Specific Lectin Research Articles

Cephalosporium curvulum lectin causes mycotic keratitis by initiating infection through MyD88 dependent cellular proliferation and apoptosis in human corneal epithelial cells.

Physiological role of a core fucose specific lectin from Cephalosporium curvulum isolated from mycotic keratitis patient in mediating pathogenesis was reported earlier. CSL has opposite effects on HCECs, at the initiation of infection when lectin concentration is low, CSL induces proinflammatory response and at higher concentration it inhibits growth as the infection progresses. Here we delineate detailed mechanism of opposing effects of CSL by confirming the binding of CSL and anti TLR 2 and 4 antibodies to TLRs 2 and 4 purified from HCECs using Galectin-3 Sepharose 4B column. Further, the expression of signaling proteins were monitored by Western blotting and apoptosis assay. At concentration of 0.3µg/ml, CSL induced the activation of TLR-2,-4 and adapter protein MyD88. CSL also induced the expression of transcription factors NFkB, C-Jun and proinflammatory cytokines like interleukins -6 and -8 essential in maintaining cell proliferation. In contrast at higher concentrations i.e. 5µg/ml CSL induces apoptotic effect as evidenced by increase in early and late apoptotic population as demonstrated by Annexin V-PI assay. Western blotting revealed that CSL treated HCECs at higher concentration lead to MyD88 dependent expression of apoptotic proteins like FADD, Caspase -8 and -3. All these results are in line with and substantiate our earlier results that indeed CSL is involved in mediating host pathogen interactions by interacting with cell surface TLRs, activating downstream signaling pathways leading to pathogenesis. Findings are of clinical significance in developing carbohydrate based therapeutic strategy to control infection and the disease.

Galectin-3: A factotum in carcinogenesis bestowing an archery for prevention.

Cancer metastasis and therapy resistance are the foremost hurdles in oncology at the moment. This review aims to pinpoint the functional aspects of a unique multifaceted glycosylated molecule in both intracellular and extracellular compartments of a cell namely galectin-3 along with its metastatic potential in different types of cancer. All materials reviewed here were collected through the search engines PubMed, Scopus, and Google scholar. Among the 15 galectins identified, the chimeric gal-3 plays an indispensable role in the differentiation, transformation, and multi-step process of tumor metastasis. It has been implicated in the molecular mechanisms that allow the cancer cells to survive in the intravascular milieu and promote tumor cell extravasation, ultimately leading to metastasis. Gal-3 has also been found to have a pivotal role in immune surveillance and pro-angiogenesis and several studies have pointed out the importance of gal-3 in establishing a resistant phenotype, particularly through the epithelial-mesenchymal transition process. Additionally, some recent findings suggest the use of gal-3 inhibitors in overcoming therapeutic resistance. All these reports suggest that the deregulation of these specific lectins at the cellular level could inhibit cancer progression and metastasis. A more systematic study of glycosylation in clinical samples along with the development of selective gal-3 antagonists inhibiting the activity of these molecules at the cellular level offers an innovative strategy for primary cancer prevention.

Cuantificación de la actividad específica de lectina en teosinte Zea diploperennis

Objective. To evaluate the specific activity of lectin in healthy and infected teosinte Zea diploperennis with Ustilago maydis. Materials and methods. Zea diploperennis seedlings of 6 days of growth were inoculated with Ustilago maydis. Total protein concentration and hemagglutinating activity of crude extracts of healthy and infected teosinte were evaluated in microtiter plates with 3% type O red cells following the technique of serial double dilutions for 6 days. Results. The concentration of total proteins increases in healthy coleoptile during each day of its growth. Not so in infected teosinte where the curve shows a downward trend from the moment of inoculation. The specific lectin activity decreases in both cases from the first day of quantification. Conclusions. The evident reduction in lectin activity in infected teosinte compared to healthy teosinte could explain the susceptibility of this teosinte to said phytopathogen. Although the participation of the teosinte and corn lectins in the defense mechanism against Ustilago maydis has not yet been clarified, the results obtained contribute to the understanding of the effect that the concentration of lectin and protein can have on resistance in teosinte.

