Tanuja Singh

Human galectin-3 (Mac-2 antigen): defining molecular switches of affinity to natural glycoproteins, structural and dynamic aspects of glycan binding by flexible ligand docking and putative regulatory sequences in the proximal promoter region.

BACKGROUND Human galectin-3 (Mac-2 antigen) is a cell-type-specific multifunctional effector owing to selective binding of distinct cell-surface glycoconjugates harboring β-galactosides. The structural basis underlying the apparent preferences for distinct glycoproteins and for expression is so far unknown. METHODS We strategically combined solid-phase assays on 43 natural glycoproteins with a new statistical approach to fully flexible computational docking and also processed the proximal promoter region in silico. RESULTS The degree of branching in N-glycans and clustering of core 1 O-glycans are positive modulators for avidity. Sialylation of N-glycans in α2-6 linkage and of core 1 O-glycans in α2-3 linkage along with core 2 branching was an unfavorable factor, despite the presence of suited glycans in the vicinity. The lectin-ligand contact profile was scrutinized for six natural di- and tetrasaccharides enabling a statistical grading by analyzing flexible docking trajectories. The computational analysis of the proximal promoter region delineated putative sites for Lmo2/c-Ets-1 binding and new sites with potential for RUNX binding. GENERAL SIGNIFICANCE These results identify new features of glycan selectivity and ligand contact by combining solid-phase assays with in silico work as well as of reactivity potential of the promoter.

Carbohydrate specificity of an insecticidal lectin isolated from the leaves of Glechoma hederacea (ground ivy) towards mammalian glycoconjugates

Preliminary studies indicated that the potent insecticidal lectin, Gleheda, from the leaves of Glechoma hederacea (ground ivy) preferentially agglutinates human erythrocytes carrying the Tn (GalNAcalpha1-Ser/Thr) antigen. However, no details have been reported yet with respect to the fine specificity of the lectin. To corroborate the molecular basis of the insecticidal activity and physiological function of Gleheda, it is necessary to identify the recognition factors that are involved in the Gleheda-glycotope interaction. In the present study, the requirement of high-density multivalent carbohydrate structural units for Gleheda binding and a fine-affinity profile were evaluated using ELLSA (enzyme-linked lectinosorbent assay) with our extended glycan/ligand collections, a glycan array and molecular modelling. From the results, we concluded that a high-density of exposed multivalent Tn-containing glycoproteins (natural armadillo and asialo ovine salivary glycoproteins) were the most potent factors for Gleheda binding. They were, on a nanogram basis, 6.5x10(5), 1.5x10(4) and 3.1x10(3) times more active than univalent Gal (galactose), GalNAc (N-acetylgalactosamine) and Tn respectively. Among mono- and oligo-saccharides examined, simple clustered Tn (molecular mass <3000 Da) from ovine salivary glycoprotein was the best, being 37.5 and 1.7x10(3) times better than GalNAc and Gal respectively. GalNAc glycosides were significantly more active than Gal glycosides, indicating that the N-acetamido group at C-2 plays an important role in Gleheda binding. The results of glycan array support the conclusions drawn with respect to the specificity of Gleheda based on the ELLSA assays. These findings combined with the results of the molecular modelling and docking indicate the occurrence of a primary GalNAcalpha1-binding site in the Gleheda monomer. However, the extraordinary binding feature of Gleheda for glycoproteins demonstrates the importance of affinity enhancement by high-density multivalent glycotopes in the ligand-lectin interactions in biological processes.

