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Banana lectin is unique in its recognition of the reducing unit of 3-O-beta-glucosyl/mannosyl disaccharides: a calorimetric study
Abstract
The binding of banana lectin (BanLec) to laminaribiose (Glcβ1,3Glc) and a series of novel synthetic analogues was measured
by titration calorimetry to assess the contribution of the hydroxyl groups of the reducing glycosyl moiety and its 3-O-β-substituent to binding. Key areas of interaction involved the 1, 2, and 6 positions of the reducing-terminal hexose unit.
The α-anomeric configuration of the reducing hexose was strongly favored over the β-anomer. The 2-hydroxyl in the axial position
(mannose) also enhanced binding, whereas the 6-hydroxymethyl group was essential, because xylopyranose in the reducing position
was inactive. The 3-O-β-glucosyl unit of methyl α-laminaribioside could be replaced by any of its monodeoxy derivatives. However, the 49-deoxy
derivative or axial hydroxy (galactosyl) substitution was somewhat detrimental to binding. 3-O-substitution with the (S)tetrahydropyranyl ring or a benzyl group had similar effect as 49-deoxyglucosyl substitution. Surprisingly, p‐nitrobenzyl or β-xylosyl 3-O-substitution greatly enhanced binding of the reducing glucosyl or mannosyl derivative. Chemical syntheses of a number of
novel disaccharides and analogues prepared for this study are described.
... BanLec is unique in its specificity for internal β-1,3 linkages as well as β-1,3 linkages at the reducing termini [26]. BanLec also explicitly binds to terminal nonreducing α-D-glucosyl/mannosyl units of oligo-/polysaccharide chain ends [21,27,28]. Mo et al. [27], performed extensive experiments in order to find out the exact specificity of BanLec (Musa acuminata) and lectin from the closely related plantain (Musa spp.) by using the techniques of quantitative precipitation, hapten inhibition of precipitation, and isothermal titration calorimetry and showed that they are mannose/glucose binding proteins with a preference for the α-anomeric form of these sugars. ...
... This behavior clearly distinguishes the banana lectin from other mannose/glucose binding lectins, such as concanavalin A and the pea, lentil and Calystegia sepium lectins. Therefore BanLec is unique in its recognition of internal glucosyl/mannosyl residues linked α-1,3-, but not β-1,3-, and especially in its recognition of the reducing sugar unit of laminaribiose (Glcβ1,3Glc) and its higher homologues [27][28][29]. ...
... Mo [27]; Winter [28] BanLec ...
Lectins are a group of proteins of non-immune origin that recognize and bind to carbohydrates without modifying them. Banana is the common name for both herbaceous plants of the genus Musa and for the fruit they produce. They are indeed a promising source for many medicinal applications. Banana lectins have the potential for inhibiting HIV-1 reverse transcriptase activity, suppressing cancer cell proliferation and stimulating macrophage activities. Nevertheless, compared to other plant lectins, there is relatively little information in the literature on banana lectins, particularly with respect to their structure and biological functions. Herein we focus our review on the structure, functions and exploitable properties of banana lectins.
... Banana lectin (BanLec) from Musa acuminata is a tetramer composed of 15 kDa subunits[34]with a somewhat unusual carbohydrate binding specificity. It recognises 2-or 3-substituted glucose or mannose residues,[35,36]including internal (α13)-linked glucose, but it will not tolerate substitution (or deoxygenation) at the 4-or 6-positions. It will, though, bind the terminal glucose residues in (α16)branched glucans and mannans.[37]BanLec ...
... Error values are for curve fitting of the single titration, not variance of replicates, which were not done. data suggest that BanLec prefers the α-Glc configuration over the β-Glc, which is consistent with the reported binding preferences for reducing terminal glucose.[36]Comparing the affinities of different 3-substituted glucose compounds: ethers 76, 79, 77 and thioether 47, we see a wide range of binding, which leads us to speculate that both monosaccharide residues are interacting with the protein. The thioether 49 does bind more strongly than the monosaccharide glucose. ...
... However, the binding affinities of these pseudodisaccharides do not come up to the level of the most strongly binding disaccharides, e.g. Glc(β13)Glc (K a = 830 –1 ), Glc(β13)GlcαMe (K a = 2400 –1 ),[36]and even some simple 3-O-alkylglucose derivatives (Table 4, Entries 5,6) bind more strongly to BanLec. Some thioether-linked 6-substituted mannose derivatives (47, 48) bind, whereas others (46, 52) do not. ...
