Lectin

Lectins was extracted from processed and unprocessed black-eyed peas, green beans, soybeans, peanuts, and red kidney beans by grinding using attrition mill and then soaked in distilled water. The proteins in the supernatant were precipitated by dissolving 10g of ammonium sulphate in 100ml of the supernatants. The lectin extracts were evaluated for hemagglutinin activity using human erythrocytes from blood group A + , B + , AB + , and O + , respectively. Result revealed that lectins from unprocessed soybean, black-eyed pea and green bean are blood group selective and may be classified as blood type specific lectins. While lectins from kidney beans and peanuts agglutinated all blood groups and may be classified as panhemagglutinin lectins, i.e. they agglutinated erythrocytes from all blood groups. Result shows that blood group O is less affected by hemagglutinin activity of lectin. Reduction/loss of hemagglutinin activity was observed with reduction in concentration of the crude lectin extracts in all unprocessed legumes. Boiling (at 100 0 C) for 30minutes completely inactivated hemagglutinin activity of lectin in Black eyed peas, soybeans, green beans and red kidney beans. Slight hemagglutinin activity was seen in lectin from roasted peanuts, indicating that dry heating may be very effective method to inactivate lectin completely. Also, in this study, lectins were extracted from rice (Oryza sativa), maize (Zea mays), sorghum (Sorghum bicolor), Acha (Digitaria excells), millet (Panicum maniceum); partial purification of the extract assayed from hemugglutinin activity using 4% of human erythrocyte in lectin buffer. Extract from these selected cereals showed positive and negative agglutination reaction with blood group A + , B + , O + and AB + depending on the concentration (dilution) and the blood group. Lectin extract from maize showed no agglutination reaction when diluted in blood group A + , B + , and AB + and even when processed and diluted. While crude lectin extract from rice showed no activity in blood group A + , B + , and AB + and when unprocessed and showed lectin agglutination activity when processed by boiling (cooking) showing that heat may encourage its activity. Blood group B + , and AB + showed no activity in all dilution. Also acha showed high agglutination activity in blood group A + , B + , AB + and O + but losses activity in dilution a 2 and a 3. Crude lectin extract from processed sorghum showed

Herein we will review the role of glycans in the immune system. Specific topics covered include: the glycosylation sites of IgE, IgM, IgD, IgE, IgA, and IgG; how glycans can encode “self” identity by functioning as either danger associated molecular patterns (DAMPs) or self-associated molecular patterns (SAMPs); the role of glycans as markers of protein integrity and age; how the glycocalyx can dictate the migration pattern of immune cells; and how the combination of Fc N-glycans and Ig isotype dictate the effector function of immunoglobulins. We speculate that the latter may be responsible for the well-documented association between alterations of the serum glycome and autoimmunity. Due to
technological limitations, the extent of these autoimmune-associated glycan alterations and their role in disease pathophysiology has not been fully elucidated. Thus, we also review the current technologies available for glycan analysis, placing an emphasis on Multiple Reaction Monitoring (MRM), a rapid high-throughput technology that has great potential for glycan biomarker research. Finally, we put forth The Altered Glycan Theory of Autoimmunity, which states that each autoimmune disease will have a unique glycan signature characterized by the site-specific relative abundances of individual glycan structures on immune cells and extracellular proteins, especially the site-specific glycosylation patterns of the different immunoglobulin(Ig) classes and subclasses.

G lucose is an important biomolecule because it is an important source of energy for cells and is used as an intermediate/ metabolic agent. Selective recognition of glucose is important for diagnosing many metabolic diseases. In this study, lectin ligand (Con A) attached p(GMA) nanopolymer was synthesized and characterized. The best interaction of p(GMA)ConA nanopolymer and glucose was determined to be 10 mM glucose concentration at pH = 8.0. In the selectivity assay, p(GMA)-Con A was found to be 2-fold selective for glucose than galactose. Lectin affinity based nanopolymeric system that is selective, with high surface area, low cost and highly biocompatible with high adsorption capacity has been developed for recognition of glucose.

