Lectin Affinity Research Articles

A Bacterial Mannose Binding Lectin as a Tool for the Enrichment of C- and O-Mannosylated Peptides.

Mass spectrometry (MS) easily detects C-mannosylated peptides from purified proteins but not from complex biological samples. Enrichment of specific glycopeptides by lectin affinity prior to MS analysis has been widely applied to support glycopeptide identification but was until now not available for C-mannosylated peptides. Here, we used the α-mannose-specific Burkholderia cenocepacia lectin A (BC2L-A) and show that, in addition to its previously demonstrated high-mannose N-glycan binding capability, this lectin is able to retain C- and O-mannosylated peptides. Besides testing binding abilities to standard peptides, we applied BC2L-A affinity to enrich C-mannosylated peptides from complex samples of tryptic digests of HEK293 and MCF10A whole cell extracts, which led to the identification of novel C-mannosylation sites. In conclusion, BC2L-A enabled specific enrichment of C- and O-mannosylated peptides and might have superior properties over other mannose binding lectins for this purpose.

Recent Strategies for Using Monolithic Materials in Glycoprotein and Glycopeptide Analysis

There is continuous effort towards developing monolithic materials as solid supports for the separation, enrichment, and digestion of glycoproteins. The intention of this review is to discuss and summarize work reported in this area during the period 2015–2021 as a follow-up to our prior review. Reports from the past three decades have already proven the advantages of monolithic materials, such as the ease with which they can be prepared and functionalized, their high permeability and low resistance to mass transfer, and their stability over a wide range of pH. Recent works on glycoprotein analysis introduce different strategies in using monolithic materials specifically in separation, enrichment, and identification of glycoproteins, glycopeptides, and free glycans. A majority of these are focused on boronic acid affinity-based technique and others on lectin affinity and HILIC-based techniques. There are also newly developed ligands that utilize different interactions with glycans, such as encapsulation into β-cyclodextrin vesicles, CH- or OH-π interactions with fullerenes, immunoaffinity with monoclonal antibodies, H-bonding interactions with metallophthalocyanines, coordination interactions with cobalt phthalocyanine tetracarboxylic acid, and hydrophilic interaction with cyclodextrin molecular tubes, zwitterionic iminodiacetic acid, and boric acid. Recent strategies for developing on-line, multidimensional systems use immobilized monolithic enzyme reactors (IMERs) for high-throughput glycoprotein analysis. These works serve as contributions to better understand glycan structure-function relationship, as glycoproteins are now widely accepted disease biomarkers.

Rapid monitoring of high-mannose glycans during cell culture process of therapeutic monoclonal antibodies using lectin affinity chromatography.

We developed a simple high‐performance liquid chromatography assay to monitor high‐mannose glycans in monoclonal antibodies by monitoring terminal alpha‐mannose as a surrogate marker. Analysis of glycan data of therapeutic monoclonal antibodies by 2‐aminobenzamide assay showed a linear relationship between high mannose and terminal mannose of Fc glycans. Concanavalin A has a strong affinity to alpha‐mannose in glycans of typical therapeutic monoclonal antibodies. To show that terminal mannose binds specifically to Concanavalin A column, exoglycosidase‐treated monoclonal antibodies were serially blended with untreated monoclonal antibodies. Linear responses of terminal‐mannose binding to the column and comparable data trending with high mannose levels by 2‐aminobenzamide assay confirmed that terminal‐mannose levels measured by the Concanavalin A column can be used as a surrogate for the prediction of high‐mannose levels in monoclonal antibodies. The assay offers a simple, fast, and specific capability for the prediction of high‐mannose content in samples compared with traditional glycan profiling by 2‐aminobenzamide or mass spectrometry‐based methods. When the Concanavalin A column was coupled with protein A column for purification of antibodies from cell culture samples in a fully automated two‐dimensional analysis, high‐mannose data could be relayed to the manufacturing team in less than 30 min, allowing near‐real‐time monitoring of high‐mannose levels in the cell culture process.

