Griffonia Simplicifolia Lectin II Research Articles

Biofilm inhibitor Con A lectin feed additive protects shrimp against pathogenic Vibrio harveyi and V. parahaemolyticus

Extracellular matrix (ECM) is a major component of all bacterial biofilms that protect the bacterial cells living within. It consists mainly of carbohydrates, proteins, lipids, and nucleic acids. We characterized the ECM of an acute hepatopancreatic necrosis disease (AHPND) causing Vibrio parahaemolyticus isolate 3HP and two luminescent shrimp disease-causing V. harveyi isolates (1114GL or VH0 and its Siphoviridae phage-lysogenized isolate, VH1) using 11 FITC-labeled plant lectins with different terminal sugar specificities. Canavalia ensiformis lectin (Con A), wheat germ lectin (Triticum vulgaris, WGA), Griffonia simplicifolia lectin II (GSL-II), and Maclura pomifera Lectin (MPL) were found to react with the biofilms of all three isolates. Based on these results and availability, Con A and WGA and their respective specific sugars (D-mannose or glucose and N-acetyl-D-glucosamine) were tested for effects on V. harveyi and V. parahaemolyticus biofilm formations. Biofilms of these bacteria were evaluated by thickness after growth with and without the two lectins and with and without their respective sugar targets at different concentrations. Only Con A at 0.1 mg/ml and 0.5 mg/ml (with and without D-mannose or d-glucose) were found to inhibit biofilms of all three Vibrio isolates in a dose-dependent manner. Con A was then tested for efficacy in protecting shrimp against V. harveyi (VH1) and V. parahaemolyticus 3-HP by its addition to shrimp feed at 0.05 mg/g and 0.5 mg/g and fed for seven days before they were immersion challenged with either VH1 or 3HP on day 8. After the challenge, the shrimp continued to be fed with their respective feed, and their health and survival were monitored for another 11 and 12 days for the VH1 and 3HP experiments, respectively. From both experiments, the groups fed with Con A-supplemented feed gave significantly higher survival rates [51.9% and 53.6% (VH1) and 52.8% and 62.9% (3HP)] than those fed with un-supplemented feed [38.3% (VH1) and 21.2% (3HP)] (p < 0.05). These results suggested that D-mannose/d-glucose plays an essential role in V. harveyi and V. parahaemolyticus biofilm formations, and Con A lectin might be helpful in monitoring and controlling biofilms to prevent Vibrio diseases.

Decreased B4GALT1 promotes hepatocellular carcinoma cell invasiveness by regulating the laminin-integrin pathway

Beta1,4-galactosyltransferases (B4GALTs) play a crucial role in several diseases, including cancer. B4GALT1 is highly expressed in the liver, and patients with mutations in B4GALT1 exhibit hepatopathy. However, the role of B4GALT1 in liver cancer remains unclear. Here, we found that B4GALT1 was significantly downregulated in hepatocellular carcinoma (HCC) tissue compared with the adjacent liver tissue, and low B4GALT1 expression was associated with vascular invasion and poor overall survival in patients with HCC. Additionally, silencing or loss of B4GALT1 enhanced HCC cell migration and invasion in vitro and promoted lung metastasis of HCC in NOD/SCID mice. Moreover, B4GALT1 knockdown or knockout increased cell adhesion to laminin, whereas B4GALT1 overexpression decreased the adhesion. Through a mass spectrometry-based approach and Griffonia simplicifolia lectin II (GSL-II) pull-down assays, we identified integrins α6 and β1 as the main protein substrates of B4GALT1 and their N-glycans were modified by B4GALT1. Further, the increased cell migration and invasion induced by B4GALT1 knockdown or knockout were significantly reversed using a blocking antibody against integrin α6 or integrin β1. These results suggest that B4GALT1 downregulation alters N-glycosylation and enhances the laminin-binding activity of integrin α6 and integrin β1 to promote invasiveness of HCC cells. Our findings provide novel insights into the role of B4GALT1 in HCC metastasis and highlight targeting the laminin-integrin axis as a potential therapeutic strategy for HCC with low B4GALT1 expression.

Binding of Helicobacter pylori to Human Gastric Mucins Correlates with Binding of TFF1.