Differential contributions of recognition factors of two plant lectins – Amaranthus caudatus lectin and Arachis hypogea agglutinin, reacting with Thomsen-Friedenreich disaccharide (Galβ1–3GalNAcα1–Ser/Thr)

Previous reports on the carbohydrate specificities of Amaranthus caudatus lectin (ACL) and peanut agglutinin (PNA, Arachis hypogea) indicated that they share the same specificity for the Thomsen-Friedenreich (T(alpha), Galbeta1-3GalNAcalpha1-Ser/Thr) glycotope, but differ in monosaccharide binding--GalNAc>>Gal (inactive) for ACL; Gal>>GalNAc (weak) with respect to PNA. However, knowledge of the recognition factors of these lectins was based on studies with a small number monosaccharides and T-related oligosaccharides. In this study, a wider range of interacting factors of ACL and PNA toward known mammalian structural units, natural polyvalent glycotopes and glycans were examined by enzyme-linked lectinosorbent and inhibition assays. The results indicate that the main recognition factors of ACL, GalNAc was the only monosaccharide recognized by ACL as such, its polyvalent forms (poly GalNAcalpha1-Ser/Thr, Tn in asialo OSM) were not recognized much better. Human blood group precursor disaccharides Galbeta1-3/4GlcNAcbeta (I(beta)/II(beta)) were weak ligands, while their clusters (multiantennary II(beta)) and polyvalent forms were active. The major recognition factors of PNA were a combination of alpha or beta anomers of T disaccharide and their polyvalent complexes. Although I(beta)/II(beta) were weak haptens, their polyvalent forms played a significant role in binding. From the 50% molar inhibition profile, the shape of the ACL combining site appears to be a cavity type and most complementary to a disaccharide of Galbeta1-3GalNAc (T), while the PNA binding domain is proposed to be Galbeta1-3GalNAcalpha or beta1--as the major combining site with an adjoining subsite (partial cavity type) for a disaccharide, and most complementary to the linear tetrasaccharide, Galbeta1-3GalNAcbeta1-4Galbeta1-4Glc (T(beta)1-4L, asialo GM(1) sequence). These results should help us understand the differential contributions of polyvalent ligands, glycotopes and subtopes for the interaction with these lectins to binding, and make them useful tools to study glycosciences, glycomarkers and their biological functions.

A novel lectin (morniga M) from mulberry (Morus nigra) bark recognizes oligomannosyl residues in N-Glycans

Morniga M is a jacalin-related and mannose-specific lectin isolated from the bark of the mulberry (Morus nigra). In order to understand the function and application of this novel lectin, the binding property of Morniga M was studied in detail using an enzyme-linked lectinosorbent assay and lectin-glycan inhibition assay with extended glycan/ligand collection. From the results, it was found that the di-, tri-, and oligomannosyl structural units of N-glycans such as those of the bovine α1-acid glycoprotein (gp) and lactoferrin were the most active gps, but not the O-glycans or polysaccharides including mannan from yeast. The binding affinity of Morniga M for ligands can be ranked in decreasing order as follows: gps carrying multiple N-glycans with oligomannosyl residues >> N-glycopeptide with a single trimannosyl core > Tri-Man oligomer [Manα1→6(Man α1→3) Man], Penta-Man oligomer [Manα1→6(Manα1→3)Manα1→6(Manα1→3) Man] ≧ Man α1→2, 3 or 6 Man > Man > GlcNAc, Glc >> L-Fuc, Gal, GalNAc (inactive), demonstrating the unique specificity of this lectin that may not only assist in our understanding of cell surface carbohydrate ligand-lectin recognition, but also provide informative guidelines for the application of this structural probe in biotechnological and clinical regimens, especially in the detection and purification of N-linked glycans.

Network Monitoring of Adhesion/Growth‐Regulatory Galectins: Localization of the Five Canonical Chicken Proteins in Embryonic and Maturing Bone and Cartilage and Their Introduction as Histochemical Tools

