Antimicrobial Studies on Garlic Lectin.

Allium sativum agglutinin (ASA) is an important lectin isolated from garlic bulbs and has shown promising therapeutic potential in earlier reports. It has a bulb-type lectin domain, and members of this protein family have been investigated for anti-cancer, antimicrobial and other effects. In our earlier study, we have reported ASA as an anti-cancer agent, and in the present study, we have evaluated it for its antifungal and antimicrobial effects. The effects of ASA on the opportunistic pathogens in humans Candida auris and Candida glabrata fungal strains have been evaluated, and efforts are made to evaluate the mechanistic basis of these antifungal effects. The antifungal activity of ASA on different strains of C. glabrata and C. auris was found with MIC50 concentration range of 30-70 µg/ml. Fungal growth was significantly suppressed upon treatment with ASA at MIC50 and 2MIC50. Hydrogen peroxide production was detected after ASA treatment in fungal cells and cell morphology, and integrity was affected when analysed through FE-SEM. Further, the anti-biofilm effect of ASA was investigated against Candida and three bacterial strains (Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae), and promising results were obtained with maximal effect in case of K. pneumoniae among the bacterial strains. These results can form the basis for the development of ASA as antimicrobial agent.

Investigations on the Biological Activity of Allium sativum Agglutinin (ASA) Isolated from Garlic.

BACKGROUND: Garlic (Allium sativum) from the family Amaryllidaceae is widely used in culinary and is reported to have potential anticancer, anti-diabetic, antimicrobial, and cardioprotective activities. Allium sativum agglutinin (ASA) is a bulb-type lectin (BTL) domaincontaining lectin isolated from garlic and has been studied for its various biological functions. Previous studies have reported the anti-cancer effects of ASA on histiocytic lymphoma (U937), promyelocytic leukemia (HL60), and oral cancer (KB). METHODS: In this study, we have purified and characterized ASA and evaluated it for its anticancer effects on other cancer cell lines. MTT assay and FACS analysis was done to corroborate the anticancer findings against cervical (HeLa) and lung cancer (A549) cell lines. RESULTS: IC50 value of 37 µg/ml in HeLa and a weak activity (26.4 ± 1.9% cellular inhibition at 100µg/ml treatment) in A549 were found in the MTT assay. FACS analysis further corroborated these findings and showed the apoptotic effects of ASA in these cell lines. CONCLUSION: Anticancer activity for members of bulb-type lectin (BTL) domain-containing lectins has been widely reported, and we hope that our study forms a basis for the development of ASA as a therapeutic agent.

Immunoadjuvant and Humoral Immune Responses of Garlic (Allium sativum L.) Lectins upon Systemic and Mucosal Administration in BALB/c Mice.

Dietary food components have the ability to affect immune function; following absorption, specifically orally ingested dietary food containing lectins can systemically modulate the immune cells and affect the response to self- and co-administered food antigens. The mannose-binding lectins from garlic (Allium sativum agglutinins; ASAs) were identified as immunodulatory proteins in vitro. The objective of the present study was to assess the immunogenicity and adjuvanticity of garlic agglutinins and to evaluate whether they have adjuvant properties in vivo for a weak antigen ovalbumin (OVA). Garlic lectins (ASA I and ASA II) were administered by intranasal (50 days duration) and intradermal (14 days duration) routes, and the anti-lectin and anti-OVA immune (IgG) responses in the control and test groups of the BALB/c mice were assessed for humoral immunogenicity. Lectins, co-administered with OVA, were examined for lectin-induced anti-OVA IgG response to assess their adjuvant properties. The splenic and thymic indices were evaluated as a measure of immunomodulatory functions. Intradermal administration of ASA I and ASA II had showed a four-fold and two-fold increase in anti-lectin IgG response, respectively, vs. the control on day 14. In the intranasal route, the increases were 3-fold and 2.4-fold for ASA I and ASA II, respectively, on day 50. No decrease in the body weights of animals was noticed; the increases in the spleen and thymus weights, as well as their indices, were significant in the lectin groups. In the adjuvanticity study by intranasal administration, ASA I co-administered with ovalbumin (OVA) induced a remarkable increase in anti-OVA IgG response (~six-fold; p < 0.001) compared to the control, and ASA II induced a four-fold increase vs. the control on day 50. The results indicated that ASA was a potent immunogen which induced mucosal immunogenicity to the antigens that were administered intranasally in BALB/c mice. The observations made of the in vivo study indicate that ASA I has the potential use as an oral and mucosal adjuvant to deliver candidate weak antigens. Further clinical studies in humans are required to confirm its applicability.

