Carbohydrate-binding Proteins Research Articles

The lectin Orysata induces phosphatase-mediated and carbohydrate-independent aggregation of insect cells

Lectins, or carbohydrate-binding proteins, can cause agglutination of particular cells. This process is mediated by the interaction of the carbohydrate-binding domain with sugar structures on the cell surface, and this binding can be inhibited by pre-incubation of the lectin with its specific sugars. However, when incubated with insect cells, Orysata, a mannose-binding lectin from rice, caused aggregation of the cells, independent from carbohydrate binding activity. This phenomenon was observed for multiple insect cell lines, confirming the robustness of this phenotype. While the carbohydrate-dependent agglutination of red blood cells happens within minutes, the carbohydrate-independent aggregation of insect cells requires longer incubation times. Further analysis with the galactose-binding lectins SSA and Jacalin, validated the robustness of this lectin-induced, carbohydrate-independent aggregation in different insect cell lines. Since proteomic analysis revealed no changes in the proteome after treatment with the lectins, this cell aggregation is likely caused by the (in) activation or re-organization of the existing surface proteins. The use of inhibitors of phosphorylation and dephosphorylation, staurosporine (STS) and a phosphatase inhibitor (PPI) cocktail, pointed to dephosphorylation as a key mechanism in the lectin-induced, carbohydrate-independent aggregation of insect cells. Similar to contact inhibition, cell proliferation in cell aggregates was decreased. Analysis of the marker for cell proliferation, cyclin E, confirmed that aggregated cells enter a quiescent state. The current data offer a new perspective on the mechanism by which lectins execute their activities, specifically through lectin-induced phosphatase-mediated cell aggregation and proliferation inhibition, independent from their carbohydrate-binding activity.

Lectin and Its Function

Lectins are carbohydrate-binding proteins that are profoundly particular for sugar bunches that are portion of other particles and so cause agglutination of specific cells or precipitation of glycoconjugates and polysaccharides. Lectins have a part in acknowledgment on the cellular and atomic level and play various parts in natural acknowledgment marvels including cells, carbohydrates, and proteins. Lectins too intervene connection and authoritative of microscopic organisms, infections, and organisms to their aiming targets.

Visualization of hydrogen atoms in a perdeuterated lectin-fucose complex reveals key details of protein-carbohydrate interactions

Carbohydrate-binding proteins from pathogenic bacteria and fungi have been shown to be implicated in various pathological processes, where they interact with glycans present on the surface of the host cells. These interactions are part of the initial processes of infection of the host and are very important to study at the atomic level. Here, we report the room temperature neutron structures of PLL lectin from Photorhabdus laumondii in its apo form and in complex with deuterated L-fucose, which is, to our knowledge, the first neutron structure of a carbohydrate-binding protein in complex with a fully deuterated carbohydrate ligand. A detailed structural analysis of the lectin-carbohydrate interactions provides information on the hydrogen bond network, the role of water molecules, and the extent of the CH-π stacking interactions between fucose and the aromatic amino acids in the binding site.

Hyaluronic acid and its benefits

Lectins are carbohydrate-binding proteins that are profoundly particular for sugar bunches that are portion of other particles and so cause agglutination of specific cells or precipitation of glycoconjugates and polysaccharides. Lectins have a part in acknowledgment on the cellular and atomic level and play various parts in natural acknowledgment marvels including cells, carbohydrates, and proteins. Lectins too intervene connection and authoritative of microscopic organisms, infections, and organisms to their aiming targets.

Lysin Motif (LysM) Proteins: Interlinking Manipulation of Plant Immunity and Fungi.

The proteins with lysin motif (LysM) are carbohydrate-binding protein modules that play a critical role in the host-pathogen interactions. The plant LysM proteins mostly function as pattern recognition receptors (PRRs) that sense chitin to induce the plant’s immunity. In contrast, fungal LysM blocks chitin sensing or signaling to inhibit chitin-induced host immunity. In this review, we provide historical perspectives on plant and fungal LysMs to demonstrate how these proteins are involved in the regulation of plant’s immune response by microbes. Plants employ LysM proteins to recognize fungal chitins that are then degraded by plant chitinases to induce immunity. In contrast, fungal pathogens recruit LysM proteins to protect their cell wall from hydrolysis by plant chitinase to prevent activation of chitin-induced immunity. Uncovering this coevolutionary arms race in which LysM plays a pivotal role in manipulating facilitates a greater understanding of the mechanisms governing plant-fungus interactions.