Mevo lectin specificity toward high-mannose structures with terminal αMan(1,2)αMan residues and its implication to inhibition of the entry of Mycobacterium tuberculosis into macrophages.

Mannose-binding lectins can specifically recognize and bind complex glycan structures on pathogens and have potential as antiviral and antibacterial agents. We previously reported the structure of a lectin from an archaeal species, Mevo lectin, which has specificity toward terminal α1,2 linked manno-oligosaccharides. Mycobacterium tuberculosis expresses mannosylated structures including lipoarabinomannan (ManLAM) on its surface and exploits C-type lectins to gain entry into the host cells. ManLAM structure has mannose capping with terminal αMan(1,2)αMan residues and is important for recognition by innate immune cells. Here, we aim to address the specificity of Mevo lectin toward high-mannose type glycans with terminal αMan(1,2)αMan residues and its effect on M. tuberculosis internalization by macrophages. Isothermal titration calorimetry studies demonstrated that Mevo lectin shows preferential binding toward manno-oligosaccharides with terminal αMan(1,2)αMan structures and showed a strong affinity for ManLAM, whereas it binds weakly to Mycobacterium smegmatis lipoarabinomannan, which displays relatively fewer and shorter mannosyl caps. Crystal structure of Mevo lectin complexed with a Man7D1 revealed the multivalent cross-linking interaction, which explains avidity-based high-affinity for these ligands when compared to previously studied manno-oligosaccharides lacking the specific termini. Functional studies suggest that M. tuberculosis internalization by the macrophage was impaired by binding of Mevo lectin to ManLAM present on the surface of M. tuberculosis. Selectivity shown by Mevo lectin toward glycans with terminal αMan(1,2)αMan structures, and its ability to compromise the internalization of M. tuberculosis in vitro, underscore the potential utility of Mevo lectin as a research tool to study host-pathogen interactions.

Variability of serum IgG sialylation and galactosylation degree in women with advanced endometriosis

Endometriosis is an inflammatory disease which diagnostics is difficult and often invasive, therefore non-invasive diagnostics methods and parameters are needed for endometriosis detection. The aim of our study was to analyse the glycosylation of native serum IgG and IgG isolated from sera of women classified as: with endometriosis, without endometriosis but with some benign ginecological disease, and control group of healthy women, in context of its utility for differentiation of advanced endometriosis from the group of healthy women. IgG sialylation and galactosylation/agalactosylation degree was determined using specific lectins: MAA and SNA detecting sialic acid α2,3- and α2,6-linked, respectively, RCA-I and GSL-II specific to terminal Gal and terminal GlcNAc, respectively. The results of ROC and cluster analysis showed that the serum IgG MAA-reactivity, sialylation and agalactosylation factor may be used as supplementary parameters for endometriosis diagnostics and could be taken into account as a useful clinical tool to elucidate women with high risk of endometriosis development. Additionally, we have shown that the analysis of native serum IgG glycosylation, without the prior time-consuming and expensive isolation of the protein, is sufficient to differentiation endometriosis from a group of healthy women.