APPLICATIONS OF PLANT LECTINS IN BIOTECHNOLOGY AND THERAPEUTICS

Lectins are carbohydrate-binding proteins broadly used in various therapeutic and biomedical applications. The variable affinity of lectins towards variety of carbohydrates has raised attention for the biologist to explore functional aspects of lectins. Lectins express specificity to simple carbohydrates for example mannose, lactose, sialic acid, complex glycan, and glycoproteins. Lectins are classified based on their sugar specificity and are used as a tool to study protein-carbohydrate interactions. Lectins are ubiquitous in nature and identified from all sources such as bacteria, fungi, algae, and animals. Plants are the most abundant source of lectins, and till now, more than three hundred lectins were characterized from plants. These are distributed to various parts of a plant according to their requirements and function. The physiological role of lectins in a plant is still not well understood. The overabundant presence of lectins in plant seeds and storage tissues indicated their role in plant development. Plant lectins shows a broad range of activities like antibacterial, antifungal, insecticidal, anticancerous, antileishmanial, antiviral, and anticoagulants. In this review, we aim to highlight the plant lectins classification and their application in various biological aspects.

Novel diagnostic and prognostic factors for the advanced melanoma based on the glycosylation-related changes studied by biophysical profiling methods

Melanoma is a life-threatening disease due to the early onset of metastasis and frequent resistance to the applied treatment. For now, no single histological, immunohistochemical or serological biomarker was able to provide a precise predictive value for the aggressive behavior in melanoma patients. Thus, the search for quantifying methods allowing a simultaneous diagnosis and prognosis of melanoma patients is highly desirable. By investigating specific molecular interactions with some biosensor-based techniques, one can determine novel prognostic factors for this tumor. In our previous study, we have shown the possibility of a qualitative in vitro distinguishing the commercially available melanoma cells at different progression stages based on the measurements of the lectin Concanavalin A interacting with surface glycans present on cells.Here, we present the results of the quantitative diagnostic and prognostic study of both commercial and patient-derived melanoma cells based on the evaluation of two novel factors: lectin affinity and glycan viscoelastic index obtained from the quartz crystal microbalance with dissipation monitoring (QCM-D) measurements. Two approaches to the QCM-D measurements were applied, the first uses the ability of melanoma cells to grow as a monolayer of cells on the sensor (cell-based sensors), and the second shortens the time of the analysis (suspension cell based-sensors). The results were confirmed by the complementary label-free (atomic force microscopy, AFM; and surface plasmon resonance, SPR) and labeling (lectin-ELISA; and microscale thermophoresis, MST) techniques. This new approach provides additional quantitative diagnosis and a personalized prognosis which can be done simultaneously to the traditional histopathological analysis.

Preliminary evidence that lectins in infant soy formula apparently bind bovine milk exosomes and prevent their absorption in healthy adults

BackgroundMilk exosomes and their microRNA (miR) cargos are bioavailable. The content of exosomes and miRs is negligible in infant formulas compared to human milk, and dietary depletion of exosomes led to changes in bacterial communities and impaired gut health in juvenile mice. Adverse effects of formula feeding may be compounded by using soy formulas due to exosome binding by abundant lectins in that matrix. The purpose of this study was to assess the bioavailability of milk exosomes and their miR cargos added to soy formula in adults, as well as the potential role of soy lectins in exosome bioavailability.MethodsEleven healthy adults (6 men, 5 women) enrolled in this randomized crossover study. Participants consumed 1.0 l of soy formula without (SF) or with (SFE) bovine milk exosomes added. Concentration-time curves of six plasma miRs were analyzed using reverse transcription quantitative PCR. Lectin affinity chromatography was used to assess the binding of exosomes by soy lectins. Data were analyzed by using paired t test. P < 0.05 was considered statistically significant.ResultsConsumption of SF and SFE did not elicit postprandial increases in plasma miRs. Approximately 39% of bovine milk exosome particles were retained by lectin columns.ConclusionsWe conclude that fortification of soy formulas with milk exosomes, in the absence of removing lectins, is not a viable strategy for delivering bioavailable exosomes and their miR cargos. Lectins in soy formulas bind glycoprotein on the surfaces of milk exosomes, thereby preventing exosome absorption.Trial registrationISRCTN registry ID: 16329971. Retrospectively registered on February 7th, 2019.

Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients.

Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells, and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. The complement-activation split products C3c, C3dg, and soluble C5b-9-associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEVs) were enriched by lectin and immunoaffinity isolation and analyzed by immunoblot analysis. Urine complement excretion increased significantly in KTRs with an albumin-to-creatinine ratio of ≥300 mg/g compared with <30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 mo after transplantation. Fractional excretion of C9 neoantigen was significantly higher than for albumin, indicating postfiltration generation. C9 neoantigen was detected in uEVs in six of the nine albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen-positive KTRs, lectin affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen, and Na+-glucose transporter 2 but only weakly for aquaporin 2. Coisolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9-associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts.NEW & NOTEWORTHY The present study proposes a mechanistic coupling between proteinuria and aberrant filtration of complement precursors, intratubular complement activation, and apical membrane attack in kidney transplant recipients. C3dg and C5b-9-associated C9 neoantigen associate with proximal tubular apical membranes as demonstrated in urine extracellular vesicles. The discovery suggests intratubular complement as a mediator between proteinuria and progressive kidney damage. Inhibitors of soluble and/or luminal complement activation with access to the tubular lumen may be beneficial.

Probing the glycans accessibility in the nanoparticle biomolecular corona

HypothesisFollowing blood administration, the pristine surface of nanoparticles (NPs) associates with biomolecules from the surrounding environment forming the so-called “biomolecular corona”. It is well accepted that the biomolecular corona dramatically affects the NP fate in the biological medium while the pristine surface is no longer available for binding. Recent studies have shown that the glycans associated with the proteins forming the corona have a role in the NP interaction with macrophages, but the glycan identities remain unknown. We aim here to identify the glycan composition of the biomolecular corona and to assess the role of these glycans in the interaction of the proteins from the corona with glycan binding biomolecules, such as lectins. ExperimentsIn this study, we have characterized the biomolecular corona of citrate stabilised gold NPs after exposure of the NPs to blood plasma at two different plasma concentrations, mimicking the in vitro and in vivo conditions. We have extensively characterized the biomolecular corona using HILIC chromatography and shotgun proteomics. Following this, a lectin binding assay was carried out using Dynamic Light Scattering (DLS) and Fluorescence Correlation Spectroscopy (FCS) to assess whether proteins with known affinity towards specific glycans would bind to the corona. FindingsOur findings highlighted that the protein corona composition is dependent on the exposing conditions. However, under both plasma concentrations, the biantennary sialylated glycans (A2G2S2) are enriched. DLS and FCS confirmed that the glycans are accessible for binding as the corona interacts with lectins with known affinity towards terminal sialic acids and the enzymatic removal of the glycans leads to a decrease in lectin affinity. This study shows for the first time that the glycans are present in the corona and that they could potentially be responsible for the modulation of NP biological processes as they can directly engage with glycan binding receptors that are highly expressed in an organism.

Elevated Fab glycosylation of anti-hinge antibodies

Objective Rheumatoid arthritis (RA) is characterized by systemic inflammation and the presence of anti-citrullinated protein antibodies (ACPAs), which contain remarkably high levels of Fab glycosylation. Anti-hinge antibodies (AHAs) recognize immunoglobulin G (IgG) hinge neoepitopes exposed following cleavage by inflammation-associated proteases, and are also frequently observed in RA, and at higher levels compared to healthy controls (HCs). Here, we investigated AHA specificity and levels of Fab glycosylation as potential immunological markers for RA. Method AHA serum levels, specificity, and Fab glycosylation were determined for the IgG1/4-hinge cleaved by matrix metalloproteinase-3, cathepsin G, pepsin, or IdeS, using enzyme-linked immunosorbent assay and lectin affinity chromatography, in patients with early active RA (n = 69) and HCs (n = 97). Results AHA reactivity was detected for all hinge neoepitopes in both RA patients and HCs. Reactivity against CatG-IgG1-F(ab´)2s and pepsin-IgG4-F(ab´)2s was more prevalent in RA. Moreover, all AHA responses showed increased Fab glycosylation levels in both RA patients and HCs. Conclusions AHA responses are characterized by elevated levels of Fab glycosylation and highly specific neoepitope recognition, not just in RA patients but also in HCs. These results suggest that extensive Fab glycosylation may develop in response to an inflammatory proteolytic microenvironment, but is not restricted to RA.

Tenofovir Hampers the Efficacy of Sorafenib in Prolonging Overall Survival in Hepatocellular Carcinoma.