Generation and Use of Recombinant Galectins.

Galectins are soluble carbohydrate binding proteins that can bind β-galactose-containing glycoconjugates by means of a conserved carbohydrate recognition domain (CRD). In mammalian systems, galectins have been shown to mediate very important roles in innate and adaptive immunity as well as facilitating host-pathogen relationships. Many of these studies have relied on purified recombinant galectins to uncover key features of galectin biology. A major limitation to this approach is that certain recombinant galectins purified using standard protocols are easily susceptible to loss of glycan-binding activity. As a result, biochemical studies that employ recombinant galectins can be misleading if the overall activity of a galectin remains unknown in a given assay condition. This article examines fundamental considerations when purifying galectins by lactosyl-sepharose and nickel-NTA affinity chromatography using human galectin-4N and -7 as examples, respectively. As other approaches are also commonly applied to galectin purification, we also discuss alternative strategies to galectin purification, using human galectin-1 and -9 as examples. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Purification of galectins using lactosyl-sepharose affinity chromatography Basic Protocol 2: Purification of human galectin-7 using a nickel-NTA affinity chromatography column Alternate Protocol 1: Iodoacetamide alkylation of free sulfhydryls on galectin-1 Alternate Protocol 2: Purification of human galectin-9 using lactosyl-sepharose column chromatography.

Antiviral Bioactive Compounds of Mushrooms and Their Antiviral Mechanisms: A Review.

Mushrooms are used in their natural form as a food supplement and food additive. In addition, several bioactive compounds beneficial for human health have been derived from mushrooms. Among them, polysaccharides, carbohydrate-binding protein, peptides, proteins, enzymes, polyphenols, triterpenes, triterpenoids, and several other compounds exert antiviral activity against DNA and RNA viruses. Their antiviral targets were mostly virus entry, viral genome replication, viral proteins, and cellular proteins and influenced immune modulation, which was evaluated through pre-, simultaneous-, co-, and post-treatment in vitro and in vivo studies. In particular, they treated and relieved the viral diseases caused by herpes simplex virus, influenza virus, and human immunodeficiency virus (HIV). Some mushroom compounds that act against HIV, influenza A virus, and hepatitis C virus showed antiviral effects comparable to those of antiviral drugs. Therefore, bioactive compounds from mushrooms could be candidates for treating viral infections.

Concanavalin A induces apoptosis in a dose-dependent manner by modulating thiol/disulfide homeostasis in C6 glioblastoma cells.

Glioma is the most common brain tumor. C6 rat glioblastoma cells provide the possibility to the scientist to study brain cancer. Concanavalin A (Con A) has a lot of antitumoral effects, especially over oxidative stress. In the present study, it was aimed to decide the impacts of various doses of Con A on C6 glioblastoma cells regarding cytotoxicity, thiol/disulfide homeostasis, apoptosis, and inflammation. We detected the cytotoxic activity of Con A (from 7.8 to 500 µg/ml) in C6 cells by utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and determined the toxic concentration of Con A. Once the optimal doses were found, the thiol-disulfide homeostasis, levels of total antioxidant and oxidant status (TAS and TOS), malondialdehyde (MDA) and glutathione (GSH), pro-inflammatory cytokines as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), apoptotic proteins as cytochrome c (CYCS), and caspase 3 (CASP3) were measured. Apoptotic and morphological changes in the C6 cells were examined with an inverted microscope and flow cytometry technique. Dose-dependent Con A triggered oxidative damage in the C6 cells, affecting the inflammatory pathway, so reducing proliferation with apoptotic proteins and morphological changes. But especially, Con A increased disulfide formation by disrupting the thiol/disulfide balance in C6 cells. This study revealed that Con A, known as carbohydrate-binding protein, generated oxidative damage, inflammation, and apoptosis in a dose-dependent manner by modulating thiol/disulfide homeostasis in C6 glioblastoma cells.