Long-Term Trypsin Treatment Promotes Stem Cell Potency of Canine Adipose-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) isolated from adipose tissue (adipose-derived stem cells [ADSCs]) are considered one of the most promising cell types for applications in regenerative medicine. However, the regenerative potency of ADSCs may vary because of heterogeneity. Long-term trypsin treatment (LTT) is known to significantly concentrate multilineage-differentiating stress-enduring (Muse) cells from human MSCs. In this study, we aimed to generate cells with high stem cell potency from canine ADSCs using LTT. After 16 h of treatment with trypsin, surviving ADSCs (LTT-tolerant cells) had significantly enhanced expression of stage-specific embryonic antigen (SSEA)-1, a mouse embryonic stem cell marker, and fucosyltransferase 9, one of several fucosyltransferases for SSEA-1 biosynthesis. However, LTT-tolerant cells did not enhance the expression of SSEA-3, a known human Muse cell marker. LTT-tolerant cells, however, showed significantly higher self-renewal capacity in the colony-forming unit fibroblast assay than ADSCs. In addition, the LTT-tolerant cells formed cell clusters similar to embryoid bodies and expressed undifferentiated markers. Moreover, these cells differentiated into cells of all three germ layers and showed significantly higher levels of α 2-6 sialic acid (Sia)-specific lectins, known as differentiation potential markers of human MSCs, than ADSCs. LTT-tolerant cells had a normal karyotype and had low telomerase activity, showing little carcinogenetic potency. LTT-tolerant cells also showed significantly increased activity of transmigration in the presence of chemoattractants and had increased expression of migration-related genes compared with ADSCs. In addition, LTT-tolerant cells had stronger suppressive activity against mitogen-stimulated lymphocyte proliferation than ADSCs. Overall, these results indicated that the LTT-tolerant cells in canine ADSCs have similar properties as human Muse cells (although one of the undifferentiated markers is different) and are expected to be a promising tool for regenerative therapy in dogs.

A biotechnological tool for glycoprotein desialylation based on immobilized neuraminidase from Clostridium perfringens

BackgroundSialic acids are widely distributed in nature and have biological relevance owing to their varied structural and functional roles. Immobilized neuraminidase can selectively remove terminal N-acetyl neuraminic acid from glycoproteins without altering the protein backbone while it can be easily removed from the reaction mixture avoiding sample contamination. This enables the evaluation of changes in glycoprotein performance upon desialylation. MethodsNeuraminidase was immobilized onto agarose activated with cyanate ester groups and further used for desialylation of model glycoproteins, a lysate from tumour cells and tumour cells. Desialylation process was analysed by lectin binding assay, determination of sialyl-Tn or flow cytometry. ResultsClostridium perfringens neuraminidase was immobilized with 91 % yield and expressed activity yield was of 41%. It was effective in the desialylation of bovine fetal serum fetuin, bovine lactoferrin and ovine submaxilar mucin. A decrease in sialic-specific SNA lectin recognition of 83% and 53 % was observed for fetuin and lactoferrin with a concomitant increase in galactose specific ECA and PNA lectin recognition. Likewise, a decrease in the recognition of a specific antibody (82%) upon mucin desialylation was observed. Moreover, desialylation of a protein lysate from the sialic acid-rich cell line TA3/Ha was also possible leading to a decrease in 47 % in SNA recognition. Immobilized neuraminidase kept 100% of its initial activity upon five desialylation cycles. ConclusionsImmobilized neuraminidase is an interesting as well as a robust biotechnological tool for enzymatic desialylation purposes. General significanceImmobilized neuraminidase would contribute to understand the role of sialic acid in biological processes.

Tailor-made recombinant prokaryotic lectins for characterisation of glycoproteins

Development of biosimilars is costly, where glycan analysis is a significant constraint on time and money. This paper provides an in-depth characterisation of several novel recombinant prokaryotic lectins (RPLs), developed through directed evolution, displaying specific binding activities to α-mannose, β-galactose, fucose and sialic acid residues, tested against major biosimilar targets. The binding characterisation of all lectins was performed employing the principles of bio-layer interferometry (BLI), with help of the streptavidin-coated sensor with the biotinylated lectins. The binding activity of the RPLs and the specificity to a broad range of glycoproteins and glycoconjugates were evaluated and compared to those of equivalent plant-derived lectins. While exhibiting better or similar specificity, RPLs displayed significantly better binding in all cases. The binding mechanisms are explained with particular focus on the role hydrogen bonding plays in the change of specificity for a galactose specific lectin. Furthermore, different sets of RPLs and their plant equivalents were assayed against the different glycoprotein targets to evaluate the analytical parameters of the lectin-glycoprotein interaction. The obtained LoDs reached by the RPLs were lower than those of their plant counterparts apart from one, exhibiting RPL:PL LoD ratios of 0.8, 2.5, 14.2 and 380 for the sets of lectins specific to fucose, α-mannose, β-galactose and sialic acid, respectively. Such enhancement in analytical parameters of RPLs shows their applicability in protein purification and as bioanalytical tools for glycan analysis and biosensor development.