Hydrolytically stable non-glycosidically linked tail-to-tail pseudodisaccharides are linked by a single bridging atom remote from the anomeric centre of the constituent monosaccharides. Some such pseudodisaccharides with sulfur or oxygen bridges were found to act as disaccharide mimetics in their binding to the Banana Lectin and to Concanavalin A. A versatile synthetic route to a small library of such compounds is described.
... In common with other glucose-mannose-specific lectins, BanLec binds to α-glucosyl and α-mannosyl terminal non-reducing units [10]. However, BanLec is unique in its specificity for the branched mannose-containing oligosaccharides which are components of the so-called core region of N-linked glycoproteins [8,11,12]. Although BanLec had been well characterised and its crystal structure had been revealed [13,14], further advances in the study of BanLec were hampered by the difficulties of purifying sufficiently large quantities; its availability in biological material is generally low and is dependent upon the stage of fruit ripeness [15]. ...
... Because the intensity of rBanLec's cellular interaction is influenced by its binding affinity for the target cell's epitopic expression, this microheterogeneity indicated that rBanlec recognised both finely and densely structured oligosaccharides existing within cell subpopulations. Natural BanLec exhibits a higher specificity towards the branched trisaccharides and branched pentamannose oligosaccharides contained within the core region of N-linked glycoproteins than it does towards the monosaccharides [11,12]. The density of the structures available for rBanLec binding is of paramount importance. ...
Lectins are widely used in many types of assay but some lectins such as banana lectin (BanLec) are recognised as potent immunostimulators. Although BanLec's structure and binding characteristics are now familiar, its immunostimulatory potential has not yet been fully explored. The synthesis by recombinant technology of a BanLec isoform (rBanLec) whose binding properties are similar to its natural counterpart has made it possible to overcome the twin problems of natural BanLec's microheterogeneity and low availability. This study's aim is to explore the immunostimulatory potential of rBanLec in the murine model. Analyses of the responses of Balb/c- and C57 BL/6-originated splenocytes to in vitro rBanLec stimulation were performed to examine the dependency of rBanLec's immunostimulatory potential upon the splenocytes' genetic background. It is shown that the responses of Balb/c- and C57 BL/6-originated splenocytes to rBanLec stimulation differ both qualitatively and in intensity. The hallmarks of the induced responses are T lymphocyte proliferation and intensive interferon-gamma secretion. Both phenomena are more marked in Balb/c-originated cultures; Balb/c-originated lymphocytes produce interleukin (IL)-4 and IL-10 following rBanLec stimulation. Our results demonstrate that any responses to rBanLec stimulation are highly dependent upon genetic background; they suggest that genetic background must be an important consideration in any further investigations using animal models or when exploring rBanLec's potential human applications.
... The recombinant lectin which is the object of this study was produced by Gavrovic-Jankulovic et al. [8]. In common with other glucose-mannose-specific lectins, BL binds to α-glucosyl and α-mannosyl terminal non-reducing units [9] as well as to the branched mannose-containing oligosaccharides which are components of the so-called core region of N-linked glycoproteins, as well as to ß-1,3-linked glucosyl oligosaccharides and gentiobiosyl groups [10][11][12]. BL had been well characterized and its crystal structure had been revealed [13,14] and to the best of our knowledge so far it had not been tested in a glycan array. ...
The surface of microorganisms is covered with polysaccharide structures which are in immediate contact with receptor structures on host’s cells and antibodies. The interaction between microorganisms and their host is dependent on surface glycosylation and in this study we have tested the interaction of plant lectins with different microorganisms. Enzyme-linked lectin sorbent assay - ELLSA was used to test the binding of recombinant Musa acuminata lectin - BL to 27 selected microorganisms and 7 other lectins were used for comparison: Soy bean agglutinin - SBA, Lens culinaris lectin - LCA, Wheat germ agglutinin - WGA, RCA120 - Ricinus communis agglutinin, Con A - from Canavalia ensiformis, Sambucus nigra agglutinin - SNA I and Maackia amurensis agglutinin - MAA. The goal was to define the microorganisms’ surface glycosylation by means of interaction with the selected plant lectins and to make a comparison with BL. Among the tested lectins most selective binding was observed for RCA120 which preferentially bound Lactobacillus casei DG. Recombinant banana lectin showed specific binding to all of the tested fungal species. The binding of BL to Candida albicans was further tested with fluorescence microscopy and flow cytometry and it was concluded that this lectin can differentiate ß-glucan rich surfaces. The binding of BL to S. boulardii could be inhibited with ß-glucan from yeast with IC50 1.81 μg mL⁻¹ and to P. roqueforti with 1.10 μg mL⁻¹. This unique specificity of BL could be exploited for screening purposes and potentially for the detection of ß-glucan in solutions.
