Altered Glycosylation Patterns Research Articles

Design, synthesis, and optimization of novel PD-L1 inhibitors and the identification of a highly potent and orally bioavailable PD-L1 inhibitor

In this paper we report the discovery of structurally novel and highly potent programmed cell death-ligand 1 (PD-L1) inhibitors targeting surface and intracellular PD-L1. A ring fusion design utilizing dimethoxyphenyl indazole derivatives was used, followed by structural extension, which further improved potency by inducing the formation of additional symmetrical interactions within the PD-L1 binding site, leading to the discovery of novel and highly active tetra-aryl-scaffold inhibitors. Key optimizations involved polar tail chain modifications that improve potency and minimize cell cytotoxicity. In addition, druggability issues that exist outside the rule-of-five chemical space were addressed. CB31, a representative compound, was found to exhibit outstanding activity in blocking programmed cell death-1 (PD-1)/PD-L1 interactions (IC50 = 0.2 nM) and enhancing T-cell functions, with minimal cell cytotoxicity. CB31 also displayed favorable oral pharmacokinetic properties, consistent with its high passive permeability and insusceptibility to efflux transporters, as well as its high metabolic stability. Additionally, CB31 demonstrated mechanistically differentiated features from monoclonal antibodies by inducing PD-L1 internalization, intracellular retention of PD-L1 with altered glycosylation pattern, and PD-L1 degradation. It also demonstrated greater effects on tumor size reduction and tumor cell killing, with enhanced T-cell infiltration, in a 3D tumor spheroid model. Overall, results show that CB31 is a promising small-molecule PD-L1 inhibitor that can inhibit PD-1/PD-L1 interactions and promote PD-L1 degradation.

Glycosylation and Characterization of Human Transferrin in an End-Stage Kidney Disease.

Chronic kidney disease (CKD) is a global health concern affecting approximately one billion individuals worldwide. End-stage kidney disease (ESKD), the most severe form of CKD, is often accompanied by anemia. Peritoneal dialysis (PD), a common treatment for ESKD, utilizes the peritoneum for solute transfer but is associated with complications including protein loss, including transferrin (Tf) a key protein involved in iron transport. This study investigated Tf characteristics in ESKD patients compared to healthy individuals using lectin microarray, spectroscopic techniques and immunocytochemical analysis to assess Tf interaction with transferrin receptors (TfRs). ESKD patients exhibited altered Tf glycosylation patterns, evidenced by significant changes in lectin reactivity compared to healthy controls. However, structural analyses revealed no significant differences in the Tf secondary or tertiary structures between the two groups. A functional analysis demonstrated comparable Tf-TfR interaction in both PD and healthy samples. Despite significant alterations in Tf glycosylation, structural integrity and Tf-TfR interaction remained preserved in PD patients. These findings suggest that while glycosylation changes may influence iron metabolism, they do not impair Tf function. The study highlights the importance of a glucose-free dialysis solutions in managing anemia exacerbation in PD patients with poorly controlled anemia, potentially offering a targeted therapeutic approach to improve patient outcomes.

IgG1 glycosylation highlights premature aging in Down syndrome.

Down syndrome (DS) is characterized by lowered immune competence and premature aging. We previously showed decreased antibody response following SARS-CoV-2 vaccination in adults with DS. IgG1 Fc glycosylation patterns are known to affect the effector function of IgG and are associated with aging. Here, we compare total and anti-spike (S) IgG1 glycosylation patterns following SARS-CoV-2 vaccination in DS and healthy controls (HC). Total and anti-Spike IgG1 Fc N-glycan glycoprofiles were measured in non-exposed adults with DS and controls before and after SARS-CoV-2 vaccination by liquid chromatography-mass spectrometry (LC-MS) of Fc glycopeptides. We recruited N = 44 patients and N = 40 controls. We confirmed IgG glycosylation patterns associated with aging in HC and showed premature aging in DS. In DS, we found decreased galactosylation (50.2% vs. 59.0%) and sialylation (6.7% vs. 8.5%) as well as increased fucosylation (97.0% vs. 94.6%) of total IgG. Both cohorts showed similar bisecting GlcNAc of total and anti-S IgG1 with age. In contrast, anti-S IgG1 of DS and HC showed highly comparable glycosylation profiles 28 days post vaccination. The IgG1 glycoprofile in DS exhibits strong premature aging. The combination of an early decrease in IgG1 Fc galactosylation and sialylation and increase in fucosylation is predicted to reduce complement activity and decrease FcγRIII binding and subsequent activation, respectively. The altered glycosylation patterns, combined with decreased antibody concentrations, help us understand the susceptibility to severe infections in DS. The effect of premature aging highlights the need for individuals with DS to receive tailored vaccines and/or vaccination schedules.