Human Galectin-1 in Multiple Cancers: A Privileged Molecular Target in Oncology

Galectin-1 (Gal-1), a 14kDa carbohydrate-binding protein of the galectin family found in humans affects intracellular signaling pathways upon interaction with β-galactosides on cell-surface, cytosol, and nucleus. The structural information reveals that it consists of a monovalent dimer composed of subunits with one Carbohydrate Recognition Domain (CRD), which is the main active site to interact with various glycoproteins, and carbohydrates in the body to regulate cellular functions. Gal-1 contributes towards different events associated with cancer biology, including tumor transformation, cell cycle regulation, apoptosis, cell adhesion, migration, and inflammation. The extracellular existence and function of Gal-1 have been well-established, and it is known to express in many tumor types, including astrocytoma, melanoma, prostate, colon, bladder, and ovarian carcinomas, etc. Several studies suggested the upregulation of Gal-1 levels in multiple cancer cells. Thus, Gal-1 is a promising molecular target for the development of new therapeutic tools. The present review focuses on the functions of Gal-1 in tumor progression in multiple cancers and structural insights.

Label-Free, Multiplex Glycan Microarray Biosensor for Influenza Virus Detection.

Newly emerging influenza viruses adapted from animal species pose significant pandemic threats to public health. An understanding of hemagglutinin (HA) receptor-binding specificity to host receptors is key to studying the adaptation of influenza viruses in humans. This information may be particularly useful for predicting the emergence of a pandemic outbreak. Therefore, high-throughput sensing technologies able to profile HA receptor binding can facilitate studies of influenza virus evolution and adaptation in humans. As a step toward this goal, we have prepared glycan-based receptor analogue microarrays on the Arrayed Imaging Reflectometry (AIR) platform. These arrays demonstrate label-free, multiplex detection and discrimination between human and avian influenza viruses. Microarrays consisting of glycan probes with 2,6 and 2,3 linkages were prepared. After first confirming their ability to capture lectins (carbohydrate-binding proteins) with known specificities, we observed that the arrays were able to discriminate between and quantify human pandemic influenza A/California/07/2009 (H1N1pdm) and avian A/Netherlands/1/2000 (H13N8) influenza viruses, respectively. As the method may be expanded to large numbers of glycans (>100) and virus subtypes (H1-H18), we anticipate it can be applied to systematically evaluate influenza virus adaptation in humans. In turn, this will facilitate global influenza surveillance and serve as a new tool enabling health organizations, governments, research institutes, and laboratories to react quickly in the face of a pandemic outbreak.

In silico characterization and expression analysis of eight C-type lectins in obscure puffer Takifugu obscurus

C-type lectins (CTLs) are a group of carbohydrate-binding proteins that play crucial roles in innate immune defense against invading pathogens. CTLs have been extensively studied in lower vertebrates, such as fish, for their roles in eliminating pathogens; however, their homologs in pufferfish are not well known. In the present study, eight CTLs from obscure puffer Takifugu obscurus (designated as ToCTL3–10 according to the order they were discovered) were obtained. All predicted ToCTL proteins contained a single carbohydrate recognition domain (CRD). ToCTL7 also contained one calcium-binding epidermal growth factor (EGF)-like domain (EGF_CA) and a transmembrane region. ToCTL9 also contained an SCP domain, an EGF domain, and an EGF-like domain. Bioinformatics analysis revealed that ToCTL3–10 mainly clustered with the corresponding CTL homologs of other pufferfish species. Tissue distribution analysis detected ToCTL3–10 in all tissues examined, including kidneys, liver, gills, spleen, intestines, and heart. Moreover, the expressions of ToCTL3–10 were significantly induced in the kidneys of obscure puffer following challenges with three Gram-negative bacterial pathogens, namely, Vibrio harveyi, Aeromonas hydrophila, and Edwardsiella tarda, and a synthetic analog of double-stranded RNA poly(I:C). The expression patterns of ToCTL3–10 in response to different immune stimulants were different. Our results indicated that the eight ToCTLs obtained herein might be involved in host defense against bacterial and poly(I:C) infections in T. obscurus.

