A database analysis of jacalin-like lectins: sequence-structure-function relationships

Lectins are known to be important for many biological processes, due to their ability to recognize cell surface carbohydrates with high specificity. Plant lectins have been model systems to study protein-carbohydrate recognition, because individually they exhibit high sensitivity and as a group large diversity in recognizing carbohydrate structures. Although extensive studies have been carried out for legume lectins that have led to interesting insights into the sequence determinants of sugar recognition in them, frameworks with such specific correlations are not available for other plant lectin families. This study reports a large-scale data acquisition and extensive analysis of sequences and structures of beta-prism-I or jacalin-related lectins (JRLs) and shows that hypervariability in the binding site loops generates carbohydrate recognition diversity, a strategy analogous to that in legume lectins. Analyses of the size, conformation, and sequence variability in key regions reveal the existence of a common theme, encoded as a set of structural features over a common scaffold, in defining specificity. This study also points to the remarkable range of domain architectures, often arising out of gene duplication events in lectins of this family. The data analyzed here also indicate a spectacular variety of quaternary associations possible in this family of lectins that have implications for glycan recognition. These results thus provide sequence-structure-function correlations, an understanding of the molecular basis of carbohydrate recognition by beta-prism-I lectins, and also a rationale for engineering specific recognition capabilities in relevant molecules.     

The crystal structure of the Calystegia sepium agglutinin reveals a novel quaternary arrangement of lectin subunits with a beta-prism fold

The high number of quaternary structures observed for lectins highlights the important role of these oligomeric assemblies during carbohydrate recognition events. Although a large diversity in the mode of association of lectin subunits is frequently observed, the oligomeric assemblies of plant lectins display small variations within a single family. The crystal structure of the mannose-binding jacalin-related lectin from Calystegia sepium (Calsepa) has been determined at 1.37-A resolution. Calsepa exhibits the same beta-prism fold as identified previously for other members of the family, but the shape and the hydrophobic character of its carbohydrate-binding site is unlike that of other members, consistent with surface plasmon resonance analysis showing a preference for methylated sugars. Calsepa reveals a novel dimeric assembly markedly dissimilar to those described earlier for Heltuba and jacalin but mimics the canonical 12-stranded beta-sandwich dimer found in legume lectins. The present structure exemplifies the adaptability of the beta-prism building block in the evolution of plant lectins and highlights the biological role of these quaternary structures for carbohydrate recognition.     

Structural and functional characterization of the GalNAc/Gal-specific lectin from the phytopathogenic ascomycete Sclerotinia sclerotiorum (Lib.) de Bary

The lectin found in mycelium and sclerotes of the phytopathogenic fungus Sclerotinia sclerotiorum is a homodimer consisting of two identical non-covalently bound subunits of 16,000 Da. CD spectra analysis revealed that the S. sclerotiorum agglutinin (SSA) contains predominantly beta-sheet structures. SSA exhibits specificity towards GalNAc whereby the hydroxyls at positions 4 and 6 of the pyranose ring play a key role in the interaction with simple sugars. The carbohydrate-binding site of SSA can also accommodate disaccharides. The N-terminal sequence of SSA shares no significant similarity with any other protein except a lectin from the Sclerotiniaceae species Ciborinia camelliae. A comparison of SSA and the lectins from C. camelliae and some previously characterized lectins indicates that the Sclerotiniaceae lectins form a homogeneous family of fungal lectins. This newly identified lectin family, which is structurally unrelated to any other family of fungal lectins, is most probably confined to the Ascomycota.     