Inhibition of Bemisia tabaci vectored, GroEL mediated transmission of tomato leaf curl New Delhi virus by garlic leaf lectin (Allium sativum leaf agglutinin).

GroEL or symbionin synthesized by the endosymbionts of whitefly (Bemisia tabaci)/ aphids play a cardinal role in the persistent, circulative transmission of plant viruses by binding to viral coat protein/ read-through protein. Allium sativum leaf agglutinin (ASAL), a Galanthus nivalis agglutinin (GNA)- related mannose-binding lectin from garlic leaf has been reported as a potent controlling agent against hemipteran insects including whitefly and aphids. GroEL related chaperonin- symbionin was previously identified as a receptor of ASAL by the present group in the brush border membrane vesicle (BBMV) of mustard aphid. In the present study similar GroEL receptor of ASAL has been identified through LC-MS/MS in the BBMV of B. tabaci which serves as a vector for several plant viruses including tomato leaf curl New Delhi virus (ToLCNDV). Ligand blot analysis of ASAL-fed B. tabaci showed that when GroEL is pre-occupied by ASAL, it completely blocks its further binding to ToLCNDV coat protein (ToLCNDV-CP). Prior feeding of ASAL hindered the co-localization of ToLCNDV-CP and GroEL in the midgut of B. tabaci. Immunoprecipitation followed by western blot with ASAL-fed B. tabaci yielded similar result. Moreover, ASAL feeding inhibited viral transmission by B. tabaci. Together, these results confirmed that the interaction of ASAL with GroEL interferes with the binding of ToLCNDV-CP and inhibits further B. tabaci mediated viral transmission.

Inferior collicular cells that project to the auditory thalamus are increasingly surrounded by perineuronal nets with age.

The age-related loss of GABA in the inferior colliculus (IC) likely plays a role in the development of age-related hearing loss. Perineuronal nets (PNs), specialized aggregates of extracellular matrix, increase with age in the IC. PNs, associated with GABAergic neurotransmission, can stabilize synapses and inhibit structural plasticity. We sought to determine whether PN expression increased on GABAergic and non-GABAergic IC cells that project to the medial geniculate body (MG). We used retrograde tract-tracing in combination with immunohistochemistry for glutamic acid decarboxylase and Wisteria floribunda agglutinin across three age groups of Fischer Brown Norway rats. Results demonstrate that PNs increase with age on lemniscal and non-lemniscal IC-MG cells, however two key differences exist. First, PNs increased on non-lemniscal IC-MG cells during middle-age, but not until old age on lemniscal IC-MG cells. Second, increases of PNs on lemniscal IC-MG cells occurred on non-GABAergic cells rather than on GABAergic cells. These results suggest that synaptic stabilization and reduced plasticity likely occur at different ages on a subset of the IC-MG pathway.

Oxidation induced by dielectric-barrier discharge (DBD) plasma treatment reduces soybean agglutinin activity.

In this study, potential of dielectric-barrier discharge (DBD) plasma treatment (40 kV, 12 kHz at 1, 2, 3 and 4 min) to eliminate soybean agglutinin (SBA) activity was investigated in a SBA model system and soymilk. The plasma treatment decreased the SBA in the model system and hemagglutination activity was decreased by 87.31%. SDS-PAGE analysis confirmed the degradation of the SBA polypeptide chain. The multi-spectroscopic analysis revealed a two-stage structure alteration in the SBA upon exposure to the plasma treatment. Oxidation of NH-/NH2- at the peptide bond disrupted the hydrogen bonds and altered the secondary structure of SBA. Further oxidation of aromatic amino acid, cleavage of peptide bonds and the breakage of polypeptide led to the SBA fragmentation and complete unfolding of the protein. The SBA inactivation by the plasma treatment was confirmed in soymilk. Plasma treatment is a promising technology for the elimination of SBA in soybean product.