Physical-Chemical and Cytotoxic Properties of Bacillus subtilis IMV B-7724 Extracellular Lectin

Aim. To study the chemical composition, sugar specificity and physicochemical properties of the extracellular lectin isolated from Bacillus subtilis ІМV В-7724. Methods. Biochemical, spectrophotometric, immunological and cultural methods were used to assess the physicochemical and a number of biological properties of lectin isolated from the culture fluid of bacteria B. subtilis ІМV В-7724. Molecular weight of the lectin was estimated in polyacrylamide gel electrophoresis. Analysis of the elemental composition was done using Perkin-Elmer 2400 CHNS analyzer. Temperature and pH stability of lectin were examined based on residual hemagglutination activity of the lectin. Cytotoxic activity was determined by the MTT-assay. The statistical analysis was made using Student’s t-test. Results. B. subtilis IMV B-7724 lectin is a glycoprotein (protein – 86.0%, carbohydrates – 7.0%) with molecular weight of 18–20 kDa (major). Analysis of the elemental composition revealed that it contains 34.00% of carbon, 7.04% of hydrogen, 16.61% of nitrogen, 42.35% of oxygen. Amino acid composition analysis determined that it is rich in leucine, tyrosine and phenylalanine. The lectin exhibited high sugar-binding specificity toward N-acetylneuraminic and N-glycolylneuraminic acids (minimal inhibitory concentration – 0.3 mM for both sugars). The lectin is heat and acid stable, has long shelf life. Conclusions. These results provide the rationale to pursue further investigation for possible ways and modes of B. subtilis IMB B-7724 lectin application in clinical settings.

In depth analysis on the carbohydrate-binding properties of a vasorelaxant lectin from Dioclea lasiophylla Mart Ex. Benth seeds

Lectins are a class of proteins or glycoproteins capable of recognizing and interacting with carbohydrates in a specific and reversible manner. Owing to this property, these proteins can interact with glycoconjugates present on the cell surface, making it possible to decipher the glycocode, as well as elicit biological effects, such as inflammation and vasorelaxation. Here, we report a structural and biological study of the mannose/glucose‐specific lectin from Dioclea lasiophylla seeds, DlyL. The study aimed to evaluate in detail the interaction of DlyL with Xman and high-mannose N-glycans (MAN3, MAN5 and MAN9) by molecular dynamics (MD) and the resultant in vitro effect on vasorelaxation using rat aortic rings. In silico analysis of molecular docking was performed to obtain the initial coordinates of the DlyL complexes with the carbohydrates to apply as inputs in MD simulations. The MD trajectories demonstrated the stability of DlyL over time as well as different profiles of interaction with Xman and N-glycans. Furthermore, aortic rings assays demonstrated that the lectin could relax pre-contracted aortic rings with the participation of the carbohydrate recognition domain (CRD) and nitric oxide (NO) when endothelial tissue is preserved. These results confirm the ability of DlyL to interact with high‐mannose N‐glycans with its expanded CRD, supporting the hypothesis that DlyL vasorelaxant activity occurs primarily through its interaction with cell surface glycosylated receptors. Communicated by Ramaswamy H. Sarma

