Changes In Glycosylation Research Articles

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 & 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 >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 (<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。

Abstract 319: MALDI-IMS of N-glycan profiles of molecular subclasses of human hepatocellular carcinoma

Abstract Introduction: Hepatocellular Carcinoma (HCC) is the second leading cause of cancer deaths globally, and the incidence rate in the US is increasing. Recent work has identified changes in N-linked glycosylation directly in HCC tissue by MALDI-IMS glycan imaging. However, there was significant inter-tumor heterogeneity, suggesting a potential correlation between the glycan changes and molecular tumor heterogeneity. Therefore, this work will analyze HCC tissues, categorized by subtype, in an effort to fit glycosylation patterns within different classes of HCC. Methods: To analyze the glycosylation of HCC tissue samples, MALDI-IMS was utilized, which uniquely allows for the analysis of spatially mapped N-glycans to paraffin embedded tissues. The sample set of HCC tissues consisted of Hoshida HCC subtypes 1, 2, and 3 tissues. Tissues were prepared through a previously published protocol that involved antigen retrieval, spraying of PNGase F Prime with a TM-Sprayer, then spraying of MALDI matrix CHCA. Each tissue is then imaged on a Bruker MALDI solariX FT-ICR, and data is analyzed using flexImaging and SCiLS software. Results: The primary goal of this work was to determine if differences within N-glycan profiles of HCC tissues are related to the differences between Hoshida genetic subtypes. Preliminary data showed that HCC tumors contain increased fucosylation of glycoproteins, yet there was significant heterogeneity within analyzed HCC tissues regarding the expressed glycan structures. With HCC tissues that were categorized into different Hoshida subtypes, glycan MALDI-IMS data showed trends that could approximately separate the subtypes. Subtype 1, which is less differentiated and is associated with poor survival, showed the most extreme tumor-associated glycan expression when compared to the surrounding normal and cirrhotic tissue. Subtype 1 tissues showed a higher number of unique glycan structures that associated with the tumor tissue. Subtype 1 showed an average of 18.00 ± 8.48 tumor-associated glycans, subtype 2 with 4.25 ± 2.21, and subtype 3 with 2.25 ± 1.71. While the identity of these glycan structures varied from tissue to tissue, trends emerged, particularly regarding how branched and fucosylated glycans associate to tumor tissue with each subtype, including decreased fucosylation in subtype 2. Overall, this data shows that some of the heterogeneity in glycan expression of HCC tumors may be attributable to differences between genetic subtypes of HCC. Conclusion: HCC tissues exhibit glycan expression that is noticeably different from normal or cirrhotic tissue, yet there is significant variation between HCC tissues regarding both the number and identity of glycan structures that are observed on the tissue. Using the Hoshida classification system, some of that variation can be explained, which could have profound impact on the utilization of N-linked glycosylation for biomarker applications. Citation Format: Andrew DelaCourt, Anand Mehta, Alyson Black, Richard Drake, Peggi Angel, Yujin Hoshida. MALDI-IMS of N-glycan profiles of molecular subclasses of human hepatocellular carcinoma [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 319.

Mucin Binding to Moraxella catarrhalis during Airway Inflammation Is Dependent on Sialic Acid.

Chronic obstructive pulmonary disease (COPD) is associated with colonization by bacterial pathogens and repeated airway infections, leading to exacerbations and impaired lung function. The highly glycosylated mucins in the mucus lining the airways are an important part of the host defense against pathogens. However, mucus accumulation can contribute to COPD pathology. Here, we examined whether inflammation is associated with glycosylation changes that affect interactions between airway mucins and pathogens. We isolated mucins from lower airway samples (n = 4-9) from long-term smokers with and without COPD and from never-smokers. The most abundant terminal glycan moiety was N-acetylneuraminic acid (Neu5Ac) among smokers with and without COPD and N-acetyl-hexoseamine among never-smokers. Moraxella catarrhalis bound to MUC5 mucins from smokers with and without COPD. M. catarrhalis binding correlated with inflammatory parameters and Neu5Ac content. M. catarrhalis binding was abolished by enzymatic removal of Neu5Ac. Furthermore, M. catarrhalis bound to α2,6 sialyl-lactose, suggesting that α2,6 sialic acid contributes to M. catarrhalis binding to mucins. Furthermore, we detected more M. catarrhalis binding to mucins from patients with pneumonia than to those from control subjects (n = 8-13), and this binding correlated with C-reactive protein and Neu5Ac levels. These results suggest a key role of inflammation-induced Neu5Ac in the adhesion of M. catarrhalis to airway mucins. The inflammation-induced ability of MUC5 mucins to bind M. catarrhalis is likely a host defense mechanism in the healthy lung, although it cannot be excluded that impaired mucociliary clearance limits the effectiveness of this defense in patients with COPD.