Helicobacter pylori binds to the gastric mucin, MUC5AC, and to trefoil factor, TFF1, which has been shown to interact with gastric mucin. We examined the interactions of TFF1 and H. pylori with purified gastrointestinal mucins from different animal species and from humans printed on a microarray platform to investigate whether TFF1 may play a role in locating H. pylori in gastric mucus. TFF1 bound almost exclusively to human gastric mucins and did not interact with human colonic mucins. There was a strong correlation between binding of TFF1 and H. pylori to human gastric mucins, and between binding of both TFF1 and H. pylori to gastric mucins with that of Griffonia simplicifolia lectin-II, which is specific for terminal non-reducing α- or β-linked N-acetyl-d-glucosamine. These results suggest that TFF1 may help to locate H. pylori in a discrete layer of gastric mucus and hence restrain their interactions with epithelial cells.

N-acetylglucosaminyltransferase IVa promotes invasion of choriocarcinoma.

Gestational trophoblastic neoplasia (GTN) results from the malignant transformation of placental trophoblasts which secrete human chorionic gonadotropin (hCG) as do normal placenta or hydatidiform mole. N-acetylglucosaminyltransferase IV (GnT-IV) is a glycosyltransferase which catalyses the formation of β1,4GlcNAc branches on the mannose core of N-glycans. Previous studies reported that β1,4GlcNAc branches on hCG were detected in GTN but not in normal pregnancy or hydatidiform mole. The aim of the present study was to understand the role of GnT-IVa in choriocarcinoma and find the target proteins for GnT-IVa glycosylation which contribute to the malignancy of choriocarcinoma. Immunohistochemistry showed that Griffonia simplicifolia lectin-II staining and GnT-IVa staining were intense in trophoblastic cells of invasive mole and choriocarcinoma. We established a choriocarcinoma cell line with GnT-IVa overexpression (Jar-GnT4a), and examined its malignant potential and target proteins for GnT-IVa glycosylation. GnT-IVa overexpression increased the cell migration and invasion (2.5- and 1.4-fold) as well as the ability to adhere to the extracellular matrix (ECM) components, including fibronectin and collagen typeI andIV. The tumour formation potential of Jar-GnT4a in mice was significantly higher than that of control (P=0.0407), and the cumulative survival rate of mice with Jar-GnT4a was relatively lower than those with control. Immunoprecipitation studies showed that β1,4GlcNAc branches of N-glycans on integrin β1 in choriocarcinoma cells were increased by GnT-IVa overexpression. Nano-LC/MS/MS analysis suggested that lysosome-associated membrane glycoprotein 2 (LAMP-2) was a target protein for glycosylation by GnT-IVa. The increase in β1,4GlcNAc branches on LAMP-2 by GnT-IVa overexpression was confirmed by lectin blot analysis using whole cell lysate and conditioned medium. Our results suggest that highly branched N-glycans generated by the action of GnT-IVa are present in trophoblastic cells of GTN in proportion to GnT-IVa expression level, and that GnT-IVa may contribute to the malignancy of choriocarcinoma by promoting cell adhesion, migration and invasion through glycosylation of integrin β1 and LAMP-2.

Specific Endocytosis Blockade of Trypanosoma cruzi Exposed to a Poly-LAcNAc Binding Lectin Suggests that Lectin-Sugar Interactions Participate to Receptor-Mediated Endocytosis.

Trypanosoma cruzi is a protozoan parasite transmitted by a triatomine insect, and causing human Chagas disease in South America. This parasite undergoes a complex life cycle alternating between non-proliferative and dividing forms. Owing to their high energy requirement, replicative epimastigotes of the insect midgut display high endocytic activity. This activity is mainly restricted to the cytostome, by which the cargo is taken up and sorted through the endosomal vesicular network to be delivered to reservosomes, the final lysosomal-like compartments. In African trypanosomes tomato lectin (TL) and ricin, respectively specific to poly-N-acetyllactosamine (poly-LacNAc) and β-D-galactose, allowed the identification of giant chains of poly-LacNAc in N-glycoproteins of the endocytic pathway. We show that in T. cruzi epimastigote forms also, glycoproteins of the endocytic pathway are characterized by the presence of N-linked glycans binding to both ricin and TL. Affinity chromatography using both TL and Griffonia simplicifolia lectin II (GSLII), specific to non-reducing terminal residue of N-acetylglucosamine (GlcNAc), led to an enrichment of glycoproteins of the trypanosomal endocytic pathway. Incubation of live parasites with TL, which selectively bound to the cytostome/cytopharynx, specifically inhibited endocytosis of transferrin (Tf) but not dextran, a marker of fluid endocytosis. Taken together, our data suggest that N-glycan modification of endocytic components plays a crucial role in receptor-mediated endocytosis of T. cruzi.