Sorafenib is a first-line treatment for patients with advanced hepatocellular carcinoma (HCC). These patients may simultaneously receive anti-hepatitis B treatment if they are viremic. The N-Acetylgalactosaminyltransferase 14 (GALNT14) gene can serve as a biomarker to guide HCC treatments. However, the enzyme substrates of its gene product, GalNAc-T14 (a glycosyltransferase), remained uncharacterized. Here, we conducted a glycoproteome-wide search for GalNAc-T14 substrates using lectin affinity chromatography followed by tandem mass spectrometry. Seventeen novel GalNAc-T14 substrates were identified. A connective map analysis showed that an antiviral drug, tenofovir, was the leading medicinal compound to down-regulate the expression of these substrates. In vitro assays showed that HCC cells were resistant to sorafenib if pretreated by tenofovir but not entecavir. Clinical analysis showed that the concomitant use of tenofovir and sorafenib was a previously unrecognized predictive factor for unfavorable overall survival (hazard ratio = 2.060, 95% confidence interval = [1.256, 3.381], p = 0.004) in a cohort of 181 hepatitis-B-related, sorafenib-treated HCC patients (concomitant tenofovir versus entecavir treatment; p = 0.003). In conclusion, by conducting a glycoproteome-wide search for GalNAc-T14 substrates, we unexpectedly found that tenofovir was a major negative regulator of GalNAc-T14 substrates and an unfavorable anti-hepatitis B drug in HCC patients receiving sorafenib.

Removal of COVID-19 Spike Protein, Whole Virus, Exosomes, and Exosomal MicroRNAs by the Hemopurifier® Lectin-Affinity Cartridge in Critically Ill Patients With COVID-19 Infection.

Coronavirus−19 (COVID-19) has rapidly spread throughout the world resulting in a significant amount of morbidity and mortality. Despite advances in therapy, social distancing, masks, and vaccination many places in the world continue to see an increase in the number of cases and deaths. Viremia is commonly present in severely ill patients with COVID-19 infections and is associated with organ dysfunction and poor outcomes. Exosomes released by activated cells have been implicated in the pathogenesis of COVID-19 infection. We report the experience of two cases of critically ill COVID-19 patients treated with the Hemopurifier; a lectin affinity cartridge designed to remove mannosylated viruses and exosomes. Both patients tolerated the Hemopurifier sessions without adverse effects. In the first patient removal of exosomes and exosomal microRNAs was associated with improved coagulopathy, oxygenation, and clinical recovery, while in a second patient removal of COVID-19 by the Hemopurifier cartridge was observed. The Hemopurifier is currently under further investigation in up to 40-patients in a safety and feasibility study in ICU patients with COVID-19 infection.

Lectin Affinity Chromatography for the Discovery of Novel Cancer Glycobiomarkers: A Case Study with PSA Glycoforms and Prostate Cancer.

Many clinical biomarkers in cancer are glycoproteins, but the majority of them only consider the protein levels. Indeed, only alfa-fetoprotein (AFP) in hepatocarcinoma and CA15-3 in breast cancer are clinically monitored for their glycoforms. Aberrant glycosylation occurs frequently in many of the glycoproteins synthesized by tumor cells and often produce changes in protein glycoforms that could be exploited as potential biomarkers for improving diagnosis, prognosis or to study the response to treatment. Ideally, the screening of potential biomarkers should be performed from noninvasive samples like serum or plasma, therefore these glycoproteins with tumor associated-glycoforms should be shed from the tumor cell membrane or secreted into the blood to be detectable. Glycosylation changes that are commonly associated with cancer transformation include fucosylation, sialylation, branching, and polylactosaminylation.Lectins are glycan-binding proteins that bind with great specificity to different glycan moieties. Lectin-based strategies to enrich or fractionate glycoproteins are being extensively used and hold promise in targeted analysis for cancer biomarker discovery. Here we describe the use of lectin chromatography to separate prostate specific antigen (PSA) glycoforms based on their sialic acid linkage from sera of patients with prostate cancer (with PSA levels in the range of 2-20ng/mL). In particular, agarose-bound Sambucus nigra agglutinin (SNA) lectin which has affinity for terminal α2,6-sialic acids on glycoproteins was used. The protocol included first a previous immunoaffinity step to enrich PSA and to avoid interferences of the most abundantserum glycoproteins. Then, the immunopurified PSA was loaded on the SNA chromatography and two fractions were obtained, the first one (unbound fraction) containing the PSA glycoforms withoutα2,6-sialic acid (basically α2,3-sialylated PSA glycoforms) and the second one (bound fraction) the α2,6-sialylated PSA glycoforms. The quantification of the PSA eluted in the two fractions allows for the determination of the relative content of both groups of PSA glycoforms. The percentage of the α2,6-sialylated PSA glycoforms is significantly decreased in aggressive prostate cancer compared to indolent prostate cancer and benign prostate hyperplasia, being a promising new glycobiomarker for prostate cancer risk stratification.