Autoimmune hemolytic anemia: current knowledge and perspectives.

Autoimmune hemolytic anemia (AIHA) is an acquired, heterogeneous group of diseases which includes warm AIHA, cold agglutinin disease (CAD), mixed AIHA, paroxysmal cold hemoglobinuria and atypical AIHA. Currently CAD is defined as a chronic, clonal lymphoproliferative disorder, while the presence of cold agglutinins underlying other diseases is known as cold agglutinin syndrome. AIHA is mediated by autoantibodies directed against red blood cells (RBCs) causing premature erythrocyte destruction. The pathogenesis of AIHA is complex and still not fully understood. Recent studies indicate the involvement of T and B cell dysregulation, reduced CD4+ and CD25+ Tregs, increased clonal expansions of CD8 + T cells, imbalance of Th17/Tregs and Tfh/Tfr, and impaired lymphocyte apoptosis. Changes in some RBC membrane structures, under the influence of mechanical stimuli or oxidative stress, may promote autohemolysis. The clinical presentation and treatment of AIHA are influenced by many factors, including the type of AIHA, degree of hemolysis, underlying diseases, presence of concomitant comorbidities, bone marrow compensatory abilities and the presence of fibrosis and dyserthropoiesis. The main treatment for AIHA is based on the inhibition of autoantibody production by mono- or combination therapy using GKS and/or rituximab and, rarely, immunosuppressive drugs or immunomodulators. Reduction of erythrocyte destruction via splenectomy is currently the third line of treatment for warm AIHA. Supportive treatment including vitamin supplementation, recombinant erythropoietin, thrombosis prophylaxis and the prevention and treatment of infections is essential. New groups of drugs that inhibit immune responses at various levels are being developed intensively, including inhibition of antibody-mediated RBCs phagocytosis, inhibition of B cell and plasma cell frequency and activity, inhibition of IgG recycling, immunomodulation of T lymphocytes function, and complement cascade inhibition. Recent studies have brought about changes in classification and progress in understanding the pathogenesis and treatment of AIHA, although there are still many issues to be resolved, particularly concerning the impact of age-associated changes to immunity.

A case report of severe nasal ischemia from cold agglutinin disease and a novel treatment protocol including HBOT.

Cold agglutinin disease (CAD) is a rare condition leading to blood agglutination and autoimmune hemolytic anemia. Cutaneous ischemia resulting from CAD in the head and neck is uncommon. Treatment regimens and outcomes vary widely in the literature and no clear protocol exists. This manuscript describes a patient with CAD who developed severe ischemia of the nose that resolved completely without sequellae following a medical regimen of aspirin, low molecular weight heparin, nitroglycerin ointment and hyperbaric oxygen therapy (HBOT). To our knowledge, this is the first reported case where nitroglycerin ointment or HBOT was successfully employed in the treatment of this complication.

Parkia platycephala lectin enhances the antibiotic activity against multi-resistant bacterial strains and inhibits the development of Haemonchus contortus.

Lectins have been studied in the past few years as an alternative to inhibit the development of pathogenic bacteria and gastrointestinal nematodes of small ruminants. The development of new antibacterial and anthelmintic compounds is necessary owing to the increase in drug resistance among important pathogens. Therefore, this study aimed to evaluate the capacity of a glucose/mannose-binding lectin from Parkia platycephala seeds (PPL) to inhibit the development of Haemonchus contortus and to modulate antibiotic activity against multi-resistant bacterial strains, thereby confirming its efficacy when used in combination with gentamicin. PPL at the concentration of 1.2 mg/mL did not show inhibitory activity on H. contortus in the egg hatch test or the exsheathment assay. However, it did show significant inhibition of H. contortus larval development with an IC50 of 0.31 mg/mL. The minimum inhibitory concentration (MIC) obtained for PPL against all tested bacterial strains was not clinically relevant (MIC ≥ 1024 µg/mL). However, when PPL was combined with gentamicin, a significant increase in antibiotic activity was observed against S. aureus and E.coli multi-resistant strains. The inhibition of hemagglutinating activity by gentamicin (MIC = 50 mM) revealed that it may be interacting with the carbohydrate-binding site of PPL. It is this interaction between the antibiotic and lectin carbohydrate-binding site that may be responsible for the enhanced activity of gentamicin against multi-resistant strains. It can be concluded that PPL showed selective anthelmintic effect, inhibiting the development of H. contortus larvae and that it increased the effect of the antibiotic gentamicin against multi-resistant bacterial strains, thus constituting a potential therapeutic resource against resistant bacterial strains and H. contortus.