Purification and characterisation of a jacalin-related, coleoptile specific lectin from Hordeum vulgare

A plant lectin was isolated from barley (Hordeum vulgare) coleoptiles using acidic extraction and different chromatographic methods. Sequencing of more than 50% of the protein sequence by Edman degradation confirmed a full-length cDNA clone. The subsequently identified open reading frame encodes for a 15 kDa protein which could be found in the soluble fraction of barley coleoptiles. This protein exhibited specificity towards mannose sugar and is therefore, accordingly named as Horcolin (Hordeum vulgare coleoptile lectin). Database searches performed with the Horcolin protein sequence revealed a sequence and structure homology to the lectin family of jacalin-related lectins. Together with its affinity towards mannose, Horcolin is now identified as a new member of the mannose specific subgroup of jacalin-related lectins in monocot species. Horcolin shares a high amino acid homology to the highly light-inducible protein HL#2 and, in addition to two methyl jasmonic acid-inducible proteins of 32.6 and 32.7 kDa where the jasmonic acid-inducible proteins are examples of bitopic chimerolectins containing a dirigent and jacalin-related domain. Immunoblot analysis with a cross-reactive anti-HL#2 antibody in combination with Northern blot analysis of the Horcolin cDNA revealed tissue specific expression of Horcolin in the coleoptiles. The function of Horcolin is discussed in the context of its particular expression in coleoptiles and is then compared to other lectins, which apparently share a related response to biotic or abiotic stress factors.     

Xenopus galectin-VIIa binds N-glycans of members of the cortical granule lectin family (xCGL and xCGL2)

We have identified members of the Xenopus cortical granule lectin (xCGL) family as candidate target glycoproteins of Xenopus galectin-VIIa (xgalectin-VIIa) in Xenopus embryos. In addition to the original xCGL, we also identified a novel member of the xCGL family, xCGL2. Expression of the mRNAs of xCGL and xCGL2, as well as that of xgalectin-VIIa, was observed throughout early embryogenesis. Two and three potential N-glycosylation sites were deduced from the amino acid sequences of xCGL and xCGL2, respectively, and xgalectin-VIIa recognizes N-glycans linked to a common site in xCGL and xCGL2 and also recognizes N-glycans linked to a site specific to xCGL2. However, interaction between xgalectin-Ia and xCGLs was not detectable. We also obtained consistent results on surface plasmon resonance analysis involving xCGLs as ligands and xgalectins as analytes. The Kd value of the interaction between xgalectin-VIIa and xCGLs was calculated to be 35.9 nM. The structures of the N-glycans of xCGLs, which were recognized by xgalectin-VIIa, were analyzed by the two-dimensional sugar map method, and three kinds of N-acetyllactosamine type, biantennary N-glycans were identified as the major neutral N-glycans. The binding specificity of oligosaccharides for xgalectin-VIIa was analyzed by frontal affinity chromatography (FAC). The oligosaccharide specificity pattern of xgalectin-VIIa was similar to that of the human homolog galectin-3, and it was also shown that the N-acetyllactosamine type, biantennary N-glycans exhibit high affinity for xgalectin-VIIa (Kd = 11 microM). These results suggest that xgalectin-VIIa interacts with xCGLs through binding to N-acetyllactosamine type N-glycans and that this interaction might make it possible to organize a lectin network involving members of different lectin families.     

The lectin from leaves of Japanese cycad, Cycas revoluta Thunb. (gymnosperm) is a member of the jacalin-related family.

A novel lectin was isolated from leaves of the Japanese cycad, Cycas revoluta Thunb. (gymnosperm), and its characteristics including amino acid composition, molecular mass, carbohydrate binding specificity and partial amino acid sequences were examined. The inhibition analysis of hemagglutinating activity with various sugars showed that the lectin has a carbohydrate-binding specificity similar to those of mannose recognizing, jacalin-related lectins. Partial amino acid sequences of the lysylendopeptic peptides shows that the lectin might have a repeating structure and belong to the jacalin-related lectin family.     