... BanLec shows specificity for molecules containing D-glucopyranosyl, D-mannopyranosyl and other related carbohydrate structures (33)(34)(35); it shares many properties with Glc/Man-recognizing legume lectins (Con A). The yield of BanLec was found to be 7 mg/kg banana pulp; its purity was confirmed by SDS-PAGE (single band of 15 kDa) and hemagglutination activity indicated glucose/mannose specificity. ...
Dietary lectins play a major role in the activation of mast cells / basophils by bridging cell surface IgE glycans to release histamine and other mediators. In the present study, the effect of mannose / glucose-specific banana lectin (BanLec) on the activation of mast cells / basophils from non-atopic and atopic subjects has been investigated. BanLec was purified from banana pulp in a yield of 7 mg/kg. Leukocytes isolated from heparinized blood of non-atopic / atopic subjects were used for quantitation of the released histamine. Approximately 28.2% of the atopics (n = 117) was positive by skin prick test (SPT) to purified BanLec (100 mg/mL concentration), and all the non-atopics (n = 20) were negative. Maximal release of histamine was seen at 2 g of BanLec. In percent histamine release, an increase of 35-40% is observed in case of atopics (n = 7) compared to non-atopics (n = 5), and the histamine release from atopic and non-atopic subjects correlates fairly well with the total serum IgE levels (R2 = 0.817). BanLec also induces release of histamine (26.7%) from mast cells present in rat peritoneal exudate cells. BanLec can significantly activate and degranulate mast cells and basophils by cross-linking the trimannosidic core mannose of IgE glycans in atopic population as compared to non-atopic population; the activation is marginal in the case of non-atopics.
... (333 mM À1 ) (Mo et al., 2001;Winter et al., 2005). Interestingly, slightly weaker affinities were observed for H84T as compared to WT when analyzing binding to dimannoside (300 versus 227 mM À1 for WT and H84T, respectively) ( Table S2). ...
Recognition of glycans by lectins leads to cell adhesion and growth regulation. The specificity and selectivity of this process are determined by carbohydrate structure (sequence and shape) and topology of its presentation. The synthesis of (neo)glycoconjugates with bi- to oligo-valency (glycoclusters) affords tools to delineate structure-activity relationships by blocking lectin binding to an artificial matrix, often a glycoprotein, or cultured cell lines. The drawback of these assays is that glycan presentation is different from that in tissues. In order to approach the natural context, we here introduce lectin histochemistry on fixed tissue sections to determine the susceptibility of binding of two plant lectins, i.e., GSA-II and WGA, to a series of 10 glycoclusters. Besides valency, this panel covers changes in the anomeric position (α/β) and the atom at the glycosidic linkage (O/S). Flanked by cell and solid-phase assays with human tumor lines and two mucins, respectively, staining (intensity and profile) was analyzed in sections of murine jejunum, stomach and epididymis as a function of glycocluster presence. The marked and differential sensitivity of signal generation to structural aspects of the glycoclusters proves the applicability of this method. This enables comparisons between data sets obtained by using (neo)glycoconjugates, cells and the tissue context as platforms. The special advantage of processing tissue sections is the monitoring of interference with lectin association at sites that are relevant for functionality. Testing glycoclusters in lectin histochemistry will especially be attractive in cases of multi-target recognition (glycans, proteins and lipids) by a tissue lectin.
... (333 mM À1 ) (Mo et al., 2001;Winter et al., 2005). Interestingly, slightly weaker affinities were observed for H84T as compared to WT when analyzing binding to dimannoside (300 versus 227 mM À1 for WT and H84T, respectively) ( Table S2). ...
A key effector route of the Sugar Code involves lectins that exert crucial regulatory controls by targeting distinct cellular glycans. We demonstrate that a single amino-acid substitution in a banana lectin, replacing histidine 84 with a threonine, significantly reduces its mitogenicity, while preserving its broad-spectrum antiviral potency. X-ray crystallography, NMR spectroscopy, and glycocluster assays reveal that loss of mitogenicity is strongly correlated with loss of pi-pi stacking between aromatic amino acids H84 and Y83, which removes a wall separating two carbohydrate binding sites, thus diminishing multivalent interactions. On the other hand, monovalent interactions and antiviral activity are preserved by retaining other wild-type conformational features and possibly through unique contacts involving the T84 side chain. Through such fine-tuning, target selection and downstream effects of a lectin can be modulated so as to knock down one activity, while preserving another, thus providing tools for therapeutics and for understanding the Sugar Code.