Characterization of progressive metaplasia in the gastric corpus mucosa of Mongolian gerbils infected with Helicobacter pylori.

Spasmolytic polypeptide-expressing metaplasia (SPEM) and intestinal metaplasia are considered neoplastic precursors of gastric adenocarcinoma in humans. Loss of parietal cells causes the development of SPEM in the gastric corpus and then chronic inflammation drives SPEM toward a more proliferative lineage. Mongolian gerbils infected with Helicobacter pylori develop chronic gastritis and metaplasia, mimicking aspects of human gastritis with H. pylori infection. We therefore examined metaplastic lineages in the gastric corpus mucosa of gerbils infected by H. pylori strain 7.13, which produces rapid onset of severe inflammation. Six weeks following H. pylori infection, Griffonia simplicifolia lectin II (GSII)-positive SPEM developed in the base of oxyntic glands in association with parietal cell loss and inflammation. In association with severe inflammation, SPEM glands evolved into aberrant phenotypes, including branched lesions, dilated lesions, and penetrating invasive glands. Mucin 4 (MUC4) was up-regulated in SPEM and progressive SPEM. Clusterin was expressed in the tips of branched and dilated lesions and throughout regions of invasive glands. Intriguingly, clusterin-positive regions in these lesions expressed Ki67 and matrix metalloproteinase 7 (MMP-7). These same regions were also positive for expression of phospho-IkBα, suggestive of activated NFkB signalling. These findings suggest that clusterin-positive regions in progressive phenotypes of SPEM have invasive characteristics. Thus, H. pylori infection in gerbils induces SPEM, which then can progress to further aberrant and invasive metaplastic phenotypes. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Transcriptomic response of cowpea bruchids toN-acetylglucosamine-specific lectins

Griffonia simplicifolia lectin II (GSII) and wheat germ agglutinin (WGA) are N-acetylglucosamine-binding lectins. Previous studies demonstrated that they have anti-insect activity, a property potentially useful in pest control. To gain some insight into the insect response to dietary lectins, we performed transcriptomic analysis using the cowpea bruchid (Callosobruchus maculatus) midgut microarray platform we built. Compared to the nonnutritional cellulose treatment, dietary lectins induced more profound changes in gene expression. Ingestion of relatively high doses of lectins for 24 h resulted in alteration of gene expression involved in sugar and lipid metabolism, transport, development, defense, and stress tolerance. Metabolic genes were largely downregulated. Moreover, we observed disorganized microvilli resulting from ingestion of WGA. This morphological change is consistent with the lectin-induced changes in genes related to midgut epithelial cell repair. In addition, suboptimal nutrient conditions may serve as a stress signal to trigger senescence processes, leading to growth arrest and developmental delay.

Lectin-based Immunoassay for Aberrant IgG Glycosylation as the Biomarker for Crohnʼs Disease

Easily measured and clinically useful biomarkers for inflammatory bowel disease (IBD) are required to advance patient care. We previously reported that the agalactosyl fraction among fucosylated IgG oligosaccharides is increased in IBD, especially Crohn's disease (CD). The present study aimed to establish a simple detection system for aberrant glycosylated IgG based on lectin-oligosaccharide interactions. Lectins with higher affinity to serum IgG from IBD patients than healthy volunteers (HV) were screened by lectin microarray. Binding of selected lectins to agalactosyl IgG was definitively confirmed using step-by-step glycosidase treatment. Using the selected lectins, a lectin-enzyme-linked immunosorbent assay system was established and its clinical utility was investigated in a total of 410 (249 Japanese and 161 American) IBD patients, disease controls, and HVs. Agaricus bisporus Agglutinin (ABA) and Griffonia simplicifolia Lectin-II (GSL-II) had higher affinity for serum agalactosyl IgG from IBD patients, especially those with CD, compared to HV. Agalactosyl IgG levels measured by a lectin-enzyme immunoassay (EIA) with ABA or GSL-II were significantly increased in CD compared with HV and disease controls. Agalactosyl IgG levels significantly correlated with disease activity, showed higher predictability of therapeutic outcomes for CD than C-reactive protein levels, and exhibited higher specificity for diagnosing IBD in combination with anti-Saccharomyces cerevisiae antibody (ASCA). Validation analysis showed that agalactosyl IgG levels were significantly increased in Japanese and American CD patients. A lectin-EIA for agalactosyl IgG is a novel biomarker for IBD, especially in patients with CD.