HCA and HML isolated from the red marine algae Hypnea cervicornis and Hypnea musciformis define a novel lectin family

HCA and HML represent lectins isolated from the red marine algae Hypnea cervicornis and Hypnea musciformis, respectively. Hemagglutination inhibition assays suggest that HML binds GalNAc/Gal substituted with a neutral sugar through 1-3, 1-4, or 1-2 linkages in O-linked mucin-type glycans, and Fuc(alpha1-6)GlcNAc of N-linked glycoproteins. The specificity of HCA includes the epitopes recognized by HML, although the glycoproteins inhibited distinctly HML and HCA. The agglutinating activity of HCA was inhibited by GalNAc, highlighting the different fine sugar epitope-recognizing specificity of each algal lectin. The primary structures of HCA (9193+/-3 Da) and HML (9357+/-1 Da) were determined by Edman degradation and tandem mass spectrometry of the N-terminally blocked fragments. Both lectins consist of a mixture of a 90-residue polypeptide containing seven intrachain disulfide bonds and two disulfide-bonded subunits generated by cleavage at the bond T50-E51 (HCA) and R50-E51 (HML). The amino acid sequences of HCA and HML display 55% sequence identity (80% similarity) between themselves, but do not show discernible sequence and cysteine spacing pattern similarities with any other known protein structure, indicating that HCA and HML belong to a novel lectin family. Alignment of the amino acid sequence of the two lectins revealed the existence of internal domain duplication, with residues 1-47 and 48-90 corresponding to the N- and C-terminal domains, respectively. The six conserved cysteines in each domain may form three intrachain cysteine linkages, and the unique cysteine residues of the N-terminal (Cys46) and the C-terminal (Cys71) domains may form an intersubunit disulfide bond.     

Unusual sugar specificity of banana lectin from Musa paradisiaca and its probable evolutionary origin. Crystallographic and modelling studies

The crystal structure of a complex of methyl-alpha-D-mannoside with banana lectin from Musa paradisiaca reveals two primary binding sites in the lectin, unlike in other lectins with beta-prism I fold which essentially consists of three Greek key motifs. It has been suggested that the fold evolved through successive gene duplication and fusion of an ancestral Greek key motif. In other lectins, all from dicots, the primary binding site exists on one of the three motifs in the three-fold symmetric molecule. Banana is a monocot, and the three motifs have not diverged enough to obliterate sequence similarity among them. Two Greek key motifs in it carry one primary binding site each. A common secondary binding site exists on the third Greek key. Modelling shows that both the primary sites can support 1-2, 1-3, and 1-6 linked mannosides with the second residue interacting in each case primarily with the secondary binding site. Modelling also readily leads to a bound branched mannopentose with the nonreducing ends of the two branches anchored at the two primary binding sites, providing a structural explanation for the lectin's specificity for branched alpha-mannans. A comparison of the dimeric banana lectin with other beta-prism I fold lectins, provides interesting insights into the variability in their quaternary structure.     

The galactose-binding and mannose-binding jacalin-related lectins are located in different sub-cellular compartments

A galactose-specific and a mannose-specific lectin of the family of the jacalin-related lectins have been localized by immunofluorescence microscopy. The present localization studies provide for the first time unambiguous evidence for the cytoplasmic location of the mannose-specific jacalin-related lectin from rhizomes of Calystegia sepium, which definitely differs from the vacuolar location of the galactose-specific jacalin from Artocarpus integrifolia. These observations support the hypothesis that the galactose-specific jacalin-related lectins evolved from their mannose-specific homologues through the acquisition of vacuolar targeting sequences.     

The carbohydrate-recognition domain of Dectin-2 is a C-type lectin with specificity for high mannose

We examined the carbohydrate-binding potential of the C-type lectin-like receptor Dectin-2 (Clecf4n). The carbohydrate-recognition domain (CRD) of Dectin-2 exhibited cation-dependent mannose/fucose-like lectin activity, with an IC(50) for mannose of approximately 20 mM compared to an IC(50) of 1.5 mM for the macrophage mannose receptor when assayed by similar methodology. The extracellular domain of Dectin-2 exhibited binding to live Candida albicans and the Saccharomyces-derived particle zymosan. This binding was completely abrogated by cation chelation and was competed by yeast mannans. We compared the lectin activity of Dectin-2 with that of two other C-type lectin receptors (mannose receptor and SIGNR1) known to bind fungal mannans. Both mannose receptor and SIGNR1 were able to bind bacterial capsular polysaccharides derived from Streptococcus pneumoniae, but interestingly they exhibited distinct binding profiles. The Dectin-2 CRD exhibited only weak interactions to some of these capsular polysaccharides, indicative of different structural or affinity requirements for binding, when compared with the other two lectins. Glycan array analysis of the carbohydrate recognition by Dectin-2 indicated specific recognition of high-mannose structures (Man(9)GlcNAc(2)). The differences in the specificity of these three mannose-specific lectins indicate that mannose recognition is mediated by distinct receptors, with unique specificity, that are expressed by discrete subpopulations of cells, and this further highlights the complex nature of carbohydrate recognition by immune cells.     