Objectives: To compare the plasma protein profiles of treated and untreated congenital hypothyroidism (CH) patients with those of normal control infants. Design and methods: Plasma samples were subjected to two-dimensional gel electrophoresis and silver staining or lectin detection. Resolved protein spots were analyzed by using computerized densitometry software. Results: The significant enhanced expression of the fibrinogen gamma-chain and reduced expression of haptoglobin beta-chain were demonstrated in the profiles of untreated CH patients using both silver staining and lectin detection methods. Lectin detection resolved only a single cluster of the haptoglobin beta-chain for the untreated patients, in contrast to five isoform clusters detected in the controls' and treated CH patients' profiles. Conclusions: Plasma from untreated CH patients demonstrated different altered expression of fibrinogen and haptoglobin polypeptide chains, which was normalized when patients were treated. Our data also suggest differences in structures of the N-glycans of haptoglobin beta-chain of the untreated CH patients. (c) 2005 The Canadian Society of Clinical Chemists. All rights reserved.

Background: ‍‍Cryopreservation has some detrimental impacts on sperms surface molecules. Modification of the sperm surface molecules can affect on fertility rate. One of the important surface molecules are glycoconjugates. Objective: The objective of this study was to evaluate the changes of content of the glycocalyx after standard cryopreservation procedure. Materials and Methods: Forty five healthy semen samples were frozen in 0.5ml plastic straws and kept in liquid nitrogen and thaw after 48 hours. Sperm smears were prepared before and after freezing and thawing. The smears were stained with the lectins and also with acridin orange. The smears were studied by fluorescents microscopy and the intensities of the reactions to lectins were measured by image analyses software. Results: The reactions of the sperm samples to Peanut agglutinin (PNA) Wheat germ agglutinin (WGA) and Dolichos biflorus (DBA) changed after cryopreservation and the percentage of samples that showed modification...

The survival in the rat small intestine of soybean agglutinin or Kunitz or Bowman-Birk trypsin inhibitors was studied by SDS-and native polyacrylamide gel electrophoresis, transblotting, trypsin inhibitor activity assays, and immunochemical determinations. As the inhibitors were bound in enzyme complexes in the small intestine, functional assays were unsuitable. However, as free and bound inhibitors reacted similarly with their antibodies, survival could be assayed by immunoblotting and ELISA. In addition to the mucosa-bound agglutinin, 8.6% of the original dose was free in the gut lumen and this, by moving further down in the small intestine, could have extended the wasteful gut growth from proximal to distal parts. Although only 4.8% of the Bowman-Birk inhibitor survived, most (76%) of the Kunitz inhibitor remained immunochemically intact. Accordingly, stimulation of pancreatic growth and enzyme secretion by the inhibitors, particularly the Kunitz, may have contributed to the total antinutritive effect of soybean.

Lectins are a class of ubiquitous proteins/glycoproteins that are abundantly found in nature. Lectins have unique carbohydrate binding property and hence have been exploited as drugs against various infectious diseases. We have isolated one such novel lectin from the fruit pulp of Aegle marmelos. The isolated lectin was partially characterised and its effect against Shigella dysenteriae infection was evaluated. The isolated lectin was found to be a dimeric protein with N-acetylgalactosamine, mannose and sialic acid binding specificity. The effect of Aegle marmelos fruit lectin on the adherence of Shigella dysenteriae to human colonic epithelial cells (HT29 cells) was evaluated by Enzyme Linked Immune Sorbent Assay and invasion was analysed. The protective nature of the Aegle marmelos fruit lectin was assessed by analyzing apoptosis through dual staining method. Aegle marmelos fruit lectin significantly inhibited hemagglutination activity of Shigella and its minimum inhibitory concentration is 0.625 µg/well. Further, at this concentration lectin inhibited Shigella dysenteriae adherence and invasion of HT29 cells and protects the HT29 cells from Shigella dysenteriae induced apoptosis. To conclude, isolated lectin dimeric protein with N-acetylgalactosamine, Mannose and sialic acid binding specificity and inhibits adherence and invasion of Shigellae to HT29 cells thus, protects the host.