Biochemical and structural characterization of a recombinant fibrinogen-related lectin from Penaeus monodon

Fibrinogen-related lectins are carbohydrate-binding proteins of the innate immune system that recognize glycan structures on microbial surfaces. These innate immune lectins are crucial for invertebrates as they do not rely on adaptive immunity for pathogen clearance. Here, we characterize a recombinant fibrinogen-related lectin PmFREP from the black tiger shrimp Penaeus monodon expressed in the Trichoplusia ni insect cell. Electron microscopy and cross-linking experiments revealed that PmFREP is a disulfide-linked dimer of pentamers distinct from other fibrinogen-related lectins. The full-length protein binds N-acetyl sugars in a Ca2+ ion-independent manner. PmFREP recognized and agglutinated Pseudomonas aeruginosa. Weak binding was detected with other bacteria, including Vibrio parahaemolyticus, but no agglutination activity was observed. The biologically active PmFREP will not only be a crucial tool to elucidate the innate immune signaling in P. monodon and other economically important species, but will also aid in detection and prevention of shrimp bacterial infectious diseases.

Systemic Galectin-3 in Smokers with Chronic Obstructive Pulmonary Disease and Chronic Bronchitis: The Impact of Exacerbations.

PurposeThe carbohydrate-binding protein Galectin-3 is increased in several inflammatory diseases and has recently been forwarded as a systemic biomarker in chronic obstructive pulmonary disease (COPD). In this longitudinal study, we characterized the level of systemic Galectin-3 using blood from smokers with a history of COPD and chronic bronchitis (COPD-CB), during stable clinical conditions and exacerbations.Patients and MethodsThe study population comprised 56 long-term smokers with COPD-CB, 10 long-term smokers without lung disease (LTS) and 10 clinically healthy never-smokers (HNS). Blood samples were analyzed for levels of Galectin-3, leukocyte populations and C-reactive protein (CRP). In addition, sputum samples from the COPD-CB group were analyzed for bacterial growth.ResultsWhen comparing stable clinical conditions and exacerbations in the COPD-CB group, we found that the level of Galectin-3, just like that of CRP, leukocytes and neutrophils, respectively, was increased during exacerbations. However, this exacerbation-associated increase of Galectin-3 was modest. During stable clinical conditions of COPD-CB, the level of Galectin-3 was not elevated in comparison with HNS or LTS. Nor did this level of Galectin-3 distinguish patients that remained in a clinically stable condition throughout the study to those that developed an exacerbation. In addition, neither during stable clinical conditions nor during exacerbations, did the presence of bacterial growth in sputum alter Galectin-3 levels. In contrast to Galectin-3, the level of CRP, leukocytes and neutrophils, respectively, were increased during clinical stable conditions in the COPD-CB group compared with the other groups and were further enhanced during exacerbations.ConclusionSystemic Galectin-3 is increased in a reproducible but modest manner during exacerbations in smokers with COPD-CB. During stable clinical conditions, the level of systemic Galectin-3 does not distinguish patients that remain clinically stable from those that develop exacerbations. This makes it less likely that systemic Galectin-3 may become a clinically useful biomarker in the current setting.

Induction of CXCL10-Mediated Cell Migration by Different Types of Galectins.

Chemokines are an extended group of chemoattractant cytokines responsible for the recruitment of leukocytes into tissues. Among them, interferon-γ-inducible protein 10 (CXCL10) is abundantly expressed following inflammatory stimuli and participates in the trafficking of monocytes and activated T cells into sites of injury. Here, we report that different members of the galectin family of carbohydrate-binding proteins promote the expression and synthesis of CXCL10 independently of interferon-γ. Interestingly, CXCL10 induction was observed when galectins came in contact with stromal fibroblasts isolated from human cornea but not other cell types such as epithelial, monocytic or endothelial cells. Induction of CXCL10 by the tandem repeat galectin-8 was primarily associated with the chemotactic migration of THP-1 monocytic cells, whereas the prototype galectin-1 promoted the CXCL10-dependent migration of Jurkat T cells. These results highlight the potential importance of the galectin signature in dictating the recruitment of specific leukocyte populations into precise tissue locations.