Differences in the glycosylation of rat submandibular kallikreins

The glycosylations of five different rat submandibular kallikreins, rK1, rK2, rK7, rK9 and rK10, vacuum-blotted onto nitrocellulose membranes, have been studied by means of labelled lectins using enhanced chemiluminescence detection. The results demonstrated that individual submandibular kallikreins are not heavily glycosylated in rats, but consistently show different patterns of glycosylation. Following digestion of slot-blotted enzymes with peptide-N-glycosidase F (PNGase): binding by lectin fromLens culinaris (Man-directed) was abolished, whilst that of lectin fromMaclura pomifera (Gal1,3GalNAc-directed) persisted (but could be abolished by periodate oxidation and endo--N-acetylgalactosaminidase digestion), revealing that there are O- as well as N-linked sugar chains on the kallikreins; a novel observation for this family of enzymes. The presence of GalNAc in addition to GlcNAc, Fuc, Gal and Man, in sugar chains of rK1 was confirmed by high pH anion exchange chromatography following acid hydrolysis. Different intensities of binding by lectin fromLimax flavus (NeuNAc-directed) suggest that sialylation of individual kallikreins differs, whilst sialidase and PNGase digestions suggest that sialic acid is the terminal residue of some N-linked but not O-linked structures.     

A new lectin family with structure similarity to actinoporins revealed by the crystal structure of Xerocomus chrysenteron lectin XCL

A newly defined family of fungal lectins displays no significant sequence similarity to any protein in the databases. These proteins, made of about 140 amino acid residues, have sequence identities ranging from 38% to 65% and share binding specificity to N-acetyl galactosamine. One member of this family, the lectin XCL from Xerocomus chrysenteron, induces drastic changes in the actin cytoskeleton after sugar binding at the cell surface and internalization, and has potent insecticidal activity. The crystal structure of XCL to 1.4 A resolution reveals the architecture of this new lectin family. The fold of the protein is not related to any of the several lectin folds documented so far. Unexpectedly, the structure similarity is significant with actinoporins, a family of pore-forming toxins. The specific structural features and sequence signatures in each protein family suggest a potential sugar binding site in XCL and a possible evolutionary relationship between these proteins. Finally, the tetrameric assembly of XCL reveals a complex network of protomer-protomer interfaces and generates a large, hydrated cavity of 1000 A3, which may become accessible to larger solutes after a small conformational change of the protein.     

Multivalent II [beta-D-Galp-(1-->4)-beta-D-GlcpNAc] and Talpha [beta-D-Galp-(1-->3)-alpha-D-GalpNAc] specific Moraceae family plant lectin from the seeds of Ficus bengalensis fruits

A galactose specific lectin was isolated from the seeds of Ficus bengalensis (Moraceae) fruits and designated as F. bengalensis agglutinin (FBA). The lectin was purified by affinity repulsion chromatography on fetuin-agarose and was a monomer of molecular mass 33kDa. Like other Moraceae family lectins, carbohydrate-binding activity of FBA was independent of any divalent cation. FBA did not bind with simple saccharides, however sugar ligands with aromatic aglycons showed pronounced binding. The combining site of FBA recognized preferably Galbeta1,4GlcNAcbeta1-(II) followed by Galbeta1,3GalNAcalpha1-(Talpha) containing glycotopes. Interaction with saccharides revealed that the combining site of FBA could well accommodate a tetrasaccharide, asialo GM1 glycan (Galbeta1,3GalNAcbeta1,4Galbeta1,4Glcbeta1-), whereas polyvalent Tn (GalNAcalpha1-Ser/Thr), one of the well-recognized ligands of Moraceae family lectin, did not interact well with FBA.     