Analysis of Molecular Genetic Variants of Lgals4 in Esophageal Cancer: A Preliminary Report.

Esophageal cancer is the eighth most common cancer worldwide and fourth most common in developing countries. Altered glycosylation pattern of cell membrane molecules along with inflammation is a characteristic attribute of oncogenesis. Galectin-4, a tandem repeat galectin, has shown effect on cancer progression/metastasis in digestive system cancers. This role of galectin-4 can be attributed to variations in LGALS4, gene encoding galectin-4. The present case-control study was designed to analyze four intronic SNPs in LGALS4 with susceptibility toward esophageal cancer.Esophageal cancer cases and age- and gender-matched apparently healthy individuals were recruited for the present study. Genotyping of rs8113319, rs4802886, rs4802887, and rs12610990 was carried out using Sanger sequencing and PCR-RFLP. MedCalc software, SNPStats and SHEsis online platform were used for statistical analysis.Genotypic analyses revealed an overall increased heterozygosity of rs12610990, rs4802886, and rs4802887, and AA genotype of rs8113319 in the study participants. Haplotypic analyses also revealed a predominance of AAAT haplotype in the cases. Moreover, combined presence of wild alleles of rs4802886 and rs4802887 could influence protection toward disease, and combined presence of wild alleles of rs12610990 and rs8113319 could influence disease susceptibility. Furthermore, a strong linkage disequilibrium was also observed between the SNPs. Further studies are underway to validate galectin-4 and its genetic variants as blood-based biomarkers in early disease diagnosis, improving treatment outcome.

An emerging role of N-glycosylation in cancer chemoresistance

Chemoresistance poses a significant obstacle in the effective treatment of cancer, limiting the success of chemotherapy regimens. N-glycosylation, the most important post-translational modification (PTM), plays multifaceted roles in the intricate landscape of cancer progression, particularly drug resistance in cancer cells. This review explores the complex relationship between N-glycosylation and chemoresistance in cancer. Altered glycosylation patterns have been proven to impact drug efflux mechanisms in cancer cells, which can further influence the intracellular concentration of chemotherapy drugs. Moreover, N-glycosylation also plays a regulatory role in cell signaling pathways and apoptosis regulators, continuously affecting the stemness and survival of cancer cells under the selective pressure of chemotherapy. Additionally, the impact of the tumor microenvironment on glycosylation patterns adds complexity to this interplay. This review discusses current research findings, challenges, and future directions based on the roles of N-glycosylation in cancer chemoresistance, emphasizing the potential for targeted therapeutic interventions to enhance the effectiveness of chemotherapy and improve patient outcomes.

Stool Glycoproteomics Signatures of Pre-Cancerous Lesions and Colorectal Cancer.

Colorectal cancer (CRC) screening relies primarily on stool analysis to identify occult blood. However, its sensitivity for detecting precancerous lesions is limited, requiring the development of new tools to improve CRC screening. Carcinogenesis involves significant alterations in mucosal epithelium glycocalyx that decisively contribute to disease progression. Building on this knowledge, we examined patient series comprehending premalignant lesions, colorectal tumors, and healthy controls for the T-antigen-a short-chain O-glycosylation of proteins considered a surrogate marker of malignancy in multiple solid cancers. We found the T-antigen in the secretions of dysplastic lesions as well as in cancer. In CRC, T-antigen expression was associated with the presence of distant metastases. In parallel, we analyzed a broad number of stools from individuals who underwent colonoscopy, which showed high T expressions in high-grade dysplasia and carcinomas. Employing mass spectrometry-based lectin-affinity enrichment, we identified a total of 262 proteins, 67% of which potentially exhibited altered glycosylation patterns associated with cancer and advanced pre-cancerous lesions. Also, we found that the stool (glyco)proteome of pre-cancerous lesions is enriched for protein species involved in key biological processes linked to humoral and innate immune responses. This study offers a thorough analysis of the stool glycoproteome, laying the groundwork for harnessing glycosylation alterations to improve non-invasive cancer detection.