Glycosylation of IgG Associates with Hypertension and Type 2 Diabetes Mellitus Comorbidity in the Chinese Muslim Ethnic Minorities and the Han Chinese.

Objectives: Hypertension and type 2 diabetes mellitus comorbidity (HDC) is common, which confers a higher risk of cardiovascular disease than the presence of either condition alone. Describing the underlying glycomic changes of immunoglobulin G (IgG) that predispose individuals to HDC may help develop novel protective immune-targeted and anti-inflammatory therapies. Therefore, we investigated glycosylation changes of IgG associated with HDC. Methods: The IgG N-glycan profiles of 883 plasma samples from the three northwestern Chinese Muslim ethnic minorities and the Han Chinese were analyzed by ultra-performance liquid chromatography instrument. Results: We found that 12 and six IgG N-glycan traits showed significant associations with HDC in the Chinese Muslim ethnic minorities and the Han Chinese, respectively, after adjustment for potential confounders and false discovery rate. Adding the IgG N-glycan traits to the baseline models, the area under the receiver operating characteristic curves (AUCs) of the combined models differentiating HDC from hypertension (HTN), type 2 diabetes mellitus (T2DM), and healthy individuals were 0.717, 0.747, and 0.786 in the pooled samples of Chinese Muslim ethnic minorities, and 0.828, 0.689, and 0.901 in the Han Chinese, respectively, showing improved discriminating performance than both the baseline models and the glycan-based models. Conclusion: Altered IgG N-glycan profiles were shown to associate with HDC, suggesting the involvement of inflammatory processes of IgG glycosylation. The alterations of IgG N-glycome, illustrated here for the first time in HDC, demonstrate a biomarker potential, which may shed light on future studies investigating their potential for monitoring or preventing the progression from HTN or T2DM towards HDC.

Asymmetric-flow field-flow fractionation of prions reveals a strain-specific continuum of quaternary structures with protease resistance developing at a hydrodynamic radius of 15 nm.

Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrPC, into a plethora of assemblies, PrPSc, associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrPSc structures. However, the degree to which intra- and inter-strain PrPSc heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrPSc subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrPSc subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrPSc particles. We found that each strain had a continuum of PrPSc assemblies, with strong correlation between PrPSc quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrPSc aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrPSc took place at a hydrodynamic radius (Rh) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrPSc subpopulations and demonstrate that while PrPSc quaternary structure is a major contributor to PrPSc structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrPSc particles from maintaining proteinase K resistance below an Rh of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype.

Robustness and repeatability of GlycoWorks RapiFluor-MS IgG N-glycan profiling in a long-term high-throughput glycomic study.

Protein glycosylation is the attachment of a carbohydrate moiety to a protein backbone affecting both structure and function of the protein. Abnormal glycosylation is associated with various diseases, and some of the changes in glycosylation are detectable even before symptom development. As such, glycans have emerged as compelling new biomarker candidates. A wide range of analytical methods exist for small-scale glycan analyses. However, there is a growing need for highly robust and reproducible high-throughput techniques that allow for large-scale glycoprofiling. Here, we describe the evaluation of robustness and repeatability of immunoglobulin G (IgG) N-glycan analysis using the GlycoWorks RapiFluor-MS N-Glycan Kit followed by hydrophilic interaction ultra-high-performance liquid chromatography (HILIC-UHPLC) from 335 technical replicates of human plasma randomly distributed across 67 96-well plates. The data was collected over a 5-month period using multiple UHPLC systems and chromatographic columns. Following relative IgG N-glycan quantification in acquired chromatograms, data analysis showed that the most abundant peaks that together made up for three-fourths of the detected IgG N-glycome all had coefficients of variation (CVs) lower than 2%. The highest CVs ranging from 16 to 29% accompanied low abundance glycan peaks with the individual relative peak area below 1% that together made up for <2% of the detected IgG N-glycome. These results show that the tested method is very robust and repeatable, making it suitable for the IgG N-glycan analysis of a large number of samples in a high-throughput manner over a longer period of time.

Activation of regulatory T cells triggers specific changes in glycosylation associated with Siglec-1-dependent inflammatory responses.