Studies on Galalpha3-binding proteins: comparison of the glycosphingolipid binding specificities of Marasmius oreades lectin and Euonymus europaeus lectin

The carbohydrate binding preferences of the Galalpha3Galbeta4 GlcNAc-binding lectins from Marasmius oreades and Euonymus europaeus were examined by binding to glycosphingolipids on thin-layer chromatograms and in microtiter wells. The M. oreades lectin bound to Galalpha3-terminated glycosphingolipids with a preference for type 2 chains. The B6 type 2 glycosphingolipid (Galalpha3[Fucalpha2]Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) was preferred over the B5 glycosphingolipid (Galalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer), suggesting that the alpha2-linked Fuc is accommodated in the carbohydrate binding site, providing additional interactions. The lectin from E. europaeus had broader binding specificity. The B6 type 2 glycosphingolipid was the best ligand also for this lectin, but binding to the B6 type 1 glycosphingolipid (Galalpha3[Fucalpha2]Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer) was also obtained. Furthermore, the H5 type 2 glycosphingolipid (Fucalpha2Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer), devoid of a terminal alpha3-linked Gal, was preferred over the the B5 glycosphingolipid, demonstrating a significant contribution to the binding affinity by the alpha2-linked Fuc. The more tolerant nature of the lectin from E. europaeus was also demonstrated by the binding of this lectin, but not the M. oreades lectin, to the x2 glycosphingolipid (GalNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer. The A6 type 2 glycosphingolipid (GalNAcalpha3[Fucalpha2]Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and GalNAcalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1-Cer were not recognized by the lectins despite the interaction with B6 type 2 glycosphingolipid and the B5 glycosphingolipid. These observations are explained by the absolute requirement of a free hydroxyl in the 2-position of Galalpha3 and that the E. europaea lectin can accommodate a GlcNAc acetamido moiety close to this position by reorienting the terminal sugar, whereas the M. oreades lectin cannot.     

A lectin and a lectin-related protein are the two most prominent proteins in the bark of yellow wood (Cladrastis lutea).

Using a combination of cDNA cloning and protein purification it is demonstrated that bark of yellow wood (Cladrastis lutea) contains two mannose/glucose binding lectins and a lectin-related protein which is devoid of agglutination activity. One of the lectins (CLAI) is the most prominent bark protein. It is built up of four 32 kDa monomers which are post-translationally cleaved into a 15 kDa and a 17 kDa polypeptide. The second lectin (CLAII) is a minor protein, which strongly resembles CLAI except that its monomers are not cleaved into smaller polypeptides. Molecular cloning of the Cladrastis lectin family revealed also the occurrence of a lectin-related protein (CLLRP) which is the second most prominent bark protein. Although CLLRP shows sequence homology to the true lectins, it is devoid of carbohydrate binding activity. Molecular modelling of the three Cladrastis proteins has shown that their three-dimensional structure is strongly related to the three-dimensional models of other legume lectins and, in addition, revealed that the presumed carbohydrate binding site of CLLRP is disrupted by an insertion of three extra amino acids. Since it is demonstrated for the first time that a lectin and a noncarbohydrate binding lectin-related protein are the two most prominent proteins in the bark of a tree, the biological meaning of their simultaneous occurrence is discussed.     

Banana lectin is unique in its recognition of the reducing unit of 3-O-beta-glucosyl/mannosyl disaccharides: a calorimetric study

The binding of banana lectin (BanLec) to laminaribiose (Glcbeta1,3Glc) 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-beta-substituent to binding. Key areas of interaction involved the 1, 2, and 6 positions of the reducing-terminal hexose unit. The alpha-anomeric configuration of the reducing hexose was strongly favored over the beta-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-beta-glucosyl unit of methyl alpha-laminaribioside could be replaced by any of its monodeoxy derivatives. However, the 4'-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 4'-deoxyglucosyl substitution. Surprisingly, p-nitrobenzyl or beta-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.     