A C-type lectin (OmCTL) in Onychostoma macrolepis: Binding ability to LPS, PGN and agglutinating activity against bacteria

C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins that mainly bind to carbohydrate-based or other ligands to mediate cell adhesion, recognize pathogens, and play important roles in the immune system. In the present study, a novel C-type lectin (OmCTL) isolated from Onychostoma macrolepis was investigated. The open reading frame of OmCTL comprises 468 bp, encoding a 155 amino acid polypeptide with an 18 amino acid putative signaling peptide. The predicted primary OmCTL structure contains a signal peptide, a single carbohydrate recognition domain (CRD) and an EPN/WND motif required for carbohydrate-binding specificity. Using tissue expression pattern analysis, OmCTL has been shownto be highly expressed in the liver, and is also detected in other tissues. OmCTL was significantly upregulated in the liver and spleen following infection with Aeromonas hydrophila, suggesting its involvement in immune response. The recombinant OmCTL protein (rOmCTL) agglutinated two gram-negative bacteria, Escherichia coli and A. hydrophila, in vitro in the presence of Ca2+, showing that it is a typical Ca2+-dependent carbohydrate-binding protein.Furthermore, rOmCTL purified from E. coli BL21 (DE3) strongly bound to LPS and PGN, as well as all tested bacteria in a Ca2+-independent manner. These results indicate that OmCTL plays a central role in the innate immune response and as a pattern recognition receptor that recognizes diverse pathogens among O. macrolepis.

40 years of glyco-polyacrylamide in glycobiology.

Polyacrylamide conjugates of glycans have long been widely used in many research areas of glycobiology, mainly for immobilizing glycans in solid-phase assays and as multivalent inhibitors. Pending biotin tag allows immobilizing Glyc-PAA quantitatively on any surface, and acts as a tracer for detection of carbohydrate-binding proteins. However, the scope of already realized capabilities of these probes is immeasurably richer than those listed above. This review is not so much about routine as about less common, but not less significant applications. Also, the data on the glycopolymers themselves, their molecular weight, size and polymer chain flexibility are presented, as well as the methods of synthesis, clusterisation and entropy factor in their interaction with proteins.

Genomic Organization and Transcriptional Regulation of Two Homologous Fucolectin Genes AjFTL-1 and AjFTL-2 in Apostichopus japonicus

F-type lectins (Fucolectins) are carbohydrate-binding proteins and play important roles in innate immune responses against pathogenic microbial invasion. In our previous research, we found that two homologous Fucolectin genes, AjFTL-1 and AjFTL-2, exhibited different expression profiles after lipopolysaccharides (LPS) challenge in Apostichopus japonicus. However, the transcriptional regulation mechanism of these two genes remains largely unknown. In this study, the 5′ flanking regions of AjFTL-1 and AjFTL-2 genes were cloned and the promoter activities were studied in epithelioma papulosum cyprinid (EPC) cell system. First, in silico analysis indicated that these two promoters both contain numerous putative transcription factor binding sites including NF-κB, CREB, and CREBP1, and both contain a TATA box. Additionally, luciferase assay and progressive 5′ truncation analysis revealed that AjFTL-1 and AjFTL-2 both possess high promoter activities in EPC cells. Moreover, the luciferase activity of AjFTL-1 promoter was significantly regulated by peptidoglycan (PGN) and mannan (MAN), while AjFTL-2 promoter was prominently regulated by LPS and MAN, indicating AjFTL-1 and AjFTL-2 genes showed different transcriptional regulation pattern under different immune stimulation. More importantly, analyses of the functional promoter regions revealed the presence of two potential NF-κB binding sites (−769 bp to −761 bp, −185 bp to −172bp) in AjFTL-1 and one potential binding site (−530 to −517bp) in AjFTL-2. Different truncated reporter vectors and expression vector co-transfection revealed that transcription factor NF-κB/Rel could significantly increase the AjFTL-2 promoter activity, but not AjFTL-1 promoter activity. These findings indicated that in marine invertebrates, different Fucolectin members differ in transcription regulations and expression patterns, and might play different roles in immune defenses during pathogen infection.