APPLICATIONS OF PLANT LECTINS IN BIOTECHNOLOGY AND THERAPEUTICS: Plant lectins application

Lectins are carbohydrate-binding proteins broadly used in various therapeutic and biomedical applications. The variable affinity of lectins towards variety of carbohydrates has raised attention for the biologist to explore functional aspects of lectins. Lectins express specificity to simple carbohydrates for example mannose, lactose, sialic acid, complex glycan, and glycoproteins. Lectins are classified based on their sugar specificity and are used as a tool to study protein-carbohydrate interactions. Lectins are ubiquitous in nature and identified from all sources such as bacteria, fungi, algae, and animals. Plants are the most abundant source of lectins, and till now, more than three hundred lectins were characterized from plants. These are distributed to various parts of a plant according to their requirements and function. The physiological role of lectins in a plant is still not well understood. The overabundant presence of lectins in plant seeds and storage tissues indicated their role in plant development. Plant lectins shows a broad range of activities like antibacterial, antifungal, insecticidal, anticancerous, antileishmanial, antiviral, and anticoagulants. In this review, we aim to highlight the plant lectins classification and their application in various biological aspects.

Improved assay system for acidic peptide: N-glycanase (aPNGase) activity in plant extracts

Plant acidic peptide: N-glycanase (aPNGase) release N-glycans from glycopeptides during the degradation process of glycoproteins in developing or growing plants. We have previously developed a new method to detect the aPNGase activity in crude extracts, which is prerequisite for the construction of aPNGase knockout or overexpression lines. However, this method has the disadvantage of requiring de-sialylation treatment and a lectin chromatography. In this study, therefore, we improved the simple and accurate method for detecting aPNGase activity using anion-exchange HPLC requiring neither the desialylation treatment nor the lectin affinity chromatography.

Recent advances in functionalized magnetic nanomaterials for glycoprotein and glycopeptide enrichment

蛋白质糖基化作为最重要的翻译后修饰之一,在生物体诸如细胞信号转导、蛋白质翻译调控、免疫应答等诸多生命过程中发挥重要作用。此外,蛋白质的异常糖基化还与肿瘤等疾病的发生发展密切相关,这为以糖蛋白为目标的疾病生物标志物的发现提供了可能。尽管质谱已经成为糖蛋白质组学的重要分析工具,但糖肽的低丰度和低电离效率使得其直接质谱分析仍面临挑战。在糖蛋白质组学研究中,从复杂的生物样品中富集糖蛋白和糖肽是重要的环节。磁性固相萃取(MSPE)是一种操作简单、成本低和萃取效率高的样品预处理方法。在磁性固相萃取中,磁性吸附剂是影响萃取效果的关键,将功能化磁性纳米材料作为吸附剂进行糖蛋白质组学研究已经得到广泛应用。该文综述了糖分子、离子液体、凝集素、硼酸亲和配体、金属有机框架、共价有机骨架等功能化磁性纳米材料的制备及其在糖蛋白及糖肽富集中的应用。上述功能化磁性纳米材料具有高比表面积、大量作用位点等特点,其富集机理包括亲水相互作用色谱、凝集素亲和作用色谱、硼酸化学法和肼化学法等,主要应用于血清、血浆、细胞、组织、唾液等样品的糖蛋白和糖肽的富集。该文引用了近十年来发表的约90篇源于科学引文索引(SCI)与中文核心期刊的相关论文,并于文末对磁性纳米材料在糖蛋白和糖肽富集领域的发展趋势进行了展望。

Proteoglycan 4 (lubricin) is a highly sialylated glycoprotein associated with cardiac valve damage in animal models of infective endocarditis.

