Structural basis for carbohydrate binding properties of a plant chitinase-like agglutinin with conserved catalytic machinery.

A new chitinase-like agglutinin, RobpsCRA, related to family GH18 chitinases, has previously been identified in black locust (Robinia pseudoacacia) bark. The crystal structure of RobpsCRA at 1.85Å resolution reveals unusual molecular determinants responsible for the lack of its ancestral chitinase activity. Unlike other chitinase-like proteins, which lack chitinase catalytic residues, RobpsCRA has conserved its catalytic machinery. However, concerted rearrangements of loop regions coupled to non-conservative substitutions of aromatic residues central to the chitin-binding groove explain the lack of hydrolytic activity against chitin and the switch toward recognition of high-mannose type N-glycans. Identification of close homologs in flowering plants with conservation of sequence motifs associated to the structural adaptations seen in RobpsCRA defines an emerging class of agglutinins, as emphasized by a phylogenetic analysis, that are likely to share a similar carbohydrate binding specificity for high-mannose type N-glycans. This study illustrates the recent evolution and molecular adaptation of a versatile TIM-barrel scaffold within the ancestral GH18 family.

NICTABA and UDA, two GlcNAc-binding lectins with unique antiviral activity profiles.

OBJECTIVES: This study aimed to assess the antiviral properties of a unique lectin (NICTABA) produced by the tobacco plant, Nicotiana tabacum. METHODS: Cellular assays were used to investigate the antiviral activity of NICTABA and Urtica dioica agglutinin (UDA). Surface plasmon resonance (SPR) studies were performed to study the sugar specificity and the interactions of both lectins with the envelope glycoproteins of HIV-1. RESULTS: The N-acetyl-d-glucosamine (GlcNAc)-binding lectins exhibited broad-spectrum activity against several families of enveloped viruses including influenza A/B, Dengue virus type 2, herpes simplex virus types 1 and 2 and HIV-1/2. The IC50 of NICTABA for various HIV-1 strains, clinical isolates and HIV-2 assessed in PBMCs ranged from 5 to 30 nM. Furthermore, NICTABA inhibited syncytium formation between persistently HIV-1-infected T cells and uninfected CD4+ T lymphