this work, we provide in-depth structural information on the glycan structures of known whey glycoproteins, namely, lactoferrin, lactoperoxidase, ?-lactalbumin, immunoglobulin-G (IgG), and glycosylation-dependent cellular adhesion molecule 1 (GlyCAM-1, PP3). The majority (?95%) of <i>N</i>-glycans present in the overall whey glycoprofile were attributed to three proteins: lactoferrin, IgG, and GlyCAM-1. We identified specific signature glycans for these main proteins; lactoferrin contributes oligomannose-type glycans, while IgG carries fucosylated di-antennary glycans with Gal-?(1,4)-GlcNAc (LacNAc) motifs. GlyCAM-1 is the sole whey glycoprotein carrying tri- and tetra-antennary structures, with a high degree of fucosylation and sialylation. Signature glycans can be used to recognize individual proteins in the overall whey glycoprofile as well as for protein concentration estimations. Application of the whey glycoprofile analysis to colostrum samples revealed dynamic protein concentration changes for IgG, lactoferrin, and GlyCAM-1 over time.
Project description:Glycosylation is a complex post-translational modification that conveys functional diversity to glycoconjugates. Cell surface glycosylation mediates several biological activities such as induction of the intracellular signaling pathway and pathogen recognition. Red blood cell (RBC) membrane N-glycans determine blood type and influence cell lifespan. Although several proteomic studies have been carried out, the glycosylation of RBC membrane proteins has not been systematically investigated. This work aims at exploring the human RBC N-glycome by high-sensitivity MALDI-MS techniques to outline a fingerprint of RBC N-glycans. To this purpose, the MALDI-TOF spectra of healthy subjects harboring different blood groups were acquired. Results showed the predominant occurrence of neutral and sialylated complex N-glycans with bisected N-acetylglucosamine and core- and/or antennary fucosylation. In the higher mass region, these species presented with multiple N-acetyllactosamine repeating units. Amongst the detected glycoforms, the presence of glycans bearing ABO(H) antigens allowed us to define a distinctive spectrum for each blood group. For the first time, advanced glycomic techniques have been applied to a comprehensive exploration of human RBC N-glycosylation, providing a new tool for the early detection of distinct glycome changes associated with disease conditions as well as for understanding the molecular recognition of pathogens.
Project description:Colorectal cancer (CRC) ranks second as the leading cause of cancer-related deaths worldwide. N-glycosylation is one of the most common posttranslational protein modifications. Therefore, we studied the total serum N-glycome (TSNG) of 13 colon cancer patients compared to healthy controls using MALDI-TOF/MS and LC-MS. N-glycosylation of cancer tumor samples from the same cohort were further quantified using a similar methodology. In total, 23 N-glycan compositions were down-regulated in the serum of colon cancer patients, mostly galactosylated forms whilst the mannose-rich HexNAc2Hex7, the fucosylated bi-antennary glycan HexNAc4Hex5Fuc1NeuAc2, and the tetra-antennary HexNAc6Hex7NeuAc3 were up-regulated in serum. Hierarchical clustering analysis of TSNG correctly singled out 85% of the patients from controls. Albeit heterogenous, N-glycosylation of tumor samples showed overrepresented oligomannosidic, bi-antennary hypogalactosylated, and branched compositions related to normal colonic tissue, in both MALDI-TOF/MS and LC-MS analysis. Moreover, compositions found upregulated in tumor tissue were mostly uncorrelated to compositions in serum of cancer patients. Mass spectrometry-based N-glycan profiling in serum shows potential in the discrimination of patients from healthy controls. However, the compositions profile in serum showed no parallel with N-glycans in tumor microenvironment, which suggests a different origin of compositions found in serum of cancer patients.
Project description:We previously showed that the expression of (Gal alpha 1-4Gal)-bearing glycoproteins among birds is related to their phylogeny. However, precise structures of (Gal alpha 1-4Gal)-containing N-glycans were only known for pigeon egg white glycoproteins and IgG. To compare structural features of (Gal alpha 1-4Gal)-containing N-glycans from other species, we analyzed N-glycans of gull egg white (GEW)-glycoproteins, ovomucoid, and ovotransferrin, and gull egg yolk IgG by HPLC, mass spectrometry (MS), and MS/MS analyses. GEW-glycoproteins included neutral, monosialyl, and disialyl N-glycans, and some of them contained Gal alpha 1-4Gal sequences. Bi-, tri-, and tetra-antennary oligosaccharides that lacked bisecting GlcNAc were the major core structures, and incomplete alpha-galactosylation and sialylation as well as the presence of diLacNAc on the branches generated microheterogeneity of the N-glycan structures. Moreover, unlike pigeon egg white glycoproteins, the major sialylation in GEW-glycoproteins is alpha2,3-, but not alpha2,6-linked sialic acids (NeuAc). In addition to the complex-type oligosaccharide, hybrid-type oligosaccharides that lack bisecting GlcNAc were also abundant in GEW-glycoproteins. Gull egg yolk IgG also contained Gal alpha 1-4Gal beta 1-4GlcNAc beta 1- sequences, but unlike pigeon IgG, no Gal alpha 1-4Gal beta 1-4Gal beta 1-4GlcNAc beta 1- sequence was detected. Bi- and tri-antennary complex-type oligosaccharides with bisecting GlcNAc and with core fucosylation as well as high-mannose-type oligosaccharides were the major structures in gull IgG. Our data indicated that some N-glycans from both GEW-glycoproteins and gull IgG contain the Gal alpha 1-4Gal beta 1-4GlcNAc beta 1- sequence, but the ratio of alpha-Gal-capped residues to non-alpha-Gal-capped residues in the nonreducing termini of N-glycans is much lower than that in those of pigeon glycoproteins.
Project description:Glycosylation and related processes play important roles in cancer development and progression, including metastasis. Several studies have shown that N-glycans have potential diagnostic value as cancer serum biomarkers. We have explored the significance of the abundance of particular serum N-glycan structures as important features of breast tumour biology by studying the serum glycome and tumour transcriptome (mRNA and miRNA) of 104 breast cancer patients. Integration of these types of molecular data allows us to study the relationship between serum glycans and transcripts representing functional pathways, such as metabolic pathways or DNA damage response. We identified tri antennary trigalactosylated trisialylated glycans in serum as being associated with lower levels of tumour transcripts involved in focal adhesion and integrin-mediated cell adhesion. These glycan structures were also linked to poor prognosis in patients with ER negative tumours. High abundance of simple monoantennary glycan structures were associated with increased survival, particularly in the basal-like subgroup. The presence of circulating tumour cells was found to be significantly associated with several serum glycome structures like bi and triantennary, di- and trigalactosylated, di- and trisialylated. The link between tumour miRNA expression levels and N-glycan production is also examined.