Background: Siglec-1 is a macrophage lectin-like receptor that mediates sialic acid-dependent cellular interactions. Its upregulation on macrophages in autoimmune disease was shown previously to promote inflammation through suppressing the expansion of regulatory T cells (Tregs). Here we investigate the molecular basis for Siglec-1 binding to Tregs using in vitro-induced cells as a model system. Methods: Glycosylation changes that affect Siglec‑1 binding were studied by comparing activated and resting Tregs using RNA-Seq, glycomics, proteomics and binding of selected antibodies and lectins. A proximity labelling and proteomics strategy was used to identify Siglec-1 counter-receptors expressed on activated Tregs. Results: Siglec-1 binding was strongly upregulated on activated Tregs, but lost under resting conditions. Glycomics revealed changes in N-glycans and glycolipids following Treg activation and we observed changes in expression of multiple 'glycogenes' that could lead to the observed increase in Siglec-1 binding. Proximity labelling of intact, living cells identified 49 glycoproteins expressed by activated Tregs that may function as Siglec-1 counter-receptors. These represent ~5% of the total membrane protein pool and were mainly related to T cell activation and proliferation. We demonstrate that several of these counter-receptors were upregulated following activation of Tregs and provide initial evidence that their altered glycosylation may also be important for Siglec-1 binding. Conclusions: We provide the first comprehensive analysis of glycan changes that occur in activated Tregs, leading to recognition by the macrophage lectin, Siglec-1 and suppression of Treg expansion. We furthermore provide insights into glycoprotein counter-receptors for Siglec-1 expressed by activated Tregs that are likely to be important for suppressing Treg expansion.

Epithelial apical glycosylation changes associated with thin endometrium in women with infertility - a pilot observational study

BackgroundLow endometrial receptivity is one of the major factors affecting successful implantation in assisted reproductive technologies (ART). Infertile patients with thin endometrium have a significantly lower cumulative clinical pregnancy rate than patients with normal endometrium. Molecular pathophysiology of low receptivity of thin endometrium remains understudied. We have investigated composition of glycocalyx of the apical surface of luminal and glandular epithelial cells in thin endometrium of infertile women.MethodsThirty-two patients with tubal-peritoneal infertility undergoing in vitro fertilization (IVF) were included in the study. Endometrial samples were obtained in a natural menstrual cycle. Patients were divided into two groups: patients with normal endometrium (≥8 mm) and with thin endometrium (< 8 mm). Histochemical and immunohistochemical analysis of paraffin-embedded endometrial samples was performed using six biotinylated lectins (UEA-I, MAL-II, SNA, VVL, ECL, Con A) and anti-LeY and MECA-79 monoclonal antibodies (MAbs).ResultsComplex glycans analysis taking into account the adjusted specificity of glycan-binding MAbs revealed 1.3 times less expression of MECA-79 glycans on the apical surface of the luminal epithelial cells of thin endometrium compared to normal endometrium; this deficiency may adversely affect implantation, since MECA-79 glycans are a ligand of L-selectin and mediate intercellular interactions. The glycans containing a type-2 unit Galβ1-4GlcNAcβ (LacNAc) but lacking sulfo-residues at 6-OH of GlcNAcβ, and binding to MECA-79 MAbs were found; they can be considered as potential markers of endometrium receptivity.Expression of the lectins-stained glycans on the apical surfaces of the luminal and glandular epithelial cells did not differ significantly. Correlation between the expression of difucosylated oligosaccharide LeY on the apical surfaces of the luminal and glandular epithelial cells was found in patients with thin endometrium and recurrent implantation failure. A similar relationship was shown for mannose-rich glycans.ConclusionsSpecific features of key glycans expression in epithelial compartments of thin endometrium may be essential for morphogenesis of the endometrial functional layer and explain its low receptivity.

Giantin is required for intracellular N-terminal processing of type I procollagen.

Knockout of the golgin giantin leads to skeletal and craniofacial defects driven by poorly studied changes in glycosylation and extracellular matrix deposition. Here, we sought to determine how giantin impacts the production of healthy bone tissue by focusing on the main protein component of the osteoid, type I collagen. Giantin mutant zebrafish accumulate multiple spontaneous fractures in their caudal fin, suggesting their bones may be more brittle. Inducing new experimental fractures revealed defects in the mineralization of newly deposited collagen as well as diminished procollagen reporter expression in mutant fish. Analysis of a human giantin knockout cell line expressing a GFP-tagged procollagen showed that procollagen trafficking is independent of giantin. However, our data show that intracellular N-propeptide processing of pro-α1(I) is defective in the absence of giantin. These data demonstrate a conserved role for giantin in collagen biosynthesis and extracellular matrix assembly. Our work also provides evidence of a giantin-dependent pathway for intracellular procollagen processing.