Morphological analysis for neuronal pathway from the hindbrain ependymocytes to the hypothalamic kisspeptin neurons.

Hindbrain ependymocytes are postulated to have a glucose-sensing role in regulating gonadal functions. Previous studies have suggested that malnutrition-induced suppression of gonadotropin secretion is mediated by noradrenergic inputs from the A2 region in the solitary tract nucleus to the paraventricular nucleus (PVN), and by corticotropin-releasing hormone (CRH) release in the hypothalamus. However, no morphological evidence to indicate the neural pathway from the hindbrain ependymocytes to hypothalamic kisspeptin neurons, a center for reproductive function in mammals, currently exists. The present study aimed to examine the existence of a neuronal pathway from the hindbrain ependymocytes to kisspeptin neurons in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). To determine this, wheat-germ agglutinin (WGA), a trans-synaptic tracer, was injected into the fourth ventricle (4V) in heterozygous Kiss1-tandem dimer Tomato (tdTomato) rats, where kisspeptin neurons were visualized by tdTomato fluorescence. 48 h after the WGA injection, brain sections were taken from the forebrain, midbrain and hindbrain and subjected to double immunohistochemistry for WGA and dopamine ß-hydroxylase (DBH) or CRH. WGA immunoreactivities were found in vimentin-immunopositive ependymocytes of the 4V and the central canal (CC), but not in the third ventricle. The WGA immunoreactivities were detected in some tdTomato-expressing cells in the ARC and AVPV, DBH-immunopositive cells in the A1-A7 noradrenergic nuclei, and CRH-immunopositive cells in the PVN. These results suggest that the hindbrain ependymocytes have neuronal connections with the kisspeptin neurons, most probably via hindbrain noradrenergic and CRH neurons to relay low energetic signals for regulation of reproduction.

p73 induction by Abrus agglutinin facilitates Snail ubiquitination to inhibit epithelial to mesenchymal transition in oral cancer.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT), a key step in oral cancer progression, is associated with invasion, metastasis, and therapy resistance, thus targeting the EMT represents a critical therapeutic strategy for the treatment of oral cancer metastasis. Our previous study showed that Abrus agglutinin (AGG), a plant lectin, induces both intrinsic and extrinsic apoptosis to activate the tumor inhibitory mechanism. OBJECTIVE: This study aimed to investigate the role of AGG in modulating invasiveness and stemness through EMT inhibition for the development of antineoplastic agents against oral cancer. METHODS: The EMT- and stemness-related proteins were studied in oral cancer cells using Western blot analysis and fluorescence microscopy. The potential mechanisms of Snail downregulation through p73 activation in FaDu cells were evaluated using Western blot analysis, immunoprecipitation, confocal microscopy, and molecular docking analysis. Immunohistochemical staining of the tumor samples of AGG-treated FaDu-xenografted nude mice was performed. RESULTS: At the molecular level, AGG-induced p73 suppressed Snail expression, leading to EMT inhibition in FaDu cells. Notably, AGG promoted the translocation of Snail from the nucleus to the cytoplasm in FaDu cells and triggered its degradation through ubiquitination. In this setting, AGG inhibited the interaction between Snail and p73 in FaDu cells, resulting in p73 activation and EMT inhibition. Moreover, in epidermal growth factor (EGF)-stimulated FaDu cells, AGG abolished the upregulation of extracellular signal-regulated kinase (ERK)1/2 that plays a pivotal role in the upregulation of Snail to regulate the EMT phenotypes. In immunohistochemistry analysis, FaDu xenografts from AGG-treated mice showed decreased expression of Snail, SOX2, and vimentin and increased expression of p73 and E-cadherin compared with the control group, confirming EMT inhibition as part of its anticancer efficacy against oral cancer. CONCLUSION: In summary, AGG stimulates p73 in restricting EGF-induced EMT, invasiveness, and stemness by inhibiting the ERK/Snail pathway to facilitate the development of alternative therapeutics for oral cancer.