Unveiling the Role of Concanavalin A in a Rodent Model of Chemical-Induced Hepatocellular Carcinoma: A Promising Guide in Understanding Liver Cancer Development

Hepatocellular carcinoma is a pressing global health issue, ranking as the third leading cause of cancer-related mortality in humans. Chronic liver diseases, such as hepatitis B and C infections and cirrhosis, are often associated with hepatocellular carcinoma, necessitating ongoing research for improved diagnostic and therapeutic strategies. Animal models, including both spontaneous and chemically induced models like diethylnitrosamine, play a pivotal role in understanding hepatocellular carcinoma mechanisms. Metabolic alterations in tumoral hepatocytes contribute significantly to cancer initiation and progression, impacting energy metabolism and cell survival. Lectins, specifically Concanavalin A, provide valuable insights into altered glycosylation patterns in cancer cells. This study employs lectin histochemistry to assess hepatic alterations in Concanavalin A expression in a murine model of diethylnitrosamine-induced hepatocellular carcinoma. Utilizing confocal laser scanning microscopy, our study unveils notable changes in Concanavalin A subcellular localization and intensity distribution in hepatocellular carcinoma compared with healthy liver tissue. A significant increase in the Concanavalin A labeling within the tumoral cells and a shifting of the expression within the perinuclear space is observed. These findings offer valuable insights into molecular changes in hepatocellular carcinoma, providing potential avenues for diagnostic and therapeutic advancements.

An Integrated Glycosylation Signature of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) Is a highly prevalent autoimmune disease that affects the joints but also various other organs. The disease is characterized by autoantibodies that are often already observed pre-disease. Since the 1980s, it has been known that antibody glycosylation is different in RA as compared to control individuals. While the literature on glycosylation changes in RA is dominated by reports on serum or plasma immunoglobulin G (IgG), our recent studies have indicated that the glycosylation changes observed for immunoglobulin A (IgA) and total serum N-glycome (TSNG) may be similarly prominent, and useful in differentiating between the RA patients and controls, or as a proxy of the disease activity. In this study, we integrated and compared the RA glycosylation signatures of IgG, IgA and TSNG, all determined in the pregnancy-induced amelioration of rheumatoid arthritis (PARA) cohort. We assessed the association of the altered glycosylation patterns with the disease, autoantibody positivity and disease activity. Our analyses indicated a common, composite glycosylation signature of RA that was independent of the autoantibody status.

Chemoenzymatic Synthesis of Glycopeptides to Explore the Role of Mucin 1 Glycosylation in Cell Adhesion.

Post-translational modifications affect protein biology under physiological and pathological conditions. Efficient methods for the preparation of peptides and proteins carrying defined, homogeneous modifications are fundamental tools for investigating these functions. In the case of mucin 1 (MUC1), an altered glycosylation pattern is observed in carcinogenesis. To better understand the role of MUC1 glycosylation in the interactions and adhesion of cancer cells, we prepared a panel of homogeneously O-glycosylated MUC1 peptides by using a quantitative chemoenzymatic approach. Cell-adhesion experiments with MCF-7 cancer cells on surfaces carrying up to six differently glycosylated MUC1 peptides demonstrated that different glycans have a significant impact on adhesion. This finding suggests a distinct role for MUC1 glycosylation patterns in cancer cell migration and/or invasion. To decipher the molecular mechanism for the observed adhesion, we investigated the conformation of the glycosylated MUC1 peptides by NMR spectroscopy. These experiments revealed only minor differences in peptide structure, therefore clearly relating the adhesion behaviour to the type and number of glycans linked to MUC1.