... [14] Based on this feature, it exhibits some uncommon binding properties as it recognizes 1,3-sugar units at the reducing termini, in addition to the internal a-1,3-linked glucosyl residues. [15] BanLec-1 has been recognized as a potential immunomodulatory molecule. [16] Swanson et al. [17] further demonstrated that BanLec-1 could inhibit HIV-1 by binding directly to gp120, HIV-1 envelope protein, and was able to block HIV-1 cellular entry. ...
It has been demonstrated that the lectin from Musa paradisiaca (BanLec-1) could inhibit the cellular entry of human immunodeficiency virus (HIV). In order to evaluate its effects on tobacco mosaic virus (TMV), the banlec-1 gene was cloned and transformed into Escherichia coli and tobacco, respectively. Recombinant BanLec-1 showed metal ions dependence, and higher thermal and pH stability. Overexpression of banlec-1 in tobacco resulted in decreased leaf size, and higher resistance to TMV infection, which includes reduced TMV cellular entry, more stable chlorophyll contents, and enhanced antioxidant enzymes. BanLec-1 was found to bind directly to the TMV capsid protein in vitro, and to inhibit TMV infection in a dose-dependent manner. In contrast to limited prevention in vivo, purified rBanLec-1 exhibited more significant effects on TMV infection in vitro. Taken together, our study indicated that BanLec-1 could prevent TMV infection in tobacco, probably through the interaction between BanLec-1 and TMV capsid protein.
... Recently, anti-HIV activity of a Man-specific jacalin-related lectin, BanLec, was also reported (Swanson et al. 2009). BanLec is unique in its specificity for internal α1-3 linkages of Man/Glc as well as β1-3 linkages Man/Glc at the reducing termini (Koshte et al. 1990;Winter et al. 2005). The binding strength of HRL to gp120 was the same as or more than BanLec. ...
A lectin was purified from the mushroom Hygrophorus russula by affinity chromatography on a Sephadex G-50 column and BioAssist S cation exchange chromatography and designated H. russula lectin (HRL). The results of sodium dodecyl sulfate-polyaclylamidegel electrophoresis, gel filtration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry of HRL indicated that it was composed of four identical 18.5 kDa subunits with no S-S linkage. Isoelectric focusing of the lectin showed bands near pI 6.40. The complete sequence of 175 amino acid residues was determined by amino acid sequencing of intact or enzyme-digested HRL. The sequence showed homology with Grifola frondosa lectin. The cDNA of HRL was cloned from RNA extracted from the mushroom. The open reading frame of the cDNA consisted of 528 bp encoding 176 amino acids. In hemagglutination inhibition assay, α1-6 mannobiose was the strongest inhibitor and isomaltose, Glcα1-6Glc, was the second strongest one, among mono- and oligosaccharides tested. Frontal affinity chromatography indicated that HRL had the highest affinity for Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc, and non-reducing terminal Manα1-6 was essential for the binding of HRL to carbohydrate chains. The sugar-binding specificity of HRL was also analyzed by using BIAcore. The result from the analysis exhibited positive correlations with that of the hemagglutination inhibition assay. All the results suggested that HRL recognized the α1-6 linkage of mannose and glucose, especially the Manα1-6 bond. HRL showed a mitogenic activity against spleen lymph cells of an F344 rat. Furthermore, an enzyme-linked immunosorbent assay showed strong binding of HRL to human immunodeficiency virus type-1 gp120.
... The internal symmetry of the sequence and its correlation with the number of carbohydrate-binding sites in β-prism I fold lectins and their possible evolutionary implications, with particular reference to the possible involvement of the lectin in defense, have been further investigated in a detailed study in this laboratory (Sharma et al. 2007). Thermodynamic parameters of banana lectin-carbohydrate interactions have been determined for a variety of carbohydrates Mo et al. 2001;Winter et al. 2005). Modeling studies along with the X-ray structure of the lectin-methyl-α-mannose complex provided the structural rationale of the lectin's specificity of branched α-mannans (Singh et al. 2005). ...