Fruit-specific lectins from banana and plantain

 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. Received: 1 December 1999 / Accepted: 31 January 2000     

Architecture of the sugar binding sites in carbohydrate binding proteins-a computer modeling study

Different sugars, Gal, GalNAc and Man were docked at the monosaccharide binding sites of Erythrina corallodenron (EcorL), peanut lectin (PNA), Lathyrus ochrus (LOLI), and pea lectin (PSL). To study the lectin-carbohydrate interactions, in the complexes, the hydroxymethyl group in Man and Gal favors, gg and gt conformations respectively, and is the dominant recognition determination. The monosaccharide binding site in lectins that are specific to Gal/GalNAc is wider due to the additional amino acid residues in loop D as compared to that in lectins specific to Man/Glc, and affects the hydrogen bonds of the sugar involving residues from loop D, but not its orientation in the binding site. The invariant amino acid residues Asp from loop A, and Asn and an aromatic residue (Phe or Tyr) in loop C provides the basic architecture to recognize the common features in C4 epimers. The invariant Gly in loop B together with one or two residues in the variable region of loop D/A holds the sugar tightly at both ends. Loss of any one of these hydrogen bonds leads to weak interaction. While the subtle variations in the sequence and conformation of peptide fragment that resulted due to the size and location of gaps present in amino acid sequence in the neighborhood of the sugar binding site of loop D/A seems to discriminate the binding of sugars which differ at C4 atom (galacto and gluco configurations). The variations at loop B are important in discriminating Gal and GalNAc binding. The present study thus provides a structural basis for the observed specificities of legume lectins which uses the same four invariant residues for binding. These studies also bring out the information that is important for the design/engineering of proteins with the desired carbohydrate specificity.     

Expression analysis of the nucleocytoplasmic lectin 'Orysata' from rice in Pichia pastoris

The Oryza sativa lectin, abbreviated Orysata, is a mannose-specific, jacalin-related lectin expressed in rice plants after exposure to certain stress conditions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin-related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS/PAGE and western blot analysis showed that the recombinant lectin exists in two molecular forms. Far western blot analysis revealed that the 23 kDa lectin polypeptide contains an N-glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high-mannose structures, Man9-11 glycans being the most abundant. Glycan array analysis showed that Orysata interacts with high-mannose as well as with more complex N-glycan structures. Orysata has potent anti-human immunodeficiency virus and anti-respiratory syncytial virus activity in cell culture compared with other jacalin-related lectins.     

Analysis of sugar chain-binding specificity of tomato lectin using lectin blot: recognition of high mannose-type N-glycans produced by plants and yeast

The sugar chain-binding specificity of tomato lectin (LEA) against glycoproteins was investigated qualitatively using lectin blot analysis. Glycoproteins containing tri- and tetra-antennary complex-type N-glycans were stained with LEA. Unexpectedly, glycoproteins containing high mannose-type N-glycans and a horseradish peroxidase were stained with LEA. LEA blot analysis of the glycoproteins accompanied by treatment with exoglycosidase revealed that the binding site of LEA for the complex-type N-glycans was the N-acetyllactosaminyl side chains, whereas the proximal chitobiose core appeared to be the binding site of LEA for high mannose-type N-glycans. Despite these results, the glycoproteins did not inhibit the hemagglutinating activity of LEA. Among the chitin-binding lectins compared, potato tuber lectin showed specificity similar to LEA on lectin blot analysis, while Datura stramonium lectin and wheat germ agglutinin (WGA) did not interact with glycoproteins containing high mannose-type N-glycans, except that RNase B was stained by WGA. Based on these observations, LEA blot analysis was applied to sugar chain analysis of tomato glycoproteins. The most abundant LEA-reactive glycoprotein was purified from the exocarp of ripe tomato fruits, and was identified as the tomato anionic peroxidase1 (TAP1). These results suggest that LEA interacts with glycoproteins produced by tomatoes, which participate in biological activities in tomato plants.