Specific Lectin Research Articles

Deciphering dynamic combinatorial libraries of glycoclusters with miniaturized weak affinity chromatography coupled with mass spectrometry (nano-FAC-MS)

We report an original methodology based on affinity chromatography coupled with mass spectrometry to decipher the complexity of dynamic combinatorial libraries (DCLs) of glycoclusters. Such libraries are intended to boost the design of potential therapeutic anti-infectious agents targeting Pseudomonas aeruginosa, which is responsible for numerous diseases, mostly found in hospitals as major a cause of nosocomial infections. Dynamic combinatorial chemistry provides a rapid access to an equilibrating mixture of glycocluster candidates through the formation of reversible covalent bonds under thermodynamic control. Identifying each molecule in the complex mixture overcomes challenges due to the dynamic process. Selection of glycoclusters candidates was first realized on a model lectin (Concanavalin A, ConA). Home-made affinity nanocolumns, containing covalently immobilized ConA and have volumes in the microliter range, were used to separate DCLs of glycoclusters with respect to their specific lectin binding properties under buffered aqueous conditions. Miniaturization facilitates the inline coupling with MS detection in such purely aqueous and buffered conditions and reduces target protein consumption. Monolithic lectin-affinity columns prepared by immobilization of ConA were first characterized using a known ligand. The amount of active binding immobilized lectin is 61 ± 5 pmol on 8.5-cm length column. We demonstrated the ability of our approach to evaluate individual dissociation constants of species directly in the complex mixture. The concept was then successfully applied to the screening of DCLs of more complex glycoclusters to identify (by mass spectrometry) and rank the ligands (by relative breakthrough curve delay) according to their affinity for the immobilized lectin in a single experiment.

The specific core fucose-binding lectin Pholiota squarrosa lectin (PhoSL) inhibits hepatitis B virus infection in vitro

Glycosylation of proteins and lipids in viruses and their host cells is important for viral infection and is a target for antiviral therapy. Hepatitis B virus (HBV) is a major pathogen that causes acute and chronic hepatitis; it cannot be cured because of the persistence of its covalently closed circular DNA (cccDNA) in hepatocytes. Here we found that Pholiota squarrosa lectin (PhoSL), a lectin that specifically binds core fucose, bound to HBV particles and inhibited HBV infection of a modified human HepG2 cell line, HepG2-hNTCP-C4, that expresses an HBV receptor, sodium taurocholate cotransporting polypeptide. Knockout of fucosyltransferase 8, the enzyme responsible for core fucosylation and that aids receptor endocytosis, in HepG2-hNTCP-C4 cells reduced HBV infectivity, and PhoSL facilitated that reduction. PhoSL also blocked the activity of epidermal growth factor receptor, which usually enhances HBV infection. HBV particles bound to fluorescently labeled PhoSL internalized into HepG2-hNTCP-C4 cells, suggesting that PhoSL might inhibit HBV infection after internalization. As PhoSL reduced the formation of HBV cccDNA, a marker of chronic HBV infection, we suggest that PhoSL could impair processes from internalization to cccDNA formation. Our finding could lead to the development of new anti-HBV agents.

Purification and Characterization of a Novel D-Galactose Binding Lectin from Seeds of Meizotropis buteiformis.