The three crystal structures reported here provide details of the interactions of mannose and the mannosyl-α-1,3-mannose component of a pentamannose with banana lectin and evidence for the binding of glucosyl-α-1,2-glucose to the lectin. The known structures involving the lectin include a complex with glucosyl-β-1,3-glucose. Modeling studies on the three disaccharide complexes with the reducing end and the nonreducing end at the primary binding site are also provided here. The results of the X-ray and modeling studies show that the disaccharides with an α-1,3 linkage prefer to have the nonreducing end at the primary binding site, whereas the reducing end is preferred at the site when the linkage is β-1,3 in mannose/glucose-specific β-prism I fold lectins. In the corresponding galactose-specific lectins, however, α-1,3-linked disaccharides cannot bind the lectin with the nonreducing end at the primary binding site on account of steric clashes with an aromatic residue that occurs only when the lectin is galactose-specific. Molecular dynamics simulations based on the known structures involving banana lectin enrich the information on lectin-carbohydrate interactions obtained from crystal structures. They demonstrate that conformational selection as well as induced fit operate when carbohydrates bind to banana lectin.
... The K d of Banlec binding to LAM is 1 mM and to XM is 0.2 mM (Winter et al., 2005). The 5-fold tighter binding to XM is interesting in that there are no additional hydrogen bonds to account for the difference. ...
Banana lectin (Banlec) is a dimeric plant lectin from the jacalin-related lectin family. Banlec belongs to a subgroup of this family that binds to glucose/mannose, but is unique in recognizing internal alpha1,3 linkages as well as beta1,3 linkages at the reducing termini. Here we present the crystal structures of Banlec alone and with laminaribiose (LAM) (Glcbeta1, 3Glc) and Xyl-beta1,3-Man-alpha-O-Methyl. The structure of Banlec has a beta-prism-I fold, similar to other family members, but differs from them in its mode of sugar binding. The reducing unit of the sugar is inserted into the binding site causing the second saccharide unit to be placed in the opposite orientation compared with the other ligand-bound structures of family members. More importantly, our structures reveal the presence of a second sugar binding site that has not been previously reported in the literature. The residues involved in the second site are common to other lectins in this family, potentially signaling a new group of mannose-specific jacalin-related lectins (mJRL) with two sugar binding sites.
Banana is one of the oldest cultivated plant known for its dietary and medicinal properties. Banana is grown in the tropical and subtropical regions of the world and constitutes the staple food of the people. They are classified as dessert or sweet bananas and cooking bananas or plantains depending on whether they can be eaten raw or not. Banana plant parts such as the roots, pseudostem, fruits, and inflorescence is used in some or the other way and therefore it is rightly called as the “Kalpatharu” in India. Banana and plantains contain important bioactive compounds such as phenolics, flavonoids, carotenoids, biogenic amines, sterols, and antimicrobial compounds which make bananas a perfect functional food for health improvement. Presently, research is focused on exploring and identifying compounds, refining the techniques of isolation and purification, and using it in modern medicines. Moreover, bananas are also being used as a platform to produce and accumulate important nutrients like vitamins and minerals by biofortification strategies.
To date, most biosensors for medical applications have focused either on small metabolites such as glucose, or on protein markers of disease (cancer, heart attack and others) or other conditions such as pregnancy. However, rapid measurement of other classes of analyte would clearly be useful for diagnosis and prognosis during treatment. These include ions, carbohydrates and microorganisms such as viruses and bacteria. We discuss the approaches taken to producing reagentless biosensors to these three types of analyte that are amenable to interrogation by AC impedance.
The catalytic regio‐ and stereoselective monoglycosylation of carbohydrates using organotin catalysts is demonstrated. The one‐step reaction affords various oligosaccharides linked at the secondary hydroxy group in high chemical yield and good regio‐ and stereoselectivities. The regioselectivity of the glycosylation is shown to depend on the spatial arrangement of the hydroxy groups in the carbohydrates.
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Banana lectin (BanLec) was isolated from slightly overripe bananas (PCI 6-7) by homogenation in NaCl solution, followed by extraction in the presence of glucose, ammonium sulfate precipitation, and affinity chromatography. Yields were approximately 10-fold greater that those of previously published methods using acidic extraction from very overripe fruit (Peel Color Index [PCI] 7+). By dilution of added isotopically labeled recombinant lectin, the content of total exchangeable BalLec was shown to be constant or to slightly decrease with increasing stage of ripeness, even though extractable BanLec increased, followed by rapid decrease in overripened fruit. In the course of this study we observed that recombinant BanLec expressed in Escherichia coli, although chemically and functionally identical to native BanLec, differed slightly in its apparent molecular size on gel filtration, probably due to differences in its native folding.