Divergence from an ancestral gene leads to a family of homologous proteins. Whether they are physiologically distinct, similar, or even redundant is an open question in each case. Defining profiles of tissue localization is a step toward giving diversity a functional meaning. Due to the significance of endogenous sugar receptors (lectins) as effectors for a wide range of cellular activities we have focused on galectins. The comparatively low level of network complexity constituted by only five canonical proteins makes chicken galectins (CGs) an attractive choice to perform comprehensive analysis, here studied on bone/cartilage as organ system. Galectin expression was monitored by Western blotting and immunohistochemistry using non‐cross‐reactive antibodies. Overall, three galectins (CG‐1B, CG‐3, CG‐8) were present with individual expression patterns, one was found exclusively in the mesenchyme (CG‐1A), the fifth (CG‐2) not being detectable. The documented extents of separation are a sign for functional divergence; in cases with overlapping stainings, as for example in the osteoprogenitor layer or periosteum, cooperation may also be possible. Recombinant production enabled the introduction of the endogenous lectins as tools for binding‐site localization. Their testing revealed developmental regulation and cell‐type‐specific staining. Of relevance for research on mammalian galectins, this study illustrates that certain cell types can express more than one galectin, letting functional interrelationships appear likely. Thus, complete network analysis irrespective of its degree of complexity is mandatory. Anat Rec, 298:2051–2070, 2015.

Induction of Defense Related Enzymes and Pathogenesis Related Proteins in Pseudomonas fluorescens-Treated Chickpea in Response to Infection by Fusarium oxysporum f. sp. ciceri

Pseudomonas fluorescens 1–94 induced systemic resistance in chickpea against Fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceri by the synthesis and accumulation of phenolic compounds, phenylalanine ammonia lyase (PAL) and pathogenesis related (PR) proteins (chitinase, β-1,3-glucanase and peroxidase). Time-course accumulation of these enzymes in chickpea plants inoculated with P. fluorescens was significantly (LSD, P =0.05) higher than control. Maximum activities of PR-proteins were recorded at 3 days after inoculation in all induced plants; thereafter, the activity decreased progressively. Five PR peroxidases detected in induced chickpea plants. Molecular mass of these purified peroxidases was 20, 29, 43, 66 and 97 kDa. Purified peroxidases showed antifungal activity against plant pathogenic fungi.

Multivalent human blood group ABH and Lewis glycotopes are key recognition factors for a LFuc>Man binding lectin from phytopathogenic Ralstonia solanacearum.

Ralstonia solanacearum lectin (RSL), that might be involved in phytopathogenicity, has been defined as LFuc>>Man specific. However, the effects of polyvalency of glycotopes and mammalian structural units on binding have not been established. In this study, recognition factors of RSL were comprehensively examined with natural multivalent glycotopes and monomeric ligands using enzyme linked lectin-sorbent and inhibition assays. Among the glycans tested, RSL reacted strongly with multivalent blood group A(h) (GalNAcalpha1-3[Fucalpha1-2]Gal) and H (Fucalpha1-2Gal) active glycotopes, followed by B(h) (Galalpha1-3[Fucalpha1-2]Gal), Le(a) (Galbeta1-3[Fucalpha1-4]GlcNAc) and Le(b) (Fucalpha1-2Galbeta1-3[Fucalpha1-4]GlcNAc) active glycotopes. But weak or negligible binding was observed for blood group precursors having Galbeta1-3/4GlcNAcbeta1- (Ibeta/IIbeta) residues or Galbeta1-3GalNAcalpha1- (Talpha), GalNAcalpha1-Ser/Thr (Tn) bearing glycoproteins. These results indicate that the density and degree of exposure of multivalent ligands of alpha1-2 linked LFuc to Gal at the non-reducing end is the most critical factor for binding. An inhibition study with monomeric ligands revealed that the combining site of RSL should be of a groove type to fit trisaccharide binding with highest complementarity to blood group H trisaccharide (H(L); Fucalpha1-2Galbeta1-4Glc). The outstandingly broad RSL saccharide-binding profile might be related to the unusually wide spectrum of plants that suffer from R. solanacearum pathogenicity and provide ideas for protective antiadhesion strategies.