Streptococcus gordonii and Streptococcus sanguinis are primary colonizers of tooth surfaces and are generally associated with oral health, but can also cause infective endocarditis (IE). These species express "Siglec-like" adhesins that bind sialylated glycans on host glycoproteins, which can aid the formation of infected platelet-fibrin thrombi (vegetations) on cardiac valve surfaces. We previously determined that the ability of S. gordonii to bind sialyl T-antigen (sTa) increased pathogenicity, relative to recognition of sialylated core 2 O-glycan structures, in an animal model of IE. However, it is unclear when and where the sTa structure is displayed, and which sTa-modified host factors promote valve colonization. In this study, we identified sialylated glycoproteins in the aortic valve vegetations and plasma of rat and rabbit models of this disease. Glycoproteins that display sTa vs. core 2 O-glycan structures were identified by using recombinant forms of the streptococcal Siglec-like adhesins for lectin blotting and affinity capture, and the O-linked glycans were profiled by mass spectrometry. Proteoglycan 4 (PRG4), also known as lubricin, was a major carrier of sTa in the infected vegetations. Moreover, plasma PRG4 levels were significantly higher in animals with damaged or infected valves, as compared with healthy animals. The combined results demonstrate that, in addition to platelet GPIbα, PRG4 is a highly sialylated mucin-like glycoprotein found in aortic valve vegetations and may contribute to the persistence of oral streptococci in this protected endovascular niche. Moreover, plasma PRG4 could serve as a biomarker for endocardial injury and infection.

Monose-modified organic electrochemical transistors for cell surface glycan analysis via competitive recognition to enzyme-labeled lectin.

A competitive strategy for glycan determination on cell surface with organic electrochemical transistors (OECTs) has been developed. The carboxylic multi-wall carbon nanotubes were firstly immobilized on the gate interface to cross-link the specific monose with adipic dihydrazide as the linker, which could then competitively recognize horseradish peroxidase (HRP)-labeled lectin with the target monose on the cell surface. The HRP captured on the gate interface through the affinity of lectin to monose finally catalyzed the reduction of hydrogen peroxide to produce the output current signal for detection of cell surface monose under the optimal gate voltage of 0.9V. Using mannose and galactose groups as the target models, HRP-labeled concanavalin A and peanut agglutinin were used to competitively recognize these groups on both cell surface and gate interface, respectively. The amounts of mannose and galactose on HeLa cells were measured to be 3.41 × 108 and 2.92 × 108 molecules per cell, respectively. The changes of the mannose and galactose expressions upon external stimulation were also observed with the proposed biosensors, which showed consistent results with flow cytometric analysis, indicating that the OECT-based biosensor is suitable for analysis of different glycan expressions on cell surface. Graphical abstract.

Glycoproteins Presenting Galactose and N-Acetylgalactosamine in Human Seminal Plasma as Potential Players Involved in Immune Modulation in the Fertilization Process.

In light of recent research, there is increasing evidence showing that extracellular semen components have a significant impact on the immune reaction of the female partner, leading to the tolerogenic response enabling the embryo development and implantation as well as further progress of healthy pregnancy. Seminal plasma glycoproteins are rich in the unique immunomodulatory glycoepitopes that may serve as ligands for endogenous lectins that decorate the surface of immune cells. Such interaction may be involved in modulation of the maternal immune response. Among immunomodulatory glycans, Lewis type antigens have been of interest for at least two decades, while the importance of T/Tn antigens and related structures is still far from understanding. In the current work, we applied two plant lectins capable of distinguishing glycoepitopes with terminal GalNAc and Gal to identify glycoproteins that are their efficient carriers. By means of lectin blotting and lectin affinity chromatography followed by LC-MS, we identified lactotransferrin, prolactin inducible protein as well as fibronectin and semenogelins 1 and 2 as lectin-reactive. Net-O-glycosylation analysis results indicated that the latter three may actually carry T and/or Tn antigens, while in the case of prolactin inducible protein and lactotransferrin LacdiNAc and lactosamine glycoepitopes were more probable. STRING bioinformatics analysis linked the identified glycoproteins in the close network, indicating their involvement in immune (partially innate) processes. Overall, our research revealed potential seminal plasma ligands for endogenous Gal/GalNAc specific lectins with a possible role in modulation of maternal immune response during fertilization.