THE EFFECTIVENESS OF PHYTOLECTINS AND LECTIN COMPOSITIONS APPLICATION FOR SPRAYING PLANTS DURING VEGETATION

The effectiveness of the soybean plants spraying with the soybean seed lectin solution during vegetation (against the background of seed inoculation with nodule bacteria and without seed inoculation), as well as the effectiveness of the winter wheat plants spraying with lectin-bacterial composition in green-house and field experiments was investigated respectively. It was found that spraying of soybeans in the phase of two trifoliate leaves development with a specific lectin against the background of pre-sowing seed inoculation with rhizobia caused a significantly positive effect on the functional activity of the symbiotic apparatus. The nitrogen-fixing activity of the rhizosphere microbiota remained unchanged, which may indicate the vector of lectin action when sprayed through the plant. At the same time, the activation of plants vegetative growth was noted, which was maximally manifested by the height of their above ground part. The activity of exogenous sprayed lectin was less pronounced on the background of seed inoculation with rhizobia compared to non-inoculated plants. Plants spraying with soybean lectin against the background of seed inoculation provided an increase in harvest compared to non-inoculated control by 12.8 g/plant, but by the factor of lectin action this increase was only 1.15 g/plant and was insignificant. Non-inoculated soybean plants when sprayed with lectin formed a harvest that was significantly higher (by 3.86 g/plant) than that of plants in the absence of lectin. At this, the increase by the factor of lectin action was 22%. The spraying of winter wheat plants in the phase of mass spring germinations with the Azolec preparation (without pre-sowing seed inoculation) contributed to a significant increase in harvest by 1.6 c/ha. Therefore, the application of soybean and wheat plants spraying, respectively, with soybean seed lectin and lectin-bacterial Azolec preparation (wheat lectin),without involving pre-sowing seed inoculation, provided a greater degree of plants productive potential realization compared to control (without pre-sowing seed inoculation and plants spraying during vegetation).

Immobilized peptide-N-glycosidase F onto magnetic nanoparticles: A biotechnological tool for protein deglycosylation under native conditions.

The elucidation of glycans biological function is essential to understand their role in biological processes, both normal and pathological. Immobilized glycoenzymes are excellent tools for this purpose as they can selectively release glycans from glycoproteins without altering their backbone. They can be easily removed from the reaction mixture avoiding their interference in subsequent experiments. Here, we describe the immobilization of peptide-N-glycosidase F (PNGase F) onto silica magnetic nanoparticles with immobilization yields of 86% and activity yields of 12%. Immobilized PNGase F showed higher thermal stability than its soluble counterpart, and could be reused for at least seven deglycosylation cycles. It was efficient in the deglycosylation of several glycoproteins (ribonuclease B, bovine fetuin, and ovalbumin) and a protein lysate from the parasite Fasciola hepatica under native conditions, with similar performance to that of the soluble enzyme. Successful deglycosylation was evidenced by a decrease in specific lectin recognition of the glycoproteins (40%-80%). Moreover, deglycosylated F. hepatica lysate allowed us to confirm the role of parasite N-glycans in the inhibition of the lipopolysaccharide-induced maturation of dendritic cells. Immobilized PNGase F probed to be a robust biotechnological tool for deglycosylation of glycoproteins and complex biological samples under native conditions.

Lentil lectin derived from Lens culinaris exhibit broad antiviral activities against SARS-CoV-2 variants

ABSTRACT The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutated continuously and newly emerging variants escape from antibody-mediated neutralization raised great concern. S protein is heavily glycosylated and the glycosylation sites are relatively conserved, thus glycans on S protein surface could be a target for the development of anti-SARS-CoV-2 strategies against variants. Here, we collected 12 plant-derived lectins with different carbohydrate specificity and evaluated their anti-SARS-CoV-2 activity against mutant strains and epidemic variants using a pseudovirus-based neutralization assay. The Lens culinaris-derived lentil lectin which specifically bind to oligomannose-type glycans and GlcNAc at the non-reducing end terminus showed most potent and broad antiviral activity against a panel of mutant strains and variants, including the artificial mutants at N-/O-linked glycosylation site, natural existed amino acid mutants, as well as the epidemic variants B.1.1.7, B.1.351, and P.1. Lentil lectin also showed antiviral activity against SARS-CoV and MERS-CoV. We found lentil lectin could block the binding of ACE2 to S trimer and inhibit SARS-CoV-2 at the early steps of infection. Using structural information and determined N-glycan profile of S trimer, taking together with the carbohydrate specificity of lentil lectin, we provide a basis for the observed broad spectrum anti-SARS-CoV-2 activity. Lentil lectin showed weak haemagglutination activity at 1 mg/mL and no cytotoxicity activity, and no weight loss was found in single injection mouse experiment. This report provides the first evidence that lentil lectin strongly inhibit infection of SARS-COV-2 variants, which should provide valuable insights for developing future anti-SARS-CoV-2 strategies.