Targeted disruption of pi\u2013pi stacking in Malaysian banana lectin reduces mitogenicity while preserving antiviral activity

Lectins, carbohydrate-binding proteins, have been regarded as potential antiviral agents, as some can bind glycans on viral surface glycoproteins and inactivate their functions. However, clinical development of lectins has been stalled by the mitogenicity of many of these proteins, which is the ability to stimulate deleterious proliferation, especially of immune cells. We previously demonstrated that the mitogenic and antiviral activities of a lectin (banana lectin, BanLec) can be separated via a single amino acid mutation, histidine to threonine at position 84 (H84T), within the third Greek key. The resulting lectin, H84T BanLec, is virtually non-mitogenic but retains antiviral activity. Decreased mitogenicity was associated with disruption of pi–pi stacking between two aromatic amino acids. To examine whether we could provide further proof-of-principle of the ability to separate these two distinct lectin functions, we identified another lectin, Malaysian banana lectin (Malay BanLec), with similar structural features as BanLec, including pi–pi stacking, but with only 63% amino acid identity, and showed that it is both mitogenic and potently antiviral. We then engineered an F84T mutation expected to disrupt pi–pi stacking, analogous to H84T. As predicted, F84T Malay BanLec (F84T) was less mitogenic than wild type. However, F84T maintained strong antiviral activity and inhibited replication of HIV, Ebola, and other viruses. The F84T mutation disrupted pi–pi stacking without disrupting the overall lectin structure. These findings show that pi–pi stacking in the third Greek key is a conserved mitogenic motif in these two jacalin-related lectins BanLec and Malay BanLec, and further highlight the potential to rationally engineer antiviral lectins for therapeutic purposes.

Examination of oestrus-dependent alterations of bovine cervico-vaginal mucus glycosylation for potential as optimum fertilisation indicators.

Oestrus is the period in the sexual cycle of female mammals where they become most receptive to mating and are most fertile. Efficient detection of oestrus is a key component in successful reproductive livestock management programmes. Oestrus detection in cattle is most often performed by visual observation, such as mounting behaviour and standing heat, to facilitate more successful prediction of optimal time points for artificial insemination. This time-consuming method requires a skilled, diligent observer. Biological measurements using easily accessible biomolecules in the cervico-vaginal mucus could provide an alternative strategy to physical methods of oestrus detection, providing an inexpensive means of rapidly and accurately assessing the onset of oestrus. In this study, glycosylation changes in cervico-vaginal mucus from three heifers following oestrus induction were investigated as a proof of concept to assess whether potential glycosylation-based trends could be useful for oestrus stage indication. Mucus collected at different time points following oestrus induction was immobilised in a microarray format and its glycosylation interrogated with a panel of fluorescently labelled lectins, carbohydrate-binding proteins with different specificities. Individual animal-specific glycosylation patterns were observed, however each pattern followed a similar trend around oestrus. This unique oestrus-associated glycosylation was identified by a combination of relative binding of the lectins SNA-I and WFA for each animal. This alteration in cervico-vaginal mucus glycosylation could potentially be exploited in future to more accurately identify optimal fertilisation intervention points compared to visual signs. More effective oestrus biomarkers will lead to more successful livestock reproductive programmes, decreasing costs and animal stress.

Mucin-Type O-GalNAc Glycosylation in Health and Disease.

Mucin-type GalNAc O-glycosylation is one of the most abundant and unique post-translational modifications. The combination of proteome-wide mapping of GalNAc O-glycosylation sites and genetic studies with knockout animals and genome-wide analyses in humans have been instrumental in our understanding of GalNAc O-glycosylation. Combined, such studies have revealed well-defined functions of O-glycans at single sites in proteins, including the regulation of pro-protein processing and proteolytic cleavage, as well as modulation of receptor functions and ligand binding. In addition to isolated O-glycans, multiple clustered O-glycans have an important function in mammalian biology by providing structural support and stability of mucins essential for protecting our inner epithelial surfaces, especially in the airways and gastrointestinal tract. Here the many O-glycans also provide binding sites for both endogenous and pathogen-derived carbohydrate-binding proteins regulating critical developmental programs and helping maintain epithelial homeostasis with commensal organisms. Finally, O-glycan changes have been identified in several diseases, most notably in cancer and inflammation, where the disease-specific changes can be used for glycan-targeted therapies. This chapter will review the biosynthesis, the biology, and the translational perspectives of GalNAc O-glycans.