The main aim of the present study is to find out the novelty and characteristics of a lectin purified from the plant Meizotropis buteiformis. Lectins are proteins that possess the ability to specifically bind glycans of glycoconjugates. Plants are considered rich sources of lectins and the determination of sugar specificity of a purified plant lectin is an important aspect in order to evaluate its potential area of application. In the present study, a novel D-Galactose specific lectin is purified from Meizotropis buteiformis through affinity chromatography and examined for its various physical and biochemical characteristics. The objective of the present study is to purify a novel lectin up to its homogeneity from the seeds of Meizotropis buteiformis and characterization of its various physical and biochemical properties. The lectin was purified by simple successive steps of lectin extraction, ammonium sulphate fractionation, and affinity chromatography. Activity of the purified lectin was determined by hemagglutination assay. Some physicochemical parameters of the purified protein were also determined along with identification of protein by LC-MS/MS and the spectra analysis using Mascot sequence matching software (Matrix Science) with the NCBI database. From the current investigation, it was found that the purified lectin has a native molecular weight of 75 kDa. Among the various sugars and sugar derivatives tested, lactose and D-galactose were found to be potent inhibitors of its activity. Its optimum pH range was found to be from 6.5 to 7.5 and also it exhibited full activity at a temperature from 0ºC to 50ºC. The purified lectin does not show any effects on its activities for metal ions tested. The protein view report of the LC-MS/MS result analysis showed a 50% sequence similarity with that of the lectin beta-chain of the Butea monosperma. In the present study, a novel D-Galactose specific lectin is purified from Meizotropis buteiformis by affinity chromatography using Sepharose 4B. The purified lectin is found to be heterodimeric and metal ion independent. The LC-MS/MS results suggested that the purified lectin has not been reported earlier.

Isolation of Momordica charantia seed lectin and glycosidases from the protein bodies: Lectin-glycosidase (β-hexosaminidase) protein body membrane interaction reveals possible physiological function of the lectin

Momordica charantia seeds are known to contain a galactose specific lectin that has been well characterized. Seed extracts also contain glycosidases such as the β-hexosaminidase, α-mannosidase and α-galactosidase. In the present study, lectin was affinity purified from the seed extracts and protein bodies isolated by sucrose density gradient centrifugation. From the protein bodies, lectin was identified and β-hexosaminidase was isolated by lectin affinity chromatography and subsequently separated from other glycosidases by gel filtration. In the native PAGE, the purified β-hexosaminidase migrated as a single band with a molecular weight of ∼235 kDa and by zymogram analysis using 4-methylumbelliferyl N-acetyl-β-D-glucosaminide substrate it was confirmed as β-hexosaminidase. Under reducing conditions in SDS-PAGE, the purified enzyme dissociated into three bands (Mr 33, 20 and 15 kDa). The prominent bands (20 and 15 kDa) showed immunological cross-reactivity with the human Hexosaminidase B antibody in a western blot experiment. In gel digestion of the purified enzyme, followed by proteomic analysis using tandom MS/MS revealed sequence identity as compared to the genomic sequence of the Momordica charantia with a score of 57 (24% sequence coverage). Additionally, by CD analysis the purified β-hexosaminidase showed 39.1% of α-helix. Furthermore, secondary structure variations were observed in presence of substrate, lectin and at different pH values. Protein body membrane prepared from the isolated protein bodies showed a pH dependent interaction with the purified lectin and mixture of glycosidases.

Polyvalent Glycan Functionalized Quantum Nanorods as Mechanistic Probes for Shape-Selective Multivalent Lectin-Glycan Recognition.

Multivalent lectin-glycan interactions (MLGIs) are widespread in biology and hold the key to many therapeutic applications. However, the underlying structural and biophysical mechanisms for many MLGIs remain poorly understood, limiting our ability to design glycoconjugates to potently target specific MLGIs for therapeutic intervention. Glycosylated nanoparticles have emerged as a powerful biophysical probe for MLGIs, although how nanoparticle shape affects the MLGI molecular mechanisms remains largely unexplored. Herein, we have prepared fluorescent quantum nanorods (QRs), densely coated with α-1,2-manno-biose ligands (QR-DiMan), as multifunctional probes to investigate how scaffold geometry affects the MLGIs of a pair of closely related, tetrameric viral receptors, DC-SIGN and DC-SIGNR. We have previously shown that a DiMan-capped spherical quantum dot (QD-DiMan) gives weak cross-linking interactions with DC-SIGNR but strong simultaneous binding with DC-SIGN. Against the elongated QR-DiMan, DC-SIGN retains similarly strong simultaneous binding of all four binding sites with a single QR-DiMan (apparent K d ≈ 0.5 nM, ∼1.8 million-fold stronger than the corresponding monovalent binding), while DC-SIGNR gives both weak cross-linking and strong individual binding interactions, resulting in a larger binding affinity enhancement than that with QD-DiMan. S/TEM analysis of QR-DiMan-lectin assemblies reveals that DC-SIGNR's different binding modes arise from the different nanosurface curvatures of the QR scaffold. The glycan display at the spherical ends presents too high a steric barrier for DC-SIGNR to bind with all four binding sites; thus, it cross-links between two QR-DiMan to maximize binding multivalency, whereas the more planar character of the cylindrical center allows the glycans to bridge all binding sites in DC-SIGNR. This work thus establishes glycosylated QRs as a powerful biophysical probe for MLGIs not only to provide quantitative binding affinities and binding modes but also to demonstrate the specificity of multivalent lectins in discriminating different glycan displays in solution, dictated by the scaffold curvature.