A novel lectin fromAgrocybe aegeritashows high binding selectivity for terminalN-acetylglucosamine

A novel lectin was isolated from the mushroom Agrocybe aegerita (designated AAL-2) by affinity chromatography with GlcNAc (N-acetylglucosamine)-coupled Sepharose 6B after ammonium sulfate precipitation. The AAL-2 coding sequence (1224 bp) was identified by performing a homologous search of the five tryptic peptides identified by MS against the translated transcriptome of A. aegerita. The molecular mass of AAL-2 was calculated to be 43.175 kDa from MS, which was consistent with the data calculated from the amino acid sequence. To analyse the carbohydrate-binding properties of AAL-2, a glycan array composed of 465 glycan candidates was employed, and the result showed that AAL-2 bound with high selectivity to terminal non-reducing GlcNAc residues, and further analysis revealed that AAL-2 bound to terminal non-reducing GlcNAc residues with higher affinity than previously well-known GlcNAc-binding lectins such as WGA (wheatgerm agglutinin) and GSL-II (Griffonia simplicifolia lectin-II). ITC (isothermal titration calorimetry) showed further that GlcNAc bound to AAL-2 in a sequential manner with moderate affinity. In the present study, we also evaluated the anti-tumour activity of AAL-2. The results showed that AAL-2 could bind to the surface of hepatoma cells, leading to induced cell apoptosis in vitro. Furthermore, AAL-2 exerted an anti-hepatoma effect via inhibition of tumour growth and prolongation of survival time of tumour-bearing mice in vivo.

Conserved oligomeric Golgi complex specifically regulates the maintenance of Golgi glycosylation machinery

Cell surface lectin staining, examination of Golgi glycosyltransferases stability and localization, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis were employed to investigate conserved oligomeric Golgi (COG)-dependent glycosylation defects in HeLa cells. Both Griffonia simplicifolia lectin-II and Galanthus nivalus lectins were specifically bound to the plasma membrane glycoconjugates of COG-depleted cells, indicating defects in activity of medial- and trans-Golgi-localized enzymes. In response to siRNA-induced depletion of COG complex subunits, several key components of Golgi glycosylation machinery, including MAN2A1, MGAT1, B4GALT1 and ST6GAL1, were severely mislocalized. MALDI-TOF analysis of total N-linked glycoconjugates indicated a decrease in the relative amount of sialylated glycans in both COG3 KD and COG4 KD cells. In agreement to a proposed role of the COG complex in retrograde membrane trafficking, all types of COG-depleted HeLa cells were deficient in the Brefeldin A- and Sar1 DN-induced redistribution of Golgi resident glycosyltransferases to the endoplasmic reticulum. The retrograde trafficking of medial- and trans-Golgi-localized glycosylation enzymes was affected to a larger extent, strongly indicating that the COG complex regulates the intra-Golgi protein movement. COG complex-deficient cells were not defective in Golgi re-assembly after the Brefeldin A washout, confirming specificity in the retrograde trafficking block. The lobe B COG subcomplex subunits COG6 and COG8 were localized on trafficking intermediates that carry Golgi glycosyltransferases, indicating that the COG complex is directly involved in trafficking and maintenance of Golgi glycosylation machinery.

Comparative Analysis by Frontal Affinity Chromatography of Oligosaccharide Specificity of GlcNAc-Binding Lectins, Griffonia simplicifolia Lectin-II (GSL-II) and Boletopsis leucomelas Lectin (BLL)