Extensive weight loss reduces glycan age by altering IgG N-glycosylation

BackgroundObesity, a major global health problem, is associated with increased cardiometabolic morbidity and mortality. Protein glycosylation is a frequent posttranslational modification, highly responsive to inflammation and ageing. The prospect of biological age reduction, by changing glycosylation patterns through metabolic intervention, opens many possibilities. We have investigated whether weight loss interventions affect inflammation- and ageing-associated IgG glycosylation changes, in a longitudinal cohort of bariatric surgery patients. To support potential findings, BMI-related glycosylation changes were monitored in a longitudinal twins cohort.MethodsIgG N-glycans were chromatographically profiled in 37 obese patients, subjected to low-calorie diet, followed by bariatric surgery, across multiple timepoints. Similarly, plasma-derived IgG N-glycan traits were longitudinally monitored in 1680 participants from the TwinsUK cohort.ResultsLow-calorie diet induced a marked decrease in the levels of IgG N-glycans with bisecting GlcNAc, whose higher levels are usually associated with ageing and inflammatory conditions. Bariatric surgery resulted in extensive alterations of the IgG N-glycome that accompanied progressive weight loss during 1-year follow-up. We observed a significant increase in digalactosylated and sialylated glycans, and a substantial decrease in agalactosylated and core fucosylated IgG N-glycans (adjusted p value range 7.38 × 10−04–3.94 × 10−02). This IgG N-glycan profile is known to be associated with a younger biological age and reflects an enhanced anti-inflammatory IgG potential. Loss of BMI over a 20 year period in the TwinsUK cohort validated a weight loss-associated agalactosylation decrease (adjusted p value 1.79 × 10−02) and an increase in digalactosylation (adjusted p value 5.85 × 10−06).ConclusionsAltogether, these findings highlight that weight loss substantially affects IgG N-glycosylation, resulting in reduced glycan and biological age.

Aberrant Immunoglobulin G Glycosylation in Multiple Sclerosis.

A hallmark of the inflammatory response in multiple sclerosis (MS) is the presence of intrathecal Immunoglobulin G (IgG) antibodies and oligoclonal bands (OCBs). The biological activity of IgGs is modulated by changes in glycosylation. Using multiple immunoassays with common lectins for sialylation and galactosylation, we investigated levels of IgG glycosylation in 28 MS and 37 control sera as well as paired CSF and serum. We demonstrated the presence of significantly lower levels of IgG sialylation in MS CSF compared to paired serum. Further, we showed that in MS there was no correlation between sialylated IgG and total IgG antibodies, or between sialylated IgG in CSF and serum. ELISA with native IgG antibodies showed significantly higher levels of sialylated and galactosylated IgG in MS compared to other neurological disorders and normal healthy controls. We conclude that lower levels of sialylated intrathecal IgG and higher levels of serum IgG galactosylation in MS may play significant role in disease pathogenesis. The unique IgG glycosylation profiles suggest a complexed nature of the IgG antibodies which may influence its effector functions in MS.

Glycomic Profiling Unveils Association Between Serum Glycosylation and Antibody Responses to Influenza Vaccines

Despite being the most powerful approach to tackle influenza infections, current influenza vaccines have a mixed record of protection across the population. In the 2019-2020 flu season, flu vaccines in the U.S. were ~ 40% effective. High-risk populations, such as the obese and elderly, tend to have weaker responses to influenza vaccines, the molecular mechanisms behind which remain poorly understood. Glycans play critical roles in the human immune system. In this study, we examined serum glycosylation of 160 Caucasian adults pre- and post-vaccination with the 2019 Fluzone® Quadrivalent Vaccine using an expanded version of our lectin microarray technology that included antibodies against innate immune lectins and select sera glycoproteins. Glycosylation profiles were examined in light of post-vaccination antibody responses. Results showed that obese participants who exhibited poor antibody responses (non-responders) to the vaccines had higher levels of pre-vaccination innate immune lectins and high mannose glycans, suggesting a potential role of pre-existing, lectin-mediated immunity in the determination of vaccination outcomes. We observed widespread shifts in glycosylation in all participants associated with vaccination, possibly a result of antibody production. Non-responders and responders showed strikingly similar degrees of glycosylation changes. However, we observed higher levels of β1,6-branched N-glycans in responders, indicating a correlation of this specific glycan epitope with effective antibody responses. Our findings reveal the association of serum glycosylation with antibody responses to vaccination for influenza and may point to new directions in the development of better vaccines.