Novel assay system for acidic Peptide:N-glycanase (aPNGase) activity in crude plant extract.

Acidic peptide:N-glycanase (aPNGase) plays a pivotal role in plant glycoprotein turnover. For the construction of aPNGase-knockout or -overexpressing plants, a new method to detect the activity in crude plant extracts is required because endogenous peptidases present in the extract hamper enzyme assays using fluorescence-labeled N-glycopeptides as a substrate. In this study, we developed a new method for measuring aPNGase activity in crude extracts from plant materials.

Molecular modeling and dynamic simulations of agglutinin-like family members from Candida albicans: New insights into potential targets for the treatment of candidiasis.

Infections by Candida albicans in immune compromised patients cause significant morbidity and mortality. In the search for potential molecular targets for drug development, the family of agglutinin-like proteins (Als) in C. albicans have been identified due to numerous attributes associated with high virulence, most prominently due to their role in adherence. Here, molecular models of individual members of the Als family illustrated common and unique structure features. Additionally, dynamic simulations were performed to display regions of high mobility. The results showed variations between Als members in the fluctuation of the A1B1 protein loop, which is located at the entrance to the peptide binding cavity, suggesting that this feature may be a factor contributing to observed differences in affinities to ligands and adhesion properties. Molecular docking results further suggested that ligand affinity could be influenced by movements in the A1B1 loop. In addition, a new site was identified in Als in an area adjacent to the peptide binding cavity that could serve as a new binding site for the design of future anti-adhesion ligands that provide increased specificity inhibiting Als proteins from C. albicans.

Detection of Abrin Holotoxin Using Novel Monoclonal Antibodies.

Abrin, a member of the ribosome-inactivating protein family, is produced by the Abrus precatorius plant. Having the potential to pose a severe threat to both human and animal health, abrin is classified as a Select Agent by the U.S. Department of Health and Human Services. However, an immunoassay that is specific for intact abrin holotoxin has not yet been reported. In this study, seven new monoclonal antibodies (mAbs), designated as Abrin-1 through Abrin-7 have been developed. Isotyping analyses indicate these mAbs have IgG1, IgG2a, or IgG2b heavy-chains and kappa light-chains. Western blot analyses identified two abrin A-chain specific mAbs, Abrin-1 and Abrin-2, and four B-chain specific mAbs (Abrin-3, -5, -6, and -7). A sandwich enzyme-linked immunosorbent assay (ELISA), capable of detecting a mixture of abrin isoforms and agglutinins was developed using B-chain specific Abrin-3 for capture and A-chain specific Abrin-2 as detector. The ELISA is highly sensitive and detects 1 ng/mL of the abrin holotoxin in phosphate-buffered saline, nonfat milk, and whole milk, significantly below concentrations that would pose a health concern for consumers. This ELISA also detects native abrin in plant extracts with a very low background signal. The new abrin mAbs and ELISA should be useful for detecting this potent toxin in the milk supply chain and other complex matrices.

Cancer cell death induced by nanomagnetolectin.