Realization of Nodulation and Nitrogen-Fixing Activities of Bradyrhizobium japonicum and Rhizosphere Microbiota through Seed Treatment with Pesticide Standak Top and Spraying Plants with Soybean Seed Lectin

The idea of the study was to use soybean lectin as a biologically active compound with a broad spectrum of action to spray soybean plants for stabilization of the formation and functioning of the soybean-rhizobium symbiosis as well as the nitrogen-fixing activity of rhizosphere microbiota against the background of seed treatment with chemical plant protection product Standak Top — an innovative pesticide with fungicidal and insecticidal activity for the control of major diseases and pests of soybean plants. Aim. To study the peculiarities of formation and functioning of soybean-rhizobium symbiosis as well as the nitrogen-fixing activity of rhizosphere microbiota under spraying plants with specific soybean seed lectin on the background of seed treatment with Standak Top and inoculation with nodule bacteria Bradyrhizobium japonicum 634b on the sowing day in the conditions of pot experiments with soil as a substrate. Methods. Physiological, microbiological, gas chromatography, and statistical methods were used. Results. It was shown that after seed treatment with Standak Top (1.5 L/ton of seeds) on the sowing day, there was observed suppression of the process of nodule formation on the roots in the period of soybean vegetative growth. The nitrogen-fixing activity of the symbiotic system was at the control level, while the functional activity of soil diazotrophs was suppressed (by 1.2—2.2 times). Spraying plants in the phase of two trifoliate leaves (V2) with soybean seed lectin (without pesticide) led to an increase in the total mass of nodules on the plant (by 1.5 and 1.9 times as well as by 2.3 and 2.0 times compared to the control of inoculation in the phase of three trifoliate leaves (V3) and beginning of pod formation (R3), respectively). The increase in the total mass of the symbiotic apparatus on soybean roots in the phases V3 and R3 respectively was by 1.4 and 1.5 times in comparison with seed treatment with Standak Top, and the mass of one nodule was higher by 1.3 and 1.6 times, respectively. Soybean seed lectin led to a signifi cant increase in the actual nitrogenase activity of the soybean-rhizobium symbiosis. It was 2.9 and 1.9 times higher compared to control of inoculation and 2.1 and 1.8 times compared to the variant of inoculation + pesticide in the V3 and R3 phases, respectively. The functional activity of soil nitrogen-fixing microorganisms did not change significantly. The use of soybean seed lectin against the background of the seed treatment with Standak Top and inoculation contributed to the stabilization and increase in the rhizobia nodulation ability, the suppression of which was due to the infl uence of such an anthropogenic factor as pesticides. Th ere was observed an increase in the number (by 1.6 and 1.2 times) and mass of root nodules (by 2.2 and 1.5 times and 1.4 and 1.2 times, respectively, compared to the controls of inoculation and inoculation + pesticide). Soybean seed lectin significantly increased the nitrogenase activity of the symbiosis against the background of Standak Top (by 1.9 and 1.6 times and 1.4 and 1.5 times, respectively, in the V3 and R3 phases of soybean ontogenesis) compared to the control of inoculation and inoculation + Standak Top. Conclusions. The obtained results suggest the possibility of using the method of spraying plants with specific lectin as a means of leveling (or mitigating) the negative effect of pesticides used for the seed treatment on the formation and functioning of the symbiosis and rhizosphere diazotrophic microbiota. This indicates the prospects of studying the biological activity of phytolectins in spraying plants in order to regulate the formation and functioning of phytobacterial systems, as well as their responses to various environmental or anthropogenic stress factors, in particular, to the effect of chemical plant-protecting products used for the seed treatment.