Functionalized High Mannose‐Specific Lectins for the Discovery of Type I Mannosidase Inhibitors

AbstractAn engineered cyanovirin‐N homologue that exhibits specificity for high mannose N‐glycans has been constructed to aid type I α 1,2‐mannosidase inhibitor discovery and development. Engineering the lectins C‐terminus permitted facile functionalization with fluorophores via a sortase and click strategy. The resulting lectin constructs exhibit specificity for cells presenting high mannose N‐glycans. Importantly, these lectin constructs can also be applied to specifically assess changes in cell surface glycosylation induced by type I mannosidase inhibitors. Testing the utility of these lectin constructs led to the discovery of type I mannosidase inhibitors with nanomolar potency. Cumulatively, these findings reveal the specificity and utility of the functionalized cyanovirin‐N homologue constructs, and highlight their potential in analytical contexts that require high mannose‐specific lectins.

Functionalized High Mannose-Specific Lectins for the Discovery of Type I Mannosidase Inhibitors.

An engineered cyanovirin-N homologue that exhibits specificity for high mannose N-glycans has been constructed to aid type I α 1,2-mannosidase inhibitor discovery and development. Engineering the lectins C-terminus permitted facile functionalization with fluorophores via a sortase and click strategy. The resulting lectin constructs exhibit specificity for cells presenting high mannose N-glycans. Importantly, these lectin constructs can also be applied to specifically assess changes in cell surface glycosylation induced by type I mannosidase inhibitors. Testing the utility of these lectin constructs led to the discovery of type I mannosidase inhibitors with nanomolar potency. Cumulatively, these findings reveal the specificity and utility of the functionalized cyanovirin-N homologue constructs, and highlight their potential in analytical contexts that require high mannose-specific lectins.

Detection of N,N-diacetyllactosamine (LacdiNAc) containing free prostate-specific antigen for early stage prostate cancer diagnostics and for identification of castration-resistant prostate cancer patients

Prostate cancer (PCa) is one of the most common cancer types among men and also acommon cause of death globally. With an increasing incidence, there is aneed for low-cost, reliable biomarkers present in samples, which could be provided non-invasively (without a need to perform prostate biopsy). Glycosylation changes of free-PSA (fPSA) are considered cancer-specific, while the level of different PSA forms can increase under other than cancerous conditions. In the present study, we investigated the role ofN,N-diacetyllactosamine (LacdiNAc) epitope of fPSA (i.e. glycoprofile of fPSA or gPSA) in combination with total-PSA (tPSA), prostate volume, and tPSA density (tPSA level divided by prostate volume i.e. PSAd) as biomarkers for monitoring of PCa development and progression in 105 men. Furthermore, we applied an genetic (evolutionary) algorithm to identify any suspicious individuals in abenign cohort having benign prostatic hyperplasia (BPH). We identified 3 suspicious men originally diagnosed with BPH using gPSA analysis. In thefollow-up we found out that two men should not be considered as BPH patients since multiparametric magnetic resonance imaging (mpMRI) identified one man with clinically significant PCa via Prostate Imaging – Reporting and Data System (PI RADS v2 = 4) and the second man was with High-gradeprostatic intraepithelial neoplasia (HG PIN), commonly described as apre-cancerous stage. Moreover, in the study we described for the first time that changed LacdiNAc on PSA can be applied to identify prostatitis patients and most importantly this is the first study suggesting that changed glycosylation on PSA can be applied to identify castration-resistant prostate cancer (CRPCa) patients.

Revealing the changes of IgG subclass-specific N-glycosylation in colorectal cancer progression by high-throughput assay.