Magnetic nanoparticles represent a new paradigm for molecular targeting therapy in cancer. However, the transformative targeting potential of magnetic nanoparticles has been stymied by a key obstacle-safe delivery to specified target cells in vivo. As cancer cells grow under nutrient deprivation and hypoxic conditions and decorate cell surface with excessive sialoglycans, sialic acid binding lectins might be suitable for targeting cancer cells in vivo. Here we explore the potential of magnetic nanoparticles functionalized with wheat germ lectin (WGA) conjugate, so-called nanomagnetolectin, as apoptotic targetable agents for prostate cancer. In the presence of magnetic field (magnetofection) for 15min, 2.46nM nanomagnetolectin significantly promoted apoptosis (∼12-fold, p value <0.01) of prostate cancer cells (LNCaP, PC-3, DU-145) compared to normal prostate epithelial cells (PrEC, PNT2, PZ-HPV-7), when supplemented with 10mM sialic acid under nutrient deprived condition. Nanomagnetolectin targets cell-surface glycosylation, particularly sialic acid as nanomagnetolectin induced apoptosis of cancer cells largely diminished (only 2 to 2.5-fold) compared to normal cells. The efficacy of magnetofected nanomagnetolectin was demonstrated in orthotopically xenografted (DU-145) mice, where tumor was not only completely arrested, but also reduced significantly (p value <0.001). This was further corroborated in subcutaneous xenograft model, where nanomagnetolectin in the presence of magnetic field and photothermal heating at ∼42°C induced apoptosis of tumor by ∼4-fold compared to tumor section heated at ∼42°C, but without magnetic field. Taken all together, the study demonstrates, for the first time, the utility of nanomagnetolectin as a potential cancer therapeutic.

Wheat germ agglutinin-conjugated liposomes incorporated with cardiolipin to improve neuronal survival in Alzheimer's disease treatment.

Curcumin (CRM) and nerve growth factor (NGF) were entrapped in liposomes (LIP) with surface wheat germ agglutinin (WGA) to downregulate the phosphorylation of kinases in Alzheimer's disease (AD) therapy. Cardiolipin (CL)-conjugated LIP carrying CRM (CRM-CL/LIP) and also carrying NGF (NGF-CL/LIP) were used with AD models of SK-N-MC cells and Wistar rats after an insult with ß-amyloid peptide (Aß). We found that CRM-CL/LIP inhibited the expression of phosphorylated p38 (p-p38), phosphorylated c-Jun N-terminal kinase (p-JNK), and p-tau protein at serine 202 and prevented neurodegeneration of SK-N-MC cells. In addition, NGF-CL/LIP could enhance the quantities of p-neurotrophic tyrosine kinase receptor type 1 and p-extracellular signal-regulated kinase 5 for neuronal rescue. Moreover, WGA-grafted CRM-CL/LIP and WGA-grafted NGF-CL/LIP significantly improved the permeation of CRM and NGF across the blood-brain barrier, reduced Aß plaque deposition and the malondialdehyde level, and increased the percentage of normal neurons and cholinergic activity in the hippocampus of AD rats. Based on the marker expressions and in vivo evidence, current LIP carriers can be promising drug delivery systems to protect nervous tissue against Aß-induced apoptosis in the brain during the clinical management of AD.

Poly - (l) - glutamic acid drug delivery system for the intravesical therapy of bladder cancer using WGA as targeting moiety.

In the management of bladder cancer, surgical resection of the tumour is usually followed by intravesical instillation of immunomodulatives and/or chemotherapeutics. The purpose of this local intravesical therapy is to eliminate residual malignant cells after surgical intervention. The main limitation of a localised adjuvant therapy is the insufficient concentration of the active pharmaceutical ingredient (API) in malignant cells due to the unique structure of the human urothelium making it an exclusively hard to overcome barrier in the human body. Different strategies such as electromotive drug administration or local hyperthermia are employed to ameliorate intravesical drug uptake. Previous studies on biorecognitive targeting showed promising results for lectin-, especially wheat germ agglutinin (WGA), mediated drug delivery. Here, we present a targeted conjugate that provides enough binding sites for a possible API as well as high cytoadhesive and cytoinvasive potential. The conjugate should comprise the following components: First WGA, as the targeting moiety, second poly-l-glutamic acid (PGA) as a polymeric backbone providing more than 300 possible binding sites for an API and third fluorescein cadaverine (Fc), a fluorescent dye we coupled to PGA rendering the conjugate traceable. After purification via size exclusion chromatography (SEC) the WGA containing and therefore binding conjugate was isolated from the reaction mix. In flow-cytometric and fluorimetric experiments with single cells and cell monolayers, respectively, binding and internalisation of the conjugate representing a high molecular weight (>100kDa) could be demonstrated. Fluorescent PGA without the WGA component showed neither binding nor internalisation potential. Microscopic colocalization studies with cell monolayers and single cells confirmed the cytoadhesive and cytoinvasive potential of the WGA containing conjugate. In accordance with the results of specificity studies the interaction between the conjugate and the cell surface depended solely on the WGA component of the conjugate. With the help of this targeted drug delivery system limiting factors of intravesical adjuvant therapy could eventually be overcome and thereby treatment efficacy of instillative chemotherapy could be improved. In addition, traceable drug conjugates might bear interesting advantages for theranostic purposes in the treatment of bladder cancer.