The advent of sugar-immobilized gold nano-particles (SGNPs), lipid-based nanoparticles, nano-chromatography and nano-electrophoresis has revolutionized the methodology for protein purification and proteomic research. This review provides an overview on the effective method developed for fast purification of protein from extracts using SGNPs. In addition, the current application of microfluidic systems for analytical purposes in biochemistry will also be explored that include the micro total analysis systems (µ-TAS) and lab-on-a-chip (LOC) analyses which are capable of isolation and detection of protein at the nanogram level. Finally, we describe why the lipid-based nano-particles (LBNPs) can enable the analysis in microchip electroseparation and how anionic and cationic LBNPs can be used for protein separation.
Frontal affinity chromatography using fluorescence detection (FAC-FD) is a versatile technique for the precise determination of dissociation constants (Kd) between glycan-binding proteins (lectins) and fluorescent-labeled glycans. A series of glycan-containing solutions is applied to a lectin-immobilized column, and the elution profile of each glycan (termed the 'elution front', V) is compared with that (V0) for an appropriate control. Here we describe our standard protocol using an automated FAC system (FAC-1), consisting of two isocratic pumps, an autosampler, a column oven and two miniature columns connected to a fluorescence detector. Analysis time for 100 sugar-protein interactions is approximately 10 h, using as little as 2.5 pmol of pyridylaminated (PA) oligosaccharide per analysis. Using FAC-FD, we have so far obtained quantitative interaction data of >100 lectins for >100 PA oligosaccharides.
The Jacalin-related lectin (JRL) family comprises galactose-binding-type (gJRLs) and mannose-binding-type (mJRLs) lectins. Although the documented occurrence of gJRLs is confined to the family Moraceae, mJRLs are widespread in the plant kingdom. A detailed comparison of sugar-binding specificity was made by frontal affinity chromatography to corroborate the structure-function relationships of the extended mJRL subfamily. Eight mJRLs covering a broad taxonomic range were used: Artocarpin from Artocarpus integrifolia (jackfruit, Moraceae), BanLec from Musa acuminata (banana, Musaceae), Calsepa from Calystegia sepium (hedge bindweed, Convolvulaceae), CCA from Castanea crenata (Japanese chestnut, Fagaceae), Conarva from Convolvulus arvensis (bindweed, Convolvulaceae), CRLL from Cycas revoluta (King Sago palm tree, Cycadaceae), Heltuba from Helianthus tuberosus (Jerusalem artichoke, Asteraceae) and MornigaM from Morus nigra (black mulberry, Moraceae). The result using 103 pyridylaminated glycans clearly divided the mJRLs into two major groups, each of which was further divided into two subgroups based on the preference for high-mannose-type N-glycans. This criterion also applied to the binding preference for complex-type N-glycans. Notably, the result of cluster analysis of the amino acid sequences clearly corresponded to the above specificity classification. Thus, marked correlation between the sugar-binding specificity of mJRLs and their phylogeny should shed light on the functional significance of JRLs.
To obtain lectins without tedious purification steps, we developed a convenient method for a one-step purification of lectins using sugar-immobilized gold nano-particles (SGNPs). Proteins in crude extracts from plant materials were precipitated with 60% ammonium sulphate, and the precipitate was re-dissolved in a small volume of phosphate buffer. The resultant solution was then mixed with appropriate SGNPs under an optimized condition. After incubating overnight at 4 degrees C, lectins in the mixture formed aggregate with SGNPs, which was visually detected and easily sedimented by centrifugation. The aggregate was dissolved by adding inhibitory sugars, which were identical to the non-reducing sugar moieties on the SGNPs. According to SDS-PAGE and MS of thus obtained proteins, it was found that SGNPs isolated lectins with a high purity. For example, a protein isolated from banana using Glcalpha-GNP (alpha-glucose-immobilized gold nano-particle) was identified as banana lectin by trypsin-digested peptide-MS finger printing method.
One of the predominant proteins in the pulp of ripe bananas (Musa acuminata L.) and plantains (Musa spp.) has been identified as a lectin. The banana and plantain agglutinins (called BanLec and PlanLec, respectively) were
purified in reasonable quantities using a novel isolation procedure, which prevented adsorption of the lectins onto insoluble
endogenous polysaccharides. Both BanLec and PlanLec are dimeric proteins composed of two identical subunits of 15 kDa. They
readily agglutinate rabbit erythrocytes and exhibit specificity towards mannose. Molecular cloning revealed that BanLec has
sequence similarity to previously described lectins of the family of jacalin-related lectins, and according to molecular modelling
studies has the same overall fold and three-dimensional structure. The identification of BanLec and PlanLec demonstrates the
occurrence of jacalin-related lectins in monocot species, suggesting that these lectins are more widespread among higher plants
than is actually believed. The banana and plantain lectins are also the first documented examples of jacalin-related lectins,
which are abundantly present in the pulp of mature fruits but are apparently absent from other tissues. However, after treatment
of intact plants with methyl jasmonate, BanLec is also clearly induced in leaves. The banana lectin is a powerful murine T-cell
mitogen. The relevance of the mitogenicity of the banana lectin is discussed in terms of both the physiological role of the
lectin and the impact on food safety.