Hydrophobicity and surface electrostatic charge of conidia of the mycoparasitic Trichoderma species

The present investigation was done to understand the fungal-fungal interactions mechanisms based on level of nonspecific adhesion of a potential fungal mycoparasite (Trichoderma) to their fungal host (Macrophomina phaseolina). The relative cell surface hydrophobicity (CSH) and cell surface electrostatic charge (CSEC) of 29 isolates of Trichoderma species, analyzed by bacterial adhesion to hydrocarbon (BATH), hydrophobic interaction chromatography (HIC), microelectrophoresis and contact angle, revealed a large degree of variability. CSH and CSEC of conidia depended on culture age, pH and temperature. Maximum CSH and CSEC were recorded in 25–28 °C range, and both declined significantly with increasing temperature. Isolate Trichoderma hazianum (Th)-23/98 expressed surface hydrophobicity at 25–28 °C and hydrophilicity at 40 °C. Surface hydrophobicity of the isolate was susceptible to various proteases (trypsin, pepsin, proteinase k and a-chymotrypsin) and inhibitors (SDS, mercaptoethanol and Triton X-100) and a significant reduction in CSH was recorded in hydrophobic conidia. Hydrophilic conidia remained more or less unaffected by such treatments. SDS-PAGE analysis of the hydrophobic and hydrophilic conidia exhibited several protein bands in the 25 to 61 kDa range. However, each protein population contained one protein that was not observed in the other population. For hydrophobic conidia, the unique protein had an apparent molecular mass of 49 kDa, while the unique protein associated with hydrophilic conidia had a molecular mass of 61 kDa. Our findings suggest that CSH and CSEC of mycoparasitic Trichoderma may contribute to non-specific adhesion on to the sclerotial surfaces of Macrophomina phaseolina that may be influenced by growth and environmental conditions.

Adhesion/Growth-Regulatory Galectins: Insights into Their Ligand Selectivity Using Natural Glycoproteins and Glycotopes

“Biochemistry text books commonly make it appear that it is a foregone conclusion that the hardware of biological information storage and transfer is confined to nucleotides and amino acids, the letters of the genetic code. However, the remarkable talents of a third class of biomolecules are often overlooked” [1]. This statement from a recent review guides the readers to look at and fully appreciate the chemical/lectinochemical characteristics of carbohydrates that underlie the concept of the sugar code [2].

Differential Binding to Glycotopes Among the Layers of Three Mammalian Retinal Neurons by Man-Containing N-linked Glycan, Tα (Galβ1–3GalNAcα1-), Tn (GalNAcα1-Ser/Thr) and Iβ/IIβ (Galβ1–3/4GlcNAcβ-) Reactive Lectins

Carbohydrate structures between retinal neurons and retinal pigment epithelium (RPE) play an important role in maintaining the integrity of retinal adhesion to underlying RPE, and in retinal detachment pathogenesis. Since relevant knowledge is still in the primary stage, glycotopes on the adult retina of mongrel canines (dog), micropigs and Sprague-Dawley rats were examined by lectino-histochemistry, using a panel of 16 different lectins. Paraffin sections of eyes were stained with biotinylated lectins, and visualized by streptavidin-peroxidase and diaminobenzidine staining. Mapping the affinity profiles, it is concluded that: (i) all sections of the retina reacted well with Morniga M, suggesting that N-linked glycans are present in all layers of the retina; (ii) no detectable human blood group ABH active glycotopes were found among retinal layers; (iii) outer and inner segments contained glycoconjugates rich in ligands reacting with Tα (Galβ1–3GalNAcα1-Ser/Thr) and Tn (GalNAcα1-Ser/Thr) specific lectins; (iv) cone cells of retina specifically bound peanut agglutinin (PNA), which recognizes Tα residues and could be used as a specific marker for these photoreceptors; (v) the retinas of rat, dog and pig, had a similar binding profile but with different intensity; (vi) each retinal layer had its own binding characteristic. This information may provide useful background knowledge for normal retinal physiology and miscellaneous retinal diseases, including retinal detachment (RD) and age-related macular degeneration (ARMD).