Gangliosides as Siglec ligands.

The structure of a sialoglycan can be translated into to a biological response when it binds to a specific endogenous lectin. Among endogenous sialic acid-binding lectins in humans are those comprising the 15-member Siglec family, most of which are expressed on overlapping sets of immune cells. Endogenous Siglec ligands are sialoglycolipids (gangliosides) and/or sialoglycoproteins, on cell surfaces or in the extracellular milieu, that bind to and initiate signaling by cell surface Siglecs. In the nervous system, where gangliosides are the predominant sialoglycans, Siglec-4 (myelin-associated glycoprotein) on myelinating cells binds to gangliosides GD1a and GT1b on nerve cell axons to ensure stable and productive axon-myelin interactions. In the immune system, Siglec-7 on natural killer cells binds to gangliosides GD3 and GD2 to inhibit immune signaling. Expression of GD3 and GD2 on cancer cells can lead to tumor immune evasion. Siglec-1 (sialoadhesin, CD169) on macrophages binds to gangliosides on tumors and enveloped viruses. This may enhance antigen presentation in some cases, or increase viral distribution in others. Several other Siglecs bind to gangliosides in vitro, the biological significance of which has yet to be fully established. Gangliosides, which are found on all human cells and tissues in cell-specific distributions, are functional Siglec ligands with varied roles driving Siglec-mediated signaling.

Identification of a type II LacNAc specific binding lectin CMRBL from Cordyceps militaris

The Cordyceps militaris gene CCM_03832 encodes a ricin-B like lectin. The gene was cloned and expressed in Escherichia coli, and its protein product, named CMRBL (C. militaris ricin-B like lectin), was purified by galactose affinity chromatography. Of nine different sources of erythrocytes, CMRBL showed only specific hemagglutinating activity against rat and rabbit erythrocytes with titers of 22 and 28, respectively. Glycan array analyses by the Consortium for Functional Glycomics showed that CMRBL possesses very high specific binding activity of glycans terminated with type II LacNAc (non-reducing Galβ1-4GlcNAc). Compared with other well-known Gal-terminated binding lectins such as Erythrina cristagalli agglutinin, Ricinus communis agglutinin, and Jacalin, CMRBL showed better binding specificity to type II LacNAc compared the other lectins. CMRBL showed lowest binding activity to ZR-75-30 and MDA-MB-468 cell lines among five tested cell lines (H22, THP-1, MDA-MB-231, ZR-75-30, and MDA-MB-468 cells). Transfection of type II LacNAc main galactosyltransferase B4GALT3 to ZR-75-30 significantly improved CMRBL binding activity compared with control. CMRBL was also applied for testing the type II LacNAc modification of Etanercept successfully. Our data suggest that CMRBL would be a useful tool to recognize type II LacNAc, especially distinguish type II from other galactose-terminated glycans in glycan biology research.

The staphylococcal biofilm protein Aap mediates cell-cell adhesion through mechanically distinct homophilic and lectin interactions.