Varieties of Mushrooms and their Nutraceutical Importance: A Systematic Review

Introduction: Around 2000 species of mushrooms exist in nature, of which hardly 25 varieties are accepted as edible in nature. “Nutraceuticals” are food substances that are associated with immense health benefits and also hinder the development of several maladies. Aim: This review article aims to explore the nutritional contents/composition of the varied types of mushrooms and their clinical importance for the human being. Materials and Methods: The methodology adopted is a comprehensive systematic literature review. The suitable electronic databases sources that were reviewed were Google Scholar, PubMed, PsycINFO, Medline, and Grey literature databases namely GreyNet International, Open Grey, and Med Nar. The literatures were screened by customising the databases based on the pre-determined inclusion and exclusion criteria and up to the most recent contents of the year, 2019. In total, 63 articles were considered for the systematic literature review. Results: The findings of the selected pieces of evidence were critically appraised and thematically presented. Mushrooms serve as a great nutritional source; as they are rich in proteins, contain essential amino acids, fibre, and an excellent source of fatty acids but poor in fat content. Their nutritional significance increases due to the presence of vitamins B1, B2, B12, C, D, and E. Significant content of carbohydrates are present in an edible variety of mushrooms. Trehalose, glucose, and mannitol are among the abundant sugars present in the cultivated edible mushrooms that are responsible for different categories of medicinal properties for instance immunomodulating properties, antitumour properties by modulating the immune response of the host. The distinguished protein substances present in mushrooms are specific immunomodulatory proteins, lectins, antimicrobial proteins, ribosome-inactivating proteins, ribonucleases, and laccases that have interesting biological activities. Mushrooms contain significant amounts of polyunsaturated fatty acid that contribute to the reduction of cholesterol levels in the serum and also certain essential fatty acids such as oleic acid, linolenic acid, arachidonic acid, palmitic acids. Conclusion: Mushrooms can thus be considered to be a reservoir of varied significant dietary composition contributing to a healthy diet and their daily consumption may hinder any development of maladies within our physiological system.

Integrated Glycoproteomics Identifies a Role of N-Glycosylation and Galectin-1 on Myogenesis and Muscle Development

AbstractMany cell surface and secreted proteins are modified by the covalent addition of glycans that play an important role in the development of multicellular organisms. These glycan modifications enable communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signaling. Aberrant protein glycosylation has been associated with the development of several muscular diseases, suggesting essential glycan- and lectin-mediated functions in myogenesis and muscle development, but our molecular understanding of the precise glycans, catalytic enzymes, and lectins involved remains only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during a time-course of myogenesis in cell culture. We observed wide-spread changes in the abundance of several important lectins and enzymes facilitating glycan biosynthesis. Glycomics-based quantification of released N-linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown digalactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labeling and analysis of enriched glycopeptides with multiple fragmentation approaches identified glycoproteins modified by these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins, most notably the upregulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation, suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in N-glycosylation and the upregulation of galectin-1 during postnatal skeletal muscle development were observed in mice. Treatment of new-born mice with recombinant adeno-associated viruses to overexpress galectin-1 in the musculature resulted in enhanced muscle mass. Our data form a valuable resource to further understand the glycobiology of myogenesis and will aid the development of intervention strategies to promote healthy muscle development or regeneration.

Antigen Delivery to DEC205+ Dendritic Cells Induces Immunological Memory and Protective Therapeutic Effects against HPV-Associated Tumors at Different Anatomical Sites.