Enzymes that hydrolyze insoluble complex polysaccharide structures contain non-catalytic carbohydrate binding modules (CBMS) that play a pivotal role in the action of these enzymes against recalcitrant substrates. Family 6 CBMs (CBM6s) are distinct from other CBM families in that these protein modules contain multiple distinct ligand binding sites, a feature that makes CBM6s particularly appropriate receptors for the beta-1,3-glucan laminarin, which displays an extended U-shaped conformation. To investigate the mechanism by which family 6 CBMs recognize laminarin, we report the biochemical and structural properties of a CBM6 (designated BhCBM6) that is located in an enzyme, which is shown, in this work, to display beta-1,3-glucanase activity. BhCBM6 binds beta-1,3-glucooligosaccharides with affinities of approximately 1 x 10(5) m(-1). The x-ray crystal structure of this CBM in complex with laminarihexaose reveals similarity with the structures of other CBM6s but a unique binding mode. The binding cleft in this protein is sealed at one end, which prevents binding of linear polysaccharides such as cellulose, and the orientation of the sugar at this site prevents glycone extension of the ligand and thus conferring specificity for the non-reducing ends of glycans. The high affinity for extended beta-1,3-glucooligosaccharides is conferred by interactions with the surface of the protein located between the two binding sites common to CBM6s and thus reveals a third ligand binding site in family 6 CBMs. This study therefore demonstrates how the multiple binding clefts and highly unusual protein surface of family 6 CBMs confers the extensive range of specificities displayed by this protein family. This is in sharp contrast to other families of CBMs where variation in specificity between different members reflects differences in the topology of a single binding site.
On reaction with tributylstannane, O-cycloalkyl thiobenzoates and O-cycloalkyl S-methyl dithiocarbonates, derived from secondary alcohols, give good yields of the corresponding hydrocarbons. The mechanism of this planned reaction is radical in character and thus rearrangements common in carbocation reactions are avoided. The particular applicability of this procedure in sugar chemistry is illustrated. The reaction takes place under neutral conditions compatible with the presence of the functional groups which normally occur in aminoglycoside antibiotics.
Evidence based on the quantitative precipitin method and hapten inhibition technique demonstrates that concanavalin A may interact with internal 2-O-linked alpha-D-mannopyranosyl residues as may occur in glycoproteins and polysaccharides.
A new titration calorimeter is described and results are presented for the binding of cytidine 2'-monophosphate (2'CMP) to the active site of ribonuclease A. The instrument characteristics include very high sensitivity, rapid calorimetric response, and fast thermal equilibration. Convenient software is available for instrument operation, data collection, data reduction, and deconvolution to obtain least-squares estimates of binding parameters n, delta H degree, delta S degree, and the binding constant K. Sample through-put for the instrument is high, and under favorable conditions binding constants as large as 10(8) M-1 can be measured. The bovine ribonuclease A (RNase)/2'CMP system was studied over a 50-fold range of RNase concentration and at two different temperatures. The binding constants were in the 10(5) to 10(6) M-1 range, depending on conditions, and heats of binding ca. -15,000 cal/mol. Repeat determinations suggested errors of only a few percent in n, delta H degree, and K values over the most favorable concentration range.
The interaction of concanavalin A, the phytohemagglutinin of the jack bean, with sophorose and several of its derivatives was studied by examining the extent to which these saccharides inhibited dextranconcanavalin A interaction. It is shown that the protein combining sites interact with the C-3, C-4, and C-6 hydroxyl groups of the reducing D-glucopy-ranose unit of sophorose in distinction to combination with similar hydroxyl groups of the nonreducing D-glucopyranosyl residue of α-linked glucose disaccharides. The data which establish this point include the observations that: (1) 2-O-β-D-galactopyranosyl- D-glucose falls on the same line of inhibition as sophorose; D-galactose and its α- and β-glycosides are noninhibitors of dextran-concanavalin A interaction. (2) Sophoritol is a noninhibitor. (3) In analogy with the glycosides of D-glucose, α-methyl sophoroside is a better and β-methyl sophoroside a poorer inhibitor than sophorose. These modifications involve only the reducing moiety of sophorose. (4) Concanavalin A interacts to form a precipitate with bovine serum albumin containing multiple p-phenylazo-β-sophorosyl residues.