Glycosylated Fibronectin as a wide-spectrum biomarker in pregnancy

Glycosylated Fibronectin (GFN), a glycoprotein involved in cell adhesion and migration, has been implicated in several physiological and pathological processes. Current literature suggests its altered glycosylation patterns might be associated with various pregnancy complications with recent studies providing new empirical evidence from a cohort study involving more than 2,000 pregnant women. The aim of this review, is to summarize the association of GFN levels and its glycosylation patterns with various outcomes including preeclampsia, gestational diabetes mellitus (GDM), spontaneous preterm birth, and intrauterine growth restriction (IUGR) in order to identify potential patterns of the use of GFN as a biomarker for predicting a wide range of pregnancy-related complications, and to provide evidence for further investigation on new diagnostic tools and therapeutic strategies.

Anti-human immunodeficiency virus-1 activity of MoMo30 protein isolated from the traditional African medicinal plant Momordica balsamina

BackgroundPlants are used in traditional healing practices of many cultures worldwide. Momordica balsamina is a plant commonly used by traditional African healers as a part of a treatment for HIV/AIDS. It is typically given as a tea to patients with HIV/AIDS. Water-soluble extracts of this plant were found to contain anti-HIV activity.MethodsWe employed cell-based infectivity assays, surface plasmon resonance, and a molecular-cell model of the gp120-CD4 interaction to study the mechanism of action of the MoMo30-plant protein. Using Edman degradation results of the 15 N-terminal amino acids, we determined the gene sequence of the MoMo30-plant protein from an RNAseq library from total RNA extracted from Momordica balsamina.ResultsHere, we identify the active ingredient of water extracts of the leaves of Momordica balsamina as a 30 kDa protein we call MoMo30-plant. We have identified the gene for MoMo30 and found it is homologous to a group of plant lectins known as Hevamine A-like proteins. MoMo30-plant is distinct from other proteins previously reported agents from the Momordica species, such as ribosome-inactivating proteins such as MAP30 and Balsamin. MoMo30-plant binds to gp120 through its glycan groups and functions as a lectin or carbohydrate-binding agent (CBA). It inhibits HIV-1 at nanomolar levels and has minimal cellular toxicity at inhibitory levels.ConclusionsCBAs like MoMo30 can bind to glycans on the surface of the enveloped glycoprotein of HIV (gp120) and block entry. Exposure to CBAs has two effects on the virus. First, it blocks infection of susceptible cells. Secondly, MoMo30 drives the selection of viruses with altered glycosylation patterns, potentially altering their immunogenicity. Such an agent could represent a change in the treatment strategy for HIV/AIDS that allows a rapid reduction in viral loads while selecting for an underglycosylated virus, potentially facilitating the host immune response.

Altered glycosylation profiles of serum IgG in Takayasu arteritis

BackgroundTakayasu arteritis (TAK) is an autoimmune inflammatory disorder with an undefined etiology. This study aimed to characterize the glycosylation profiles of serum immunoglobulin G (IgG) in patients with TAK.MethodsLectin microarrays containing 56 types of lectins were used to detect the glycan levels of serum IgG in 164 patients with TAK, 128 patients with atherosclerosis used as disease controls (DCs), and 100 healthy controls (HCs). Differentially altered glycosylation patterns between TAK and control groups as well as between TAK subgroups were identified and further validated by lectin blot. The classification performance of the TAK-specific glycosylation change was measured by receiver-operating characteristic (ROC) curve analysis.ResultsLectin microarray analysis revealed significantly increased N-Acetylgalactosamine (GalNAc) levels in the TAK group compared to the DC and HC groups (all p < 0.01). For TAK subgroups, significantly decreased mannosylation was observed in patients with active TAK compared to patients with inactive disease (p < 0.01). These differences were validated by lectin blot. In addition, GalNAc levels exhibited a considerable potential for discriminating patients with TAK from patients with atherosclerosis, with an area under the curve of 0.749 (p < 0.001), a sensitivity of 71.7%, and a specificity of 73.8%.ConclusionsSerum IgG in patients with TAK displayed disease-specific glycosylation alterations. Aberrant GalNAc glycosylation showed substantial value as a diagnostic biomarker. The potential proinflammatory properties of the abnormal glycans may provide new insights into the role of humoral immunity in the pathogenesis of TAK.