A new TRAF-like protein from B. oleracea ssp. botrytis with lectin activity and its effect on macrophages.

Lectins are involved in a wide range of biological mechanisms, like immunomodulatory agent able to activate the innate immunity. In this study, we purified and characterized a new lectin from cauliflower (Brassica oleracea ssp. botrytis - BOL) by three sequential chromatographic steps and confirmed the purity by SDS-PAGE. Additionally, we evaluated the role of the lectin in innate immunity by a phagocytosis assay, production of H2O2 and NO. BOL was characterized like a non-glycosylated protein that showed a molecular mass of ∼34kDa in SDS-PAGE. Its N-terminal sequence (ETRAFREERPSSKIVTIAG) did not reveal any similarity to the other lectins; nevertheless, it showed 100% homology to a putative TRAF-like protein from Brassica rapa and Brassica napus. This is a first report of the TRAF-protein with lectinic activity. The BOL retained its complete hemagglutination activity from 4°C up to 60°C, with stability being more apparent between pH 7.0 and 8.0. Moreover, the lectin was able to stimulate phagocytosis and induce the production of H2O2 and NO. Therefore, BOL can be explored as an immunomodulatory agent by being able to activate the innate immunity and favor antigen removal.

Affinity chromatography matrices for depletion and purification of casein glycomacropeptide from bovine whey.

Casein glycomacropeptide (CMP) is a 64- amino acid peptide found in cheese whey, which is released after κ-casein specific cleavage by chymosin. CMP lacks aromatic amino acids, a characteristic that makes it usable as a nutritional supplement for people with phenylketonuria. CMP consists of two nonglycosylated isoforms (aCMP A and aCMP B) and its different glycosylated forms (gCMP A and gCMP B). The most predominant carbohydrate of gCMP is N-acetylneuraminic acid (sialic acid). Here, we developed a CMP purification process based on the affinity of sialic acid for wheat germ agglutinin (WGA). After formation of chitosan beads and adsorption of WGA, the agglutinin was covalently attached with glutaraldehyde. Two matrices with different WGA density were assayed for CMP adsorption. Maximum adsorption capacities were calculated according to the Langmuir model from adsorption isotherms developed at pH 7.0, being 137.0 mg/g for the matrix with the best performance. In CMP reduction from whey, maximum removal percentage was 79% (specifically 33.7% of gCMP A and B, 75.8% of aCMP A, and 93.9% of aCMP B). The CMP was recovered as an aggregate with an overall yield of 64%. Therefore, the matrices developed are promising for CMP purification from cheese whey. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:171-180, 2017.

Lectins: a primer for histochemists and cell biologists.

An experimental observation on selecting binding partners underlies the introduction of the term 'lectin'. Agglutination of erythrocytes depending on their blood-group status revealed the presence of activities in plant extracts that act in an epitope-specific manner like antibodies. As it turned out, their binding partners on the cell surface are carbohydrates of glycoconjugates. By definition, lectins are glycan-specific (mono- or oligosaccharides presented by glycoconjugates or polysaccharides) receptors, distinguished from antibodies, from enzymes using carbohydrates as substrates and from transporters of free saccharides. They are ubiquitous in Nature and structurally widely diversified. More than a dozen types of folding pattern have evolved for proteins that bind glycans. Used as tool, this capacity facilitates versatile mapping of glycan presence so that plant/fungal and also animal/human lectins have found a broad spectrum of biomedical applications. The functional pairing with physiological counterreceptors is involved in a wide range of cellular activities from cell adhesion, glycoconjugate trafficking to growth regulation and lets lectins act as sensors/effectors in host defense.