Lectin-based structural glycomics requires a search for useful lectins and their biochemical characterization to profile complex features of glycans. In this paper, two GlcNAc-binding lectins are reported with their detailed oligosaccharide specificity. One is a classic plant lectin, Griffonia simplicifolia lectin-II (GSL-II), and the other is a novel fungal lectin, Boletopsis leucomelas lectin (BLL). Their sugar-binding specificity was analyzed by frontal affinity chromatography using 146 glycans (125 pyridylaminated and 21 p-nitrophenyl saccharides). As a result, it was found that both GSL-II and BLL showed significant affinity toward complex-type N-glycans, which are either partially or completely agalactosylated. However, their branch-specific features differed significantly: GSL-II strongly bound to agalacto-type, tri- or tetra-antennary N-glycans with its primary recognition of a GlcNAc residue transferred by GlcNAc-transferase IV, while BLL preferred N-glycans with fewer branches. In fact, the presence of a GlcNAc residue transferred by GlcNAc-transferase V abolishes the binding of BLL. Thus, GSL-II and BLL forms a pair of complementally probes to profile a series of agalacto-type N-glycans.

Calcium modulates protease resistance and carbohydrate binding of a plant defense legume lectin, Griffonia simplicifolia lectin II (GSII)

Site-directed mutagenesis previously identified the residues responsible for the biological activity of the plant defense legume lectin, Griffonia simplicifolia lectin II (GSII) [Proc. Natl. Acad. Sci. USA 95, (1998) 15123–15128]. However, these results were inconclusive as to whether these residues function as direct defense determinants through carbohydrate binding, or whether substantial changes of the protein structure had occurred in mutated proteins, with this structural disruption actually causing the loss of biochemical and biological functions. Evidence shown here supports the former explanation: circular dichroism and fluorescence spectra showed that mutations at carbohydrate-binding residues of GSII do not render it disfunctional because of substantial secondary or tertiary structure modifications; and trypsin treatment confirmed that rGSII structural integrity is retained in these mutants. Reduced biochemical stability was observed through papain digestion and urea denaturation in mutant versions that had lost carbohydrate-binding ability, and this was correlated with lower Ca 2+ content. Accordingly, the re-addition of Ca 2+ to demetalized proteins could recover resistance to papain in the carbohydrate-binding mutant, but not in the non-binding mutant. Thus, both carbohydrate binding (presumably to targets in the insect gut) and biochemical stability to proteolytic degradation in situ indeed contribute to anti-insect activity, and these activities are Ca 2+-dependent.

Functional mechanics of the plant defensive Griffonia simplicifolia lectin II: resistance to proteolysis is independent of glycoconjugate binding in the insect gut.

Griffonia simplicifolia lectin II (GSII) is a plant defensive protein that significantly delays development of the cowpea bruchid Callosobruchus maculatus (F.). Previous structure/function analysis by site-directed mutagenesis indicated that carbohydrate binding and resistance to insect gut proteolysis are required for the anti-insect activity of this lectin. However, whether there is a causal link between carbohydrate binding and resistance to insect metabolism remains unknown. Two proteases principally responsible for digestive proteolysis in third and fourth instar larvae of C. maculatus were purified by activated thiol sepharose chromatography and resolved as cathepsin L-like proteases, based on N-terminal amino acid sequence analysis. Digestion of bacterially expressed recombinant GSII (rGSII) and its mutant protein variants with the purified gut proteases indicates that carbohydrate binding, presumably to a target ligand in insect gut, and proteolytic resistance are independent properties of rGSII, and that both facilitate its efficacy as a plant defensive molecule.

Identification of N-acetylglucosamine binding residues in Griffonia simplicifolia lectin II

Primary structure and crystallographic data of several legume lectins were used to predict the involvement in carbohydrate binding of six amino acid residues (Asp 88, Glu 108, Tyr 134, Asn 136, Leu 226 and Gln 227) in Griffonia simplicifolia lectin II (GS-II). The functional involvement of these residues was evaluated by assessing GlcNAc binding of modified forms of GS-II in which these residues were eliminated in truncated peptides or systematically substituted with other amino acids by site-specific mutations. Mutations at (Asp 88, Tyr 134 or Asn 136 eliminated GlcNAc binding activity by GS-II, while those at Glut 108, Leu 226 or Gln 227 did not alter the activity. The former three amino acids were functionally essential for carbohydrate binding by GS-II presumably through hydrogen bonding to and hydrophobic interactions with GlcNAc. Although an Asp or Gly substitution for Tyr 134 eliminated GlcNAc affinity, substitution with Phe did not appreciably affect binding. Despite the fact that mutations to Leu 226 and Gln 227 did not alter carbohydrate binding, a truncated form of GS-II lacking these residues no longer exhibited carbohydrate binding affinity.