Enteroaggregative Escherichia coli induces altered glycosylation in membrane proteins of cultured human intestinal epithelial cells

Emerging evidences have suggested that pathogens are capable of manipulating the glycosylation pattern of host-cell glycoconjugates, which may promote their attachment to these cells. Several enteric pathogens are known to induce such altered glycosylation in intestinal epithelium thereby, facilitating the disease process. Enteroaggregative Escherichia coli (EAEC), is one of such pathogens, known to cause acute and persistent diarrhea worldwide. However, glycosylation modulation due to EAEC infection has not been explored so far. In this study, EAEC-induced glycosylation changes in membrane proteins of human small-intestinal and colonic epithelial cell lines were found as revealed by lectin-overlay transblotting using four lectins, among which Sambucus nigra agglutinin (SNA) was selected for subsequent experiments. Several differentially expressed membrane-proteins were detected on SNA-overlay transblots following 2D-PAGE and identified by MALDI-TOF/TOF mass spectrometric analysis. Among these, voltage-dependent anion-selective channel-protein 2 (VDAC2) and prohibitin 2 (PHB2), common to both the cell lines were chosen for further characterization. Reactivity of these proteins to SNA was substantiated by their presence in SNA-agarose affinity chromatography eluted fractions. The plasma membrane localization of VDAC2 and PHB2 in EAEC infected cell lines was validated by confocal microscopy. These proteins were characterized as sialoglycoproteins by SNA-overlay transblots in presence a specific SNA inhibitor i.e., 6'sialyl lactose and deglycosylation using PNGase F, O-glycosidase and neuraminidase. Membrane localization of these sialoglycoproteins was found to facilitate EAEC adherence to human intestinal epithelial cells. SignificanceOur findings regarding EAEC induced altered glycosylation pattern of host cell membrane proteins may help in better understanding of the disease pathogenesis.

The Role of Fc Receptors on the Effectiveness of Therapeutic Monoclonal Antibodies.

Since the approval of the first monoclonal antibody (mAb) in 1986, a huge effort has been made to guarantee safety and efficacy of therapeutic mAbs. As of July 2021, 118 mAbs are approved for the European market for a broad range of clinical indications. In order to ensure clinical efficacy and safety aspects, (pre-)clinical experimental approaches evaluate the respective modes of action (MoA). In addition to antigen-specificity including binding affinity and -avidity, MoA comprise Fc-mediated effector functions such as antibody dependent cellular cytotoxicity (ADCC) and the closely related antibody dependent cellular phagocytosis (ADCP). For this reason, a variety of cell-based assays have been established investigating effector functions of therapeutic mAbs with different effector/target-cell combinations and several readouts including Fcγ receptor (FcγR)-mediated lysis, fluorescence, or luminescence. Optimized FcγR-mediated effector functions regarding clinical safety and efficacy are addressed with modification strategies such as point mutations, altered glycosylation patterns, combination of different Fc subclasses (cross isotypes), and Fc-truncation of the mAb. These strategies opened the field for a next generation of therapeutic mAbs. In conclusion, it is of major importance to consider FcγR-mediated effector functions for the efficacy of therapeutic mAbs.

Characterization of drug binding with alpha1-acid glycoprotein in clinical samples using ultrafast affinity extraction