Diagnosis of prostate cancer (PCa) is currently much reliant on the invasive and time-consuming transrectal ultrasound-guided biopsy of the prostate gland, particularly in light of the inefficient use of prostate-specific antigen as its biomarker. In the present study, we have profiled the sera of patients with PCa and benign prostatic hyperplasia (BPH) using the gel- and lectin-based proteomics methods and demonstrated the significant differential expression of apolipoprotein AII, complement C3 beta chain fragment, inter-alpha-trypsin inhibitor heavy chain 4 fragment, transthyretin, alpha-1-antitrypsin, and high molecular weight kininogen (light chain) between the two groups of patients’ samples. Our data are suggestive of the potential use of the serum proteins as complementary biomarkers to effectively discriminate PCa from BPH, although this requires further extensive validation on clinically representative populations.

Our group has previously reported the isolation, partial characterisation, and application of a Galbeta1-3GalNAc- and IgA1-reactive lectin from the seeds of champedak (Artocarpus integer). In the present study, we have subjected the purified lectin to reverse-phase high performance liquid chromatography and sequenced its subunits. Determination of the N-terminal sequence of the first 47 residues of the large subunit demonstrated at least 95% homology to the N-terminal sequence of the alpha chains of a few other galactose-binding Artocarpus lectins, The two smaller subunits of the lectin, each comprised of 21 amino acid residues, demonstrated minor sequence variability. Their sequences were generally comparable to the beta chains of the other galactose-binding Artocarpus lectins. When used to probe human serum glycopeptides that were separated by two-dimensional gel electrophoresis, the lectin demonstrated strong apparent interactions with glycopeptides of IgA1, hemopexin, alpha(2)-HS glycoprotein, alpha(1)-antichymotrypsin, and a few unknown glycoproteins. Immobilisation of the lectin to Sepharose generated an affinity column that may be used to isolate the O-glycosylated serum glycoproteins. (C) 2002 Elsevier Science (USA). All rights reserved.

Envenomation of humans by snakes is a complex and continuously evolving medical emergency, and treatment is made that much more difficult by the diverse biochemical composition of many venoms. Venomous snakes and their venoms also provide models for the study of molecular evolutionary processes leading to adaptation and genotype-phenotype relationships. To compare venom complexity and protein sequences, venom gland transcriptomes are assembled, which usually requires the sacrifice of snakes for tissue. However, toxin transcripts are also present in venoms, offering the possibility of obtaining cDNA sequences directly from venom. This study provides evidence that unknown full-length venom protein transcripts can be obtained from the venoms of multiple species from all major venomous snake families. These unknown venom protein cDNAs are obtained by the use of primers designed from conserved signal peptide sequences within each venom protein superfamily. This technique was used to asse...

A new lectin (BvcL) from seeds of a primitive Brazilian Caesalpinoideae, the Bauhinia variegata candida was purified and biochemical characterized. BvcL was isolated by gel filtration chromatography on Sephadex G75 and affinity chromatography on immobilized d-lactose column. SDS-PAGE showed that BvcL under non-reducing condition presents two bands of 68 and 32 kDa and a single band of 32 kDa in reducing condition. However, only one band was seen in native PAGE. The hemagglutination activity of BvcL was not specific for any human blood group trypsin-treated erythrocytes. Carbohydrate inhibition analysis indicated that BvcL is inhibited by lactose, galactose, galactosamine and other galactoside derivates. Amino acid analysis revealed a large content of Ser, Gly, Thr, Asp and Glu and low concentrations of Met, Cys and His. Intrinsic fluorescence of BvcL was not significantly affected by sugar binding galactose; and aromatic-region CD is unusually high for plant lectins. The N-terminal ...