Identification of N-linked Glycoproteins in Silkworm Serum Using Con A Lectin Affinity Chromatography and Mass Spectrometry.

Glycosylation is one of the most common post-translational modifications to occur during protein biosynthesis, but remains poorly understood in insects. In this study, we collected serum proteins from two silkworm developmental stages, namely day 7 of the fifth instar larval stage and day 2 of the pupal stage. Results of SDS–PAGE and periodic acid-Schiff staining revealed that most serum proteins with high abundance were putative glycoproteins. LC-MS/MS identified 149 larval and 303 pupal serum proteins in the Con A lectin-enriched fractions. GO analysis revealed that many serum proteins were involved in the proteolysis and carbohydrate metabolic process. 82 N-linked glycoproteins with at least one glycosylation site were identified. N-Linked glycosylation occurred at the sequon, Asn-X-Ser/Thr, and the proportions of Ser and Thr glycosylation at the hydroxy position were found 39.6% and 60.3%, respectively. The N-glycan structures found in serum glycoproteins were mainly Man2FucGlcNAc2 (67.9%). Since storage protein 1 and transferrin had a relatively high abundance in the serum and could be significantly enriched by Con A lectin, their glycosylation was analyzed in detail. Glycoside hydrases, serine proteases and serpins were found to form three interacting glycoprotein networks using the website STRING. This study provides important clues for the understanding of the function of N-linked glycosylation in metabolism, immunity, and metamorphosis.

Glycoproteomics identifies HOMER3 as a potentially targetable biomarker triggered by hypoxia and glucose deprivation in bladder cancer

BackgroundMuscle invasive bladder cancer (MIBC) remains amongst the deadliest genitourinary malignancies due to treatment failure and extensive molecular heterogeneity, delaying effective targeted therapeutics. Hypoxia and nutrient deprivation, oversialylation and O-glycans shortening are salient features of aggressive tumours, creating cell surface glycoproteome fingerprints with theranostics potential.MethodsA glycomics guided glycoproteomics workflow was employed to identify potentially targetable biomarkers using invasive bladder cancer cell models. The 5637 and T24 cells O-glycome was characterized by mass spectrometry (MS), and the obtained information was used to guide glycoproteomics experiments, combining sialidase, lectin affinity and bottom-up protein identification by nanoLC-ESI-MS/MS. Data was curated by a bioinformatics approach developed in-house, sorting clinically relevant molecular signatures based on Human Protein Atlas insights. Top-ranked targets and glycoforms were validated in cell models, bladder tumours and metastases by MS and immunoassays. Cells grown under hypoxia and glucose deprivation disclosed the contribution of tumour microenvironment to the expression of relevant biomarkers. Cancer-specificity was validated in healthy tissues by immunohistochemistry and MS in 20 types of tissues/cells of different individuals.ResultsSialylated T (ST) antigens were found to be the most abundant glycans in cell lines and over 900 glycoproteins were identified potentially carrying these glycans. HOMER3, typically a cytosolic protein, emerged as a top-ranked targetable glycoprotein at the cell surface carrying short-chain O-glycans. Plasma membrane HOMER3 was observed in more aggressive primary tumours and distant metastases, being an independent predictor of worst prognosis. This phenotype was triggered by nutrient deprivation and concomitant to increased cellular invasion. T24 HOMER3 knockdown significantly decreased proliferation and, to some extent, invasion in normoxia and hypoxia; whereas HOMER3 knock-in increased its membrane expression, which was more pronounced under glucose deprivation. HOMER3 overexpression was associated with increased cell proliferation in normoxia and potentiated invasion under hypoxia. Finally, the mapping of HOMER3-glycosites by EThcD-MS/MS in bladder tumours revealed potentially targetable domains not detected in healthy tissues.ConclusionHOMER3-glycoforms allow the identification of patients’ subsets facing worst prognosis, holding potential to address more aggressive hypoxic cells with limited off-target effects. The molecular rationale for identifying novel bladder cancer molecular targets has been established.Graphical abstract