The changes of glycosylation of different IgG subclass in colorectal cancer (CRC) were rarely investigated. The authors aimed to use a simple and high-throughput analytical method to explore the changes of subclass-specific IgG glycosylation in CRC, and to find the specific glyco-biomarkers for early detection of this disease. Serum samples from 71 cancer patients and 22 benign patients with 50 age- and sex-matched healthy controls were collected from two independent cohorts. Subclass-specific IgG glycosylation was profiled by MALDI-MS followed by the structural identification through MALDI-MS/MS. The exported MS data was automatically and rapidly processed by the self-developed MATLAB code. Statistical analysis suggested the significantly decreased galactosylation and remarkably increased agalactosylation of IgG1 or IgG2 in the malignant transformation of CRC, which enables the differentiation between cancer patients and healthy controls. The changes of glycan features were elucidated by the exploration of individual glycopeptides, showing the biantennary fucosylated glycan without galactose (H3N4F1) or with two galactose (H5N4F1) of IgG1 and IgG2 could distinguish cancer group from both benign and control groups. Through the simple and high-throughput procedures, this study revealed the important role of IgG glycopeptides in the premature pathology of CRC.

The Importance of Platelet Glycoside Residues in the Haemostasis of Patients with Immune Thrombocytopaenia.

Loss of sialic acid from the carbohydrate side chains of platelet glycoproteins can affect platelet clearance, a proposed mechanism involved in the etiopathogenesis of immune thrombocytopaenia (ITP). We aimed to assess whether changes in platelet glycosylation in patients with ITP affected platelet counts, function, and apoptosis. This observational, prospective, and transversal study included 82 patients with chronic primary ITP and 115 healthy controls. We measured platelet activation markers and assayed platelet glycosylation and caspase activity, analysing samples using flow cytometry. Platelets from patients with ITP with a platelet count <30 × 103/µL presented less sialic acid. Levels of α1,6-fucose (a glycan residue that can directly regulate antibody-dependent cellular cytotoxicity) and α-mannose (which can be recognised by mannose-binding-lectin and activate the complement pathway) were increased in the platelets from these patients. Platelet surface exposure of other glycoside residues due to sialic acid loss inversely correlated with platelet count and the ability to be activated. Moreover, loss of sialic acid induced the ingestion of platelets by human hepatome HepG2 cells. Changes in glycoside composition of glycoproteins on the platelets’ surface impaired their functional capacity and increased their apoptosis. These changes in platelet glycoside residues appeared to be related to ITP severity.

Characterization and statistical modeling of glycosylation changes in sickle cell disease

Sickle cell disease is an inherited genetic disorder that causes anemia, pain crises, organ infarction, and infections in 13 million people worldwide. Previous studies have revealed changes in sialic acid levels associated with red blood cell sickling and showed that stressed red blood cells bare surface-exposed clustered terminal mannose structures mediating hemolysis, but detailed glycan structures and anti-glycan antibodies in sickle cell disease remain understudied. Here, we compiled results obtained through lectin arrays, glycan arrays, and mass spectrometry to interrogate red blood cell glycoproteins and glycan-binding proteins found in the plasma of healthy individuals and patients with sickle cell disease and sickle cell trait. Lectin arrays and mass spectrometry revealed an increase in α2,6 sialylation and a decrease in α2,3 sialylation and blood group antigens displayed on red blood cells. Increased binding of proteins to immunogenic asialo and sialyl core 1, Lewis A, and Lewis Y structures was observed in plasma from patients with sickle cell disease, suggesting a heightened anti-glycan immune response. Data modeling affirmed glycan expression and plasma protein binding changes in sickle cell disease but additionally revealed further changes in ABO blood group expression. Our data provide detailed insights into glycan changes associated with sickle cell disease and refer glycans as potential therapeutic targets.

Glycoproteins as diagnostic and prognostic biomarkers for neurodegenerative diseases: A glycoproteomic approach.

Neurodegenerative diseases (NDs) are incurable and can develop progressively debilitating disorders, including dementia and ataxias. Alzheimer's disease and Parkinson's disease are the most common NDs that mainly affect the elderly people. There is an urgent need to develop new diagnostic tools so that patients can be accurately stratified at an early stage. As a common post-translational modification, protein glycosylation plays a key role in physiological and pathological processes. The abnormal changes in glycosylation are associated with the altered biological pathways in NDs. The pathogenesis-related proteins, like amyloid-β and microtubule-associated protein tau, have altered glycosylation. Importantly, specific glycosylation changes in cerebrospinal fluid, blood and urine are valuable for revealing neurodegeneration in the early stages. This review describes the emerging biomarkers based on glycoproteomics in NDs, highlighting the potential applications of glycoprotein biomarkers in the early detection of diseases, monitoring of the disease progression, and measurement of the therapeutic responses. The mass spectrometry-based strategies for characterizing glycoprotein biomarkers are also introduced.