The accumulation phase of staphylococcal biofilms relies on both the production of an extracellular polysaccharide matrix and the expression of bacterial surface proteins. A prototypical example of such adhesive proteins is the long multidomain protein Aap (accumulation-associated protein) from Staphylococcus epidermidis, which mediates zinc-dependent homophilic interactions between Aap B-repeat regions through molecular forces that have not been investigated yet. Here, we unravel the remarkable mechanical strength of single Aap-Aap homophilic bonds between living bacteria and we demonstrate that intercellular adhesion also involves sugar binding through the lectin domain of the Aap A region. We find that the mechanical force needed to unfold individual β-sheet-rich G5-E domains from the Aap B-repeat regions is very high, ranging from 300 up to 1,000 pN at high loading rates, indicating these are extremely stable. This high mechanostability provides a means to the cells to form highly adhesive and cohesive biofilms capable of sustaining high physiological shear stress. Importantly, we identify a previously undescribed role of Aap in bacterial-bacterial adhesion, that is, heterophilic sugar binding by a specific lectin domain located in the N-terminal A region, which might be important to establish initial contacts between cells before strong homophilic bonds come into play. This study emphasizes the remarkable mechanical and binding properties of Aap as well as its wide diversity of adhesive functions.

A galactoside-specific Dalbergieae legume lectin from seeds of Vataireopsis araroba (Aguiar) Ducke.

The Dalbergieae lectin group encompasses several lectins with significant differences in their carbohydrate specificities and biological properties. The current work reports on the purification and characterization of a GalNAc/Gal-specific lectin from Vataireopsis araroba (Aguiar) Ducke, designated as VaL. The lectin was purified from the seeds in a single step using guar gum affinity chromatography. The lectin migrated as a single band of about 35kDa on SDS-PAGE and, in native conditions, occurs as a homodimer. The purified lectin is stable at temperatures up to 60°C and in a pH range from 7 to 8 and requires divalent cations for its activity. Sugar-inhibition assays demonstrate the lectin specificity towards N-acetyl-D-galactosamine, D-galactose and related sugars. Furthermore, glycan array analyses show that VaL interacts preferentially with glycans containing terminal GalNAc/Galβ1-4GlcNAc. Biological activity assays were performed using three insect cell lines: CF1 midgut cells from the spruce budworm Choristoneura fumiferana, S2 embryo cells from the fruit fly Drosophila melanogaster, and GutAW midgut cells from the corn earworm Helicoverpa zea. In vitro assays indicated a biostatic effect for VaL on CF1 cells, but not on S2 and GutAW cells. The lectin presented a biostatic effect by reducing the cell growth and inducing cell agglutination, suggesting an interaction with glycans on the cell surface. VaL has been characterized as a galactoside-specific lectin of the Dalbergieae tribe, with sequence similarity to lectins from Vatairea and Arachis.

CD169-mediated restrictive SARS-CoV-2 infection of macrophages induces pro-inflammatory responses.

Exacerbated and persistent innate immune response marked by pro-inflammatory cytokine expression is thought to be a major driver of chronic COVID-19 pathology. Although macrophages are not the primary target cells of SARS-CoV-2 infection in humans, viral RNA and antigens in activated monocytes and macrophages have been detected in post-mortem samples, and dysfunctional monocytes and macrophages have been hypothesized to contribute to a protracted hyper-inflammatory state in COVID-19 patients. In this study, we demonstrate that CD169, a myeloid cell specific I-type lectin, facilitated ACE2-independent SARS-CoV-2 fusion and entry in macrophages. CD169-mediated SARS-CoV-2 entry in macrophages resulted in expression of viral genomic and subgenomic RNAs with minimal viral protein expression and no infectious viral particle release, suggesting a post-entry restriction of the SARS-CoV-2 replication cycle. Intriguingly this post-entry replication block was alleviated by exogenous ACE2 expression in macrophages. Restricted expression of viral genomic and subgenomic RNA in CD169+ macrophages elicited a pro-inflammatory cytokine expression (TNFα, IL-6 and IL-1β) in a RIG-I, MDA-5 and MAVS-dependent manner, which was suppressed by remdesivir treatment. These findings suggest that de novo expression of SARS-CoV-2 RNA in macrophages contributes to the pro-inflammatory cytokine signature and that blocking CD169-mediated ACE2 independent infection and subsequent activation of macrophages by viral RNA might alleviate COVID-19-associated hyperinflammatory response.