The generation of successful anticancer vaccines relies on the ability to induce efficient and long-lasting immune responses to tumor antigens. In this scenario, dendritic cells (DCs) are essential cellular components in the generation of antitumor immune responses. Thus, delivery of tumor antigens to specific DC populations represents a promising approach to enhance the efficiency of antitumor immunotherapies. In the present study, we employed antibody-antigen conjugates targeting a specific DC C-type lectin receptor. For that purpose, we genetically fused the anti-DEC205 monoclonal antibody to the type 16 human papillomavirus (HPV-16) E7 oncoprotein to create a therapeutic vaccine to treat HPV-associated tumors in syngeneic mouse tumor models. The therapeutic efficacy of the αDEC205-E7 mAb was investigated in three distinct anatomical tumor models (subcutaneous, lingual and intravaginal). The immunization regimen comprised two doses of the αDEC205-E7 mAb coadministered with a DC maturation stimulus (Polyinosinic:polycytidylic acid, poly (I:C)) as an adjuvant. The combined immunotherapy produced robust antitumor effects on both the subcutaneous and orthotopic tumor models, stimulating rapid tumor regression and long-term survival. These outcomes were related to the activation of tumor antigen-specific CD8+ T cells in both systemic compartments and lymphoid tissues. The αDEC205-E7 antibody plus poly (I:C) administration induced long-lasting immunity and controlled tumor relapses. Our results highlight that the delivery of HPV tumor antigens to DCs, particularly via the DEC205 surface receptor, is a promising therapeutic approach, providing new opportunities for the development of alternative immunotherapies for patients with HPV-associated tumors at different anatomical sites.

Specific lectin binding sites during in vitro capacitation and acrosome reaction in boar spermatozoa

Sperm glycocalyx and plasma membrane undergo outstanding modifications during fertilization. However, it is unclear how in vitro capacitation time and acrosome reaction affect the specific location of boar sperm glycoconjugates. This study aimed to identify lectin binding patterns and to describe the sequential changes during different in vitro capacitation times (1 and 4 h) and acrosome reaction in boar spermatozoa. With Aleuria aurantia agglutinin (AAA), most uncapacitated cells were labelled in the postacrosomal region. Nevertheless, after 1 h of in vitro capacitation and the acrosome reaction, most AAA binding sites were in the acrosomal region. With Concanavalin A (ConA), most sperm were labeled in the postacrosomal region before and after capacitation. After the acrosome reaction induction, this pattern changed to a highly stained acrosomal and postacrosomal regions. Peanut agglutinin (PNA) binding sites were in the acrosomal region in uncapacitated and capacitated sperm. In acrosome reacted sperm after 4h capacitation, the most frequent pattern showed remaining positive labeling in the central area of the head. With Pisum sativum agglutinin (PSA), most uncapacitated cells showed a postacrosomal region staining. Nevertheless, faint stained all over the head and highly acrosomal region labelling was observed in the major part of capacitated and acrosome reacted sperm respectively. With Wheat germ agglutinin (WGA), the most representative pattern in uncapacitated, capacitated and acrosome reacted sperm was labelled in the acrosomal region. Regarding capacitation time, the most significant changes in the most representative pattern were observed in acrosome reacted spermatozoa after 4 h of in vitro capacitation. Highlights Lectins allow detailed descriptions of eight different populations in boar sperm. Changes in lectin binding patterns during capacitation are time dependent. Lectin binding patterns significantly change after 4 h of in vitro capacitation.

A facile strategy for quantitative sensing of glycans on cell surface using organic electrochemical transistors

This work designed a facile sensing strategy for quantitation of glycans on cell surface using organic electrochemical transistors (OECTs). The sensing strategy was performed by covalently binding target cells on mercaptopropionic acid modified gate electrode for the recognition of horseradish peroxidase (HRP) labeled lectins to specific glycans on cell surface, which led to sensitive channel current signal responding to the captured HRP and thus the expression of cell surface glycans. The quantitation of glycans on cell surface was achieved by using glycan modified microspheres to simulate the cells, on which the amounts of glycans were detected with enzyme linked immunosorbent assay. The proposed sensing strategy had been used for the detection of mannose and galactose on HeLa cells with Concanavalin A and peanut agglutinin as the specific lectins, which showed 1.37 × 108 mannose and 1.13 × 108 galactose on each cell, respectively. By treating HeLa cells with exoglycosidases, the mannose and galactose expression levels showed the same changes as those detected with flow cytometric analysis, indicating the practical application of the OECT-based biosensor in cell surface glycan expression analysis.