Laminaribiose () was coupled to bovine serum albumin (BSA) by an azophenyl linkage to provide the synthetic antigen BSA-p-phenylazo-β-laminaribioside. The behavior of antisera prepared in rabbits immunized with the β-laminaribiosyl conjugate was examined by immunodiffusion, quantitative precipitation and hapten inhibition. Anticonjugate absorbed with carrier protein showed the greatest reactivity with the homologous β-laminaribiosyl-BSA antigen, but also showed some cross precipitation with β-cellobiosyl, β-sophorosyl and β-gentiobiosyl-BSA conjugates. Glucobioses linked through the β-(1 → 2), β-(1 → 3), β-(1 → 4) and β-(1 → 6) positions, as well as laminaridextrins and tri and tetrasaccharides of β-linked glucose possessing a laminaribiose moiety either at a nonreducing end location or at a subterminal location, were assayed for their ability to inhibit antilaminaribioside precipitation. Hapten inhibition data showed anticonjugate a possess a high degree of specificity directed against the terminal nonreducing β-laminaribiosyl end group.
This paper extends our knowledge of the rather bizarre carbohydrate binding poperties of the banana lectin (Musa acuminata). Although a glucose/mannose binding protein which recognizes alpha-linked gluco-and manno-pyranosyl groups of polysaccharide chain ends, the banana lectin was shown to bind to internal 3-O-alpha-D-glucopyranosyl units. Now we report that this lectin also binds to the reducing glucosyl groups of beta-1,3-linked glucosyl oligosaccharides (e.g. laminaribiose oligomers). Additionally, banana lectin also recognizes beta1,6-linked glucosyl end groups (gentiobiosyl groups) as occur in many fungal beta1,3/1,6-linked polysaccharides. This behavior clearly distinguishes the banana lectin from other mannose/glucose binding lectins, such as concanavalin A and the pea, lentil and Calystegia sepium lectins.
Examination of lectins of banana (Musa acuminata) and the closely related plantain (Musa spp.) by the techniques of quantitative precipitation, hapten inhibition of precipitation, and isothermal titration calorimetry showed that they are mannose/glucose binding proteins with a preference for the alpha-anomeric form of these sugars. Both generate precipitin curves with branched chain alpha-mannans (yeast mannans) and alpha-glucans (glycogens, dextrans, and starches), but not with linear alpha-glucans containing only alpha1,4- and alpha1,6-glucosidic bonds (isolichenan and pullulan). The novel observation was made that banana and plantain lectins recognize internal alpha1,3-linked glucosyl residues, which occur in the linear polysaccharides elsinan and nigeran. Concanavalin A and lectins from pea and lentil, also mannose/glucose binding lectins, did not precipitate with any of these linear alpha-glucans. This is, the authors believe, the first report of the recognition of internal alpha1,3-glucosidic bonds by a plant lectin. It is possible that these lectins are present in the pulp of their respective fruit, complexed with starch.
Over the last decade isothermal titration calorimetry (ITC) has developed from a specialist method which was largely restricted in its use to dedicated experts, to a major, commercially available tool in the arsenal directed at understanding molecular interactions. The number of those proficient in this field has multiplied dramatically, as has the range of experiments to which this method has been applied. This has led to an overwhelming amount of new data and novel applications to be assessed. With the increasing number of publications in this field comes a need to highlight works of interest and impact. In this overview of the literature we have attempted to draw attention to papers and issues for which both the experienced calorimetrist and the interested dilettante hopefully will share our enthusiasm.
Banana lectin (Banlec) is a dimeric plant lectin from the jacalin-related lectin family. Banlec belongs to a subgroup of this family that binds to glucose/mannose, but is unique in recognizing internal alpha1,3 linkages as well as beta1,3 linkages at the reducing termini. Here we present the crystal structures of Banlec alone and with laminaribiose (LAM) (Glcbeta1, 3Glc) and Xyl-beta1,3-Man-alpha-O-Methyl. The structure of Banlec has a beta-prism-I fold, similar to other family members, but differs from them in its mode of sugar binding. The reducing unit of the sugar is inserted into the binding site causing the second saccharide unit to be placed in the opposite orientation compared with the other ligand-bound structures of family members. More importantly, our structures reveal the presence of a second sugar binding site that has not been previously reported in the literature. The residues involved in the second site are common to other lectins in this family, potentially signaling a new group of mannose-specific jacalin-related lectins (mJRL) with two sugar binding sites.
Stereochemistry of Carbohydrates
- J F Stoddart