Lectins from plants and algae act as anti-viral against HIV, influenza and coronaviruses.

BackgroundCarbohydrate–lectin interactions are extremely specific as the lectin is capable of recognising monomeric and oligomeric sugars in a reversible manner. It has been known for a long time that lectins have antibacterial, antifungal, and insecticidal activities. Recently, it has been reported that many lectins can prevent the virus growth by interacting with the viral envelop surface glycoprotein. Spike protein, which is found on the surface of some enveloped viruses, is heavily mannosylated and will have strong affinity for mannose specific lectins. According to the findings, lectins have a high binding affinity for the glycans of the SARS-CoV-2 spike glycoprotein, which contains N-glycosylation sites. As a result, various lectins are being researched and developed as anti-viral agents.ResultsAccording to our in silico studies, the amino acid residues Asn487, Tyr489, Gln493, Lys417, and Tyr505 of the receptor binding domain (RBD) of SARS-CoV-2 formed an interaction with the model lectin Lablab purpureus lectin. Similar interaction for SARS-CoV-2 spike protein was observed with Griffithsin lectin (algal source) as well. These observations demonstrate that lectins could be one of the potential molecules for neutralising coronavirus infection.ConclusionThis review focuses on anti-viral lectins isolated and characterized from plants and algae (last 5 years) and showed anti-viral properties against HIV, Influenza, and coronaviruses.

Initiation of the alternative pathway of complement and the history of "tickover".

The evolutionary history of complement suggests that the alternative pathway arose prior to the arrival of the classical and lectin pathways. In these pathways, target specificity is provided by antibodies and sugar specific lectins. While these efficient initiation systems dominate activation on most targets, the alternative pathway produces most of the C3b and 80%-90% of the C5b-9. While the tickover process, originally proposed by Peter Lachmann, provided ancient hosts with a crude self/non-self-discriminatory system that initiated complement attack on everything foreign, tickover clearly plays a more minor role in complement activation in modern organisms possessing classical and lectin pathways. Spontaneous activation of the alternative pathway via tickover may play a major role in human pathologies where tissue damage is complement-mediated. The molecular mechanism of tickover is still not convincingly proven. Prevailing hypotheses include (a) spontaneous hydrolysis of the thioester in C3 forming the C3b-like C3(H2 O) in solution and (b) "enhanced tickover" in which surfaces cause specific or non-specific contact activated conformational changes in C3. Theoretical considerations, including computer simulations, suggest that the latter mechanism is more likely and that more research needs to be devoted to understanding interactions between biological surfaces and C3.

Multicomponent reaction derived small di- and tri-carbohydrate-based glycomimetics as tools for probing lectin specificity.

Lectins, carbohydrate-binding proteins, play important functions in all forms of life from bacteria and viruses to plants, animals, and humans, participating in cell-cell communication and pathogen binding. In an attempt to modify lectin functions, artificial lectin ligands were made usually as big dendrimeric or cluster multivalent glycomimetic structures. Here we synthesized a novel set of glycomimetic ligands through protection/deprotection multicomponent reactions (MCR) approach. Multivalent di-and tri-carbohydrate glycomimetics containing D-fructose, D-galactose, and D-allose moieties were prepared in 63-96% yield. MCR glycomimetics demonstrated different binding abilities for plant lectins Con A and UEA I, and human galectin-3. Information gained about the influence of molecule structure, multivalency and optical purity on the lectin binding ability can be used in lectin detection and sensitivity measurements to further facilitate understanding of carbohydrate recognition process.

Loci and motifs of the GalNAcα1 → 3/O related glycotopes in the mammalian glycoconjugates and their lectin recognition roles.