Many drugs bind to serum transport proteins, which can affect both drug distribution and activity in the body. α1-Acid glycoprotein (AGP) is a key transport protein for basic and neutral drugs. Both elevated levels and altered glycosylation patterns of AGP have been seen in clinical conditions such as systemic lupus erythematosus (SLE). This study developed, optimized, and used the method of ultrafast affinity extraction (UAE) to examine whether these changes in AGP are associated with changes in the binding by some drugs to this transport protein. This approach used affinity microcolumns to capture and measure, in serum, the free fractions of several drugs known to bind AGP. These measurements were made with pooled normal control serum and serum samples from individuals with SLE. Immunoaffinity chromatography was used to obtain the content of AGP and HSA in these samples, and CE was used to examine the glycoform pattern for AGP in each serum sample. The free drug fractions measured for normal control serum ranged from 3.5 to 29.1%, in agreement with the results of ultrafiltration, and provided binding constants of ~105–106 M−1 for the given drugs with AGP at 37⁰C. Analysis of a screening set of SLE serum samples by UAE gave decreased free fractions (relative change, 12-55%) vs normal serum when spiked with the same types and amounts of drugs. These changes were related in some cases to AGP content, with some SLE samples having AGP levels 1.3- to 2.1-fold above the upper end of the normal range. In other cases, the changes in free fractions appeared to be linked to alterations in the glycoforms and binding constants of AGP, with some affinities differing by 1.2- to 1.5-fold vs normal AGP. This approach can be employed with other solute-protein systems and to investigate binding by other drugs or transport proteins directly in clinical samples.

Crumbs2 Is an Essential Slit Diaphragm Protein of the Renal Filtration Barrier.

Crumbs2 is expressed at embryonic stages as well as in the retina, brain, and glomerular podocytes. Recent studies identified CRB2 mutations as a novel cause of steroid-resistant nephrotic syndrome (SRNS). To study the function of Crb2 at the renal filtration barrier, mice lacking Crb2 exclusively in podocytes were generated. Gene expression and histologic studies as well as transmission and scanning electron microscopy were used to analyze these Crb2podKO knockout mice and their littermate controls. Furthermore, high-resolution expansion microscopy was used to investigate Crb2 distribution in murine glomeruli. For pull-down experiments, live cell imaging, and transcriptome analyses, cell lines were applied that inducibly express fluorescent protein-tagged CRB2 wild type and mutants. Crb2podKO mice developed proteinuria directly after birth that preceded a prominent development of disordered and effaced foot processes, upregulation of renal injury and inflammatory markers, and glomerulosclerosis. Pull-down assays revealed an interaction of CRB2 with Nephrin, mediated by their extracellular domains. Expansion microscopy showed that in mice glomeruli, Crb2 and Nephrin are organized in adjacent clusters. SRNS-associated CRB2 protein variants and a mutant that lacks a putative conserved O-glycosylation site were not transported to the cell surface. Instead, mutants accumulated in the ER, showed altered glycosylation pattern, and triggered an ER stress response. Crb2 is an essential component of the podocyte's slit diaphragm, interacting with Nephrin. Loss of slit diaphragm targeting and increasing ER stress are pivotal factors for onset and progression of CRB2-related SRNS.

Schizophrenia: Four new hypotheses

IntroductionSchizophrenia is a chronic and debilitating psychiatric disorder. Affecting social, emotional, perceptive, and cognitive domains, its clinical phenotype can be subdivided into positive and negative symptoms, and those of cognitive impairment. As the knowledge base behind the social and environmental origins accumulates, the etiological and neuropathophysiological mechanisms behind them remain elusive.ObjectivesTo review the latest developments in potential etiological hypotheses linked to schizophrenia.MethodsA non-systematic review was performed, searching Pubmed for articles published between the years of 2019 and 2020.Results (1) Common genetic variants alter brain glycosylation and may play a fundamental role in the development of schizophrenia. The strongest coding variant in schizophrenia is a missense mutation in the manganese transporter SLC39A8, which is associated with altered glycosylation patterns in humans, resulting in modification of a subset of schizophrenia-associated proteins. (2) Failure of oligodendrocytes and astrocytes to differentiate contributes to several of the key characteristics of schizophrenia, including hypomyelination and abnormalities in glutamate and potassium homoeostasis. (3) Diglossia was hypothesized as a risk factor, as it could constitute a neurodevelopmental insult. This relationship may be mediated by the reduced lateralization of language in the brain. (4) The first brain-wide resting state effective-connectivity neuroimaging analysis proposed going beyond the disconnectivity hypothesis, drawing attention to differences between back projections and forward connections, with the backward connections from the precuneus and posterior cingulate cortex implicated in memory stronger in schizophrenia.ConclusionsThese novel insights may be a promising step in the right direction, presenting not only new approaches towards the complex pathogenesis of schizophrenia, but also eventual early interventions.DisclosureNo significant relationships.