INVESTIGATION OF EMBRYONIC HISTOGENESIS OF THE PAROTID GLAND

Lectinohistochemistry methods are very sensitive and allow to identify certain types and subpopulations of cells, to characterize non-cellular tissue structures in morphological studies, when they cannot be differentiated by using traditional methods of carbohydrate histochemistry. In many diseases, changes in the carbohydrate component of various glycoconjugates are observed, which contribute to the modification of morphofunctional characteristics of the cell and changes in its interaction with other cells and extracellular factors. Most studies are devoted to the study of the existing pathology of individual organs and systems (or their norms) in adults and animals. Data from the scientific literature on the histotopography of lectin receptors in the first months of human prenatal ontogenesis are few, and regarding the peculiarities of the expression of carbohydrate determinants of the rudiments of the human parotid gland in early prenatal ontogenesis - are absent. Glycopolymers compounds make up structural and functional basis of cells and tissues of a living organism. The necessity of anatomical-lectinohistochemical examination of the parotid gland in early prenatal period of ontogenesis is substantiated, as the evidences concerning its topography are fragmentary and unsystematized, and certain aspects of its ontogenesis are disputable. A natural redistribution of glycopolimers of the cytolemma and cytoplasm of the cells of the epithelial anlage of the parotid salivary gland and the mesenchyma adjacent to it in the course of investigating 50 human embryos and prefetuses aged up 21 days to 12 weeks at stages 9-23 and the beginning of the fetal period according to the classification of Carnegy’s institute has been revealed. Invagination of epithelial cells in the regions bucco-alveolar pockets into the underlying mesenchyme and their transformation into epithelial cords due to the accumulation of specific glycopolymers lectin WGA, SNA, HPA, RCA, LABA. The results of lectin-histochemical examination of the early prenatal ontogenesis of the parotid gland can form the basis for the work of laboratories dealing with screening of morphological material in order to assess the degree of maturation and prognosis of fetus viability and diagnostics of deviations from normal development.

Sites of vulnerability on ricin B chain revealed through epitope mapping of toxin-neutralizing monoclonal antibodies.

Ricin toxin's B subunit (RTB) is a multifunctional galactose (Gal)-/N-acetylgalactosamine (GalNac)-specific lectin that promotes uptake and intracellular trafficking of ricin's ribosome-inactivating subunit (RTA) into mammalian cells. Structurally, RTB consists of two globular domains (RTB-D1, RTB-D2), each divided into three homologous sub-domains (α, β, γ). The two carbohydrate recognition domains (CRDs) are situated on opposite sides of RTB (sub-domains 1α and 2γ) and function non-cooperatively. Previous studies have revealed two distinct classes of toxin-neutralizing, anti-RTB monoclonal antibodies (mAbs). Type I mAbs, exemplified by SylH3, inhibit (~90%) toxin attachment to cell surfaces, while type II mAbs, epitomized by 24B11, interfere with intracellular toxin transport between the plasma membrane and the trans-Golgi network (TGN). Localizing the epitopes recognized by these two classes of mAbs has proven difficult, in part because of RTB's duplicative structure. To circumvent this problem, RTB-D1 and RTB-D2 were expressed as pIII fusion proteins on the surface of filamentous phage M13 and subsequently used as "bait" in mAb capture assays. We found that SylH3 captured RTB-D1 (but not RTB-D2) in a dose-dependent manner, while 24B11 captured RTB-D2 (but not RTB-D1) in a dose-dependent manner. We confirmed these domain assignments by competition studies with an additional 8 RTB-specific mAbs along with a dozen a single chain antibodies (VHHs). Collectively, these results demonstrate that type I and type II mAbs segregate on the basis of domain specificity and suggest that RTB's two domains may contribute to distinct steps in the intoxication pathway.