Galα1 → and GalNAcα1 → are the two essential key sugars in human blood group AB active glycotopes, in which GalNAcα1 → related sequences are located at both sides of the nonreducing and the reducing ends of human blood group A active O-glycans. It is also found at the nonreducing ends of GlcNAc N-glycans and glycosphingolipid(GSL) of human blood group A active glycotopes (Ah) and Forssman antigen (Fp). When monosaccharides and their α, β anomers are involved in basic units to express the complex size of the combining sites of the GalNAcα1 → specific lectins, they can be divided into a cavity site to accommodate the GalNAcα → key sugar and a subsite with a wide and broad range of recognition area to adopt the rest part of sugar sequences or glycotopes. The function of the subsite is assumed to act as an enhancement factor to increase its affinity power. The following three points are the theme of this mini review: (1) the loci and distribution of the GalNAcα1 → related glycotopes in mammalian glycoconjugates are illustrated and their chemical structures are advanced by the expression of the disaccharide units and code system; (2) the sizes and motifs of GalNAcα1 → specific lectin-glycan interactions are given and (3) the role of the polyvalent blood group Ah and Bh glycotopes as blood group AB antigens are proposed. These three highlights should provide an essential background required for the advances in this field.

Sialylated and sulfated N-Glycans in MDCK and engineered MDCK cells for influenza virus studies

The Madin-Darby canine kidney (MDCK) cell line is an in vitro model for influenza A virus (IAV) infection and propagation. MDCK-SIAT1 (SIAT1) and humanized MDCK (hCK) cell lines are engineered MDCK cells that express N-glycans with elevated levels of sialic acid (Sia) in α2,6-linkage (α2,6-Sia) that are recognized by many human IAVs. To characterize the N-glycan structures in these cells and the potential changes compared to the parental MDCK cell line resulting from engineering, we analyzed the N-glycans from these cells at different passages, using both mass spectrometry and specific lectin and antibody binding. We observed significant differences between the three cell lines in overall complex N-glycans and terminal galactose modifications. MDCK cells express core fucosylated, bisected complex-type N-glycans at all passage stages, in addition to expressing α2,6-Sia on short N-glycans and α2,3-Sia on larger N-glycans. By contrast, SIAT1 cells predominantly express α2,6-Sia glycans and greatly reduced level of α2,3-Sia glycans. Additionally, they express bisected, sialylated N-glycans that are scant in MDCK cells. The hCK cells exclusively express α2,6-Sia glycans. Unexpectedly, hCK glycoproteins bound robustly to the plant lectin MAL-1, indicating α2,3-Sia glycans, but such binding was not Sia-dependent and closely mirrored that of an antibody that recognizes glycans with terminal 3-O-sulfate galactose (3-O-SGal). The 3-O-SGal epitope is highly expressed in N-glycans on multiple hCK glycoproteins. These results indicate vastly different N-glycomes between MDCK cells and the engineered clones that could relate to IAV infectivity.

O-Glycosylation Changes in Serum Immunoglobulin G Are Associated with Inflammation Development in Advanced Endometriosis.

Endometriosis is a gynecological disease, the pathogenesis of which seems to be directly related to inflammatory processes with an immune basis. Our study aimed to analyze the O-glycosylation of native serum IgG and IgG isolated from sera of women with advanced endometriosis, without endometriosis but with benign gynecological diseases, and from a control group of healthy women, in the context of its utility for differentiation of advanced endometriosis from the other two groups of women studied. For the analysis of serum IgG O-glycosylation and the expression of multi-antennary N-glycans, lectin-ELISA with lectins specific to O-glycans (MPL, VVL, and Jacalin) and highly branched N-glycans (PHA-L) was used. The relative reactivities of isolated serum IgG O-linked glycans with specific lectins as well as the MPL/VVL O-glycosylation ratio were significantly higher in patients with advanced endometriosis and those with other gynecological diseases when compared to the control group of healthy women. We also showed significantly higher expression of PHA-L-reactive multi-antennary N-glycans in isolated IgG in the advanced endometriosis and the non-endometriosis groups in comparison to the control group. Additionally, significantly higher expression of Jacalin-reactive O-glycans in isolated IgG was observed in the non-endometriosis than in the advanced endometriosis group. The results of the ROC curve and cluster analysis additionally confirmed that the lectin-based analysis of isolated serum IgG O-glycosylation and the expression of highly branched N-glycans may help distinguish women with advanced endometriosis from healthy women. Moreover, the analysis of the expression of Jacalin-reactive i-IgG O-glycans may be helpful in differentiation between women with advanced endometriosis and patients with other gynecological diseases with an inflammatory background. In the case of non-endometriosis patients, the observed differences were most probably caused by increased expression of core 3 type O-glycans.