The role of altered glycosylation in human nucleus pulposus cells in inflammation and degeneration.

Intervertebral disc (IVD) degeneration causes low-back pain through disc compression, prolapse and herniation. Inflammation of the IVD and subsequent degeneration produce altered glycosylation profiles in several animal models of IVD injury and ageing, although the function of this altered glycosylation pattern in a human is unknown. Altered N-glycome, specifically sialylated and fucosylated N-glycosylation motif expression, might play a role in inflammation and disease progression. Healthy (foetal and adolescent idiopathic scoliosis) and degenerated (lumbar degeneration) human IVD glycosylation patterns were studied using lectin histochemistry. Small-molecule fluorinated sugar analogues (3Fax-Peracetyl Neu5Ac; 2F-Peracetyl-Fucose) were used to inhibit sialylation and fucosylation in an in vitro model of inflammation, to investigate their effects on the glycosignature, cell metabolism, extracellular matrix synthesis and cell migration. The effects of interleukin (IL)-1β, tumour necrosis factor (TNF)-α and IL-6 on glycosylation in human nucleus pulposus cells were investigated by lectin histochemistry, PCR and enzyme-linked immunosorbent assay (ELISA). In the in vitro model of IVD degeneration, cytokine-induced inflammation-induced hypersialylation was observed, as indicated by Sambucus nigra I binding. However, this modification was inhibited by the sialyltransferase inhibitor. Inhibition of sialylation and fucosylation modulates cell migration and protein translation of catabolic enzymes in response to inflammation. The altered patterns of glycosylation in human tissue in degeneration was consistent with previous IVD studies in murine, bovine and ovine models. The present study was the first functional investigation of glycosylation in human degenerated IVD, elucidating the role of the glycome in disease progression and identified potential therapeutic targets for future regenerative therapies.

In-depth Site-specific Analysis of N-glycoproteome in Human Cerebrospinal Fluid and Glycosylation Landscape Changes in Alzheimer's Disease

As the body fluid that directly interchanges with the extracellular fluid of the central nervous system (CNS), cerebrospinal fluid (CSF) serves as a rich source for CNS-related disease biomarker discovery. Extensive proteome profiling has been conducted for CSF, but studies aimed at unraveling site-specific CSF N-glycoproteome are lacking. Initial efforts into site-specific N-glycoproteomics study in CSF yield limited coverage, hindering further experimental design of glycosylation-based disease biomarker discovery in CSF. In the present study, we have developed an N-glycoproteomic approach that combines enhanced N-glycopeptide sequential enrichment by hydrophilic interaction chromatography (HILIC) and boronic acid enrichment with electron transfer and higher-energy collision dissociation (EThcD) for large-scale intact N-glycopeptide analysis. The application of the developed approach to the analyses of human CSF samples enabled identifications of a total of 2893 intact N-glycopeptides from 511 N-glycosites and 285 N-glycoproteins. To our knowledge, this is the largest site-specific N-glycoproteome dataset reported for CSF to date. Such dataset provides molecular basis for a better understanding of the structure–function relationships of glycoproteins and their roles in CNS-related physiological and pathological processes. As accumulating evidence suggests that defects in glycosylation are involved in Alzheimer's disease (AD) pathogenesis, in the present study, a comparative in-depth N-glycoproteomic analysis was conducted for CSF samples from healthy control and AD patients, which yielded a comparable N-glycoproteome coverage but a distinct expression pattern for different categories of glycoforms, such as decreased fucosylation in AD CSF samples. Altered glycosylation patterns were detected for a number of N-glycoproteins including alpha-1-antichymotrypsin, ephrin-A3 and carnosinase CN1 etc., which serve as potentially interesting targets for further glycosylation-based AD study and may eventually lead to molecular elucidation of the role of glycosylation in AD progression.

Glycans to improve efficacy and solubility of protein aggregation inhibitors.

Glycans to improve efficacy and solubility of protein aggregation inhibitors.