Blood flow acts parallel to the coronary luminal endothelial surface layer (LESL) and modulates multiple parenchymal functions via the release of paracrine agents. Evidence indicates that the LESL may be a flow-sensing organelle and that perhaps through flow-induced lectin (L)·oligosaccharide (O) complex formation (L·O) participates in this process. LESL integrins and selectins are both lectinic and flow sensitive, but the L properties of flow-sensitive G protein-coupled receptors (GPCRs) are unknown. Therefore, we investigated the presence of L in the LESL and hypothesized that if flow-sensitive GPCRs are L, flow and O will determine their response to receptor activation. The LESL protein fraction isolated from guinea pig hearts was passed through an affinity chromatography column made of three sugars, mannose, galactose, and N-acetylglucosamine, and the lectinic fraction was eluted. Immune dot blot was used to identify L proteins in the LESL fraction. Our results indicate the foll...

Mannose-binding lectin (MBL) is a protein of the innate immune system that participates in host defense and the tissue injury/repair process, enhancing the clearance of apoptotic cells by macrophages. The aim is to characterize the relationship between pre-transplant MBL levels, histological lesions and number of apoptotic cells in early surveillance renal allograft biopsies. Consecutive renal transplant recipients were recruited and MBL levels were classified into tertiles. The first tertile was considered the low MBL group. Surveillance biopsies were done during the first 6 months and were evaluated according to Banff criteria. Renal inflammatory infiltrates were studied by immunohistochemical techniques. Apoptosis was studied using morphological methods in renal tubular cells and was expressed as the number of apoptotic cells/mm(2). MBL was determined in 126 patients and a surveillance biopsy with sufficient tissue was obtained in 41 of them. Patients with low pre-transplant MBL ...

Introduction Recognition between proteins and carbohydrates is of prime importance in many biological processes. Legume lectins are well-studied proteins because they are not only easy to purify in large quantities but also exhibit a wide variety of carbohydrate specificities despite strong sequence conservations. But lectins from algae are quite hard to purify and even its existence was not known for a long time. For example, the review of “Lectin”, which was published in 1986, did not describe lectins from algae. Hori et al. first succeeded the purification of algae lectin, and now the number of species is over 20. Lectin from algae was also found to have the anti-cancer effect in the medical experiment for the mouse.

Protein glycosylation is one of the most studied post-translational modifications and has received considerable attention for its critical role in the cell biology of eukaryotic cells. The genus Trichoderma has been extensively studied in the biocontrol of soil-borne fungal phytopathogens. The aim of this study was to identify the proteins secreted from Trichoderma harzianum after interacting with the cell walls of two phytopathogens, Sclerotinia sclerotiorum and Fusarium oxysporum. This study used N-glycoprotein enrichment with a concanavalin A (Con A) affinity column, staining detection differential SDS-PAGE, sequencing by mass spectrometric, and protein identification by comparison with the NCBI database to detect the protein expression of the two resulting secretome samples. The majority of the proteins found in both enriched secretomes belonged to a specific class of carbohydrate-active enzymes (CAZymes), within which glycosyl hydrolases (GHs), glycosyltransferases (GTs), and a...

Background: ‍ ‍ Cryopreservation has some detrimental impacts on sperms surface molecules. Modification of the sperm surface molecules can affect on fertility rate. One of the important surface molecules are glycoconjugates. Objective: The objective of this study was to evaluate the changes of content of the glycocalyx after standard cryopreservation procedure. Materials and Methods: Forty five healthy semen samples were frozen in 0.5ml plastic straws and kept in liquid nitrogen and thaw after 48 hours. Sperm smears were prepared before and after freezing and thawing. The smears were stained with the lectins and also with acridin orange. The smears were studied by fluorescents microscopy and the intensities of the reactions to lectins were measured by image analyses software. Results: The reactions of the sperm samples to Peanut agglutinin (PNA), Wheat germ agglutinin (WGA) and Dolichos biflorus (DBA) changed after cryopreservation and the percentage of samples that showed modificat...

We have developed a simple enzyme-linked immunosorbent assay (ELISA) for the direct determination of chitosan-like polysaccharide (CP) produced in the cultures of Citrobacter strains. CP in sample solutions was reacted with anti-CP rabbit polyclonal antibody immobilized in a 96-well microtiter plate and sandwiched with horseradish peroxidase-conjugated lectin, followed by color development by the enzyme activities. Using standard chitosan solutions, a good standard curve
(R2 =0.977) was established between 0 and 250 μg l-1 with the average coefficient of variation of 7.0%, and CP in the diluted culture supernatants could be directly determined. This method can apply for the direct determination of other polysaccharides in solution by selecting an inexpensive custom-made polyclonal antibody and an appropriate lectin, both of which may be commercially available.

ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs), as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergis-tically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4 + T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4 + T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4 + T cells under ArtinM stimulus augmented the expression of TGF-β mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4 + T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4 + T cells were pre-incubated with anti-CD3 antibody. In conclusion , ArtinM stimulates the production of IL-17 by CD4 + T cells in two major ways: (I) through the induction of IL-23 and IL-1 by APCs and (II) through the direct interaction with CD3 on the CD4 + T cells. This study contributes to elucidation of mechanisms accounting for the property of ArtinM in inducing Th17 immunity and opens new perspectives in designing strategies for modulating immunity by using carbohydrate recognition agents.

We investigated the effects of a calcium-dependent mannose-binding lectin isolated from the serum of Atlantic salmon on
Aeromonas salmonicida viability and the anti-A. salmonicida activity of Atlantic salmon macrophages. In the absence of other factors, binding of this lectin at concentrations of 0.8, 4.0 and 20.0 ng ml1 to virulent A. salmonicida failed to significantly reduce
(P\0.05) cell viability. However, binding of the lectin to A. salmonicida did result in significant (P50.05) dose-dependent
increases in phagocytosis, and bactericidal activity. Significant increases (P50.05) were also observed in phagocyte respiratory
burst activity within the lectin concentration range of 4.0–20.0 ng ml1 but the stimulation was not dose dependent at these lectin concentrations. At the lowest lectin concentration tested (0.32 ng ml1), a significant decrease (P50.05) in respiratory burst was observed. The structure and activity of this lectin are similar to that of mammalian mannose-binding lectins, which are known to play a pivotal role in innate immunity. The presence of this lectin may be an important defense mechanism against Gram-negative bacteria such as A. salmonicida.

The expression of high-abundance serum proteins in newly diagnosed patients with endometrial adenocarcinoma (EACa), squarnous cell cervical carcinoma (SCCa) and cervical adenocarcinorna (ACCa), relative to control female subjects, was analyzed by subjecting serum samples to 2-DE followed by image analysis of the silver-stained protein profiles. The three cohorts of cancer patients demonstrated different altered expression of serum high-abundance proteins compared to negative control women. Tl-ie expression of alpha(1)-antitrypsin, alpha(1)-B glycoprotein, cleaved high-molecular-weight kininogen (light chain) and antithrombin III were consistently altered in all the patients. However, clusterin was upregulated only in the patients with EACa, while those with SCCa and ACCa were typically characterized by the upregulated expression of zinc alpha-2-glycoprotein. The aberrant expression of selective serum proteins in the various cohorts of cancer patients was validated by competitive ELISA as well as by lectin detection. Analysis by using the charnpedak galactose binding lectin further highlighted an unidentified protein that may be differently glycosylated in the sera of the EACa patients that were studied.

A mannose-binding lectin, termed champedak lectin-M, was isolated from an extract of the crude seeds of champedak (Artocarpus integer). On gel filtration chromatography, the lectin eluted in a single peak at elution volumes corresponding to 64 kDa, SDS-PAGE showed the mannose-binding lectin to be composed of 16.8 kDa polypeptides with some of the polypeptides being disulphide-linked to give dimers. When tested with all isotypes of immunoglobulins, champedak lectin-M demonstrated a selective strong interaction with human IgE and IgM, and a weak interaction with IgA2, The binding interactions of lectin-M were metal ion independent. The lectin was also shown to interact with horseradish peroxidase, ovalbumin, porcine thyroglobulin, human alpha(1)-acid glycoprotein, transferrin and alpha(1)-antitrypsin. It demonstrated a binding preference to Man alpha 1-3Man ligands in comparison to Man alpha 1-6Man or Man alpha 1-2Man. (C) 1997 Elsevier Science B.V.