Growing Maize Root: Lectins Involved in Consecutive Stages of Cell Development

Proteins that carry specific carbohydrate-binding lectin domains have a great variety and are ubiquitous across the plant kingdom. In turn, the plant cell wall has a complex carbohydrate composition, which is subjected to constant changes in the course of plant development. In this regard, proteins with lectin domains are of great interest in the context of studying their contribution to the tuning and monitoring of the cell wall during its modifications in the course of plant organ development. We performed a genome-wide screening of lectin motifs in the Zea mays genome and analyzed the transcriptomic data from five zones of primary maize root with cells at different development stages. This allowed us to obtain 306 gene sequences encoding putative lectins and to relate their expressions to the stages of root cell development and peculiarities of cell wall metabolism. Among the lectins whose expression was high and differentially regulated in growing maize root were the members of the EUL, dirigent–jacalin, malectin, malectin-like, GNA and Nictaba families, many of which are predicted as cell wall proteins or lectin receptor-like kinases that have direct access to the cell wall. Thus, a set of molecular players was identified with high potential to play important roles in the early stages of root morphogenesis.

Optimal regeneration and repair of critical size articular cartilage driven by endogenous CLECSF1

Due to the lack of blood supply and nerve in articular cartilage, it is a great challenge to repair critical size cartilage defect, to maintain the phenotype of new chondrocytes and to restore joint function. We find that the endogenous cartilage extracellular matrix specific protein C-type lectin (CLECSF1) can be applied for the repair of cartilage defect. The endogenous expression of CLECSF1 can be achieved by integrating its coding gene into the recombinant lentiviral vectors. These vectors are used to transfect into bone marrow mesenchymal stem cells (BMSCs) and then these transfected BMSCs are seeded in a scaffold. We reveal that such scaffold can promote chondrogenic differentiation, enhance cartilage extracellular matrix deposition and maintain chondrocyte phenotype. Furthermore, such scaffold can repair critical size cartilage defect and finally achieve a full recovery of both structure and function. Therefore, we believe that such scaffold can be an ideal alternative strategy for the treatment of critical size cartilage defect in future.

Comparison of Lectins as Staining Biomarkers for GNE Myopathy Gene Therapy

GNE myopathy (GNEM) is an autosomal recessive disease in which mutations in the GNEgene lead to skeletal muscle weakness and progressive wasting. GNE encodes an essential enzyme of the sialic acid (SA) biosynthetic pathway; thus, decreased SA levels in muscle is a hallmark of GNEM. While several therapeutic approaches to increase SA levels in GNEM patients are under development, these efforts have been hindered by the lack of robust biomarkers to assess SA incorporation into affected muscle tissue at the cellular and molecular level. We sought to address this unmet need by comparing the capacity of glycan‐binding proteins known as lectins to effectively monitor SA levels via fluorescent staining. Here, we compare the efficacy of a panel of SA‐detecting lectins by staining samples from multiple models of GNEM. These models include skeletal muscle of the GNED207VTgGne‐/‐ mouse model of GNEM and cell‐based in vitro models, such as those in which SA is removed by enzymatic activity or its production is precluded by GNE mutation. Together, these results indicate the relative ability of each lectin to detect SA alterations in models of GNEM, revealing specific lectins that can act as predictive biomarkers for this disease that may facilitate the development of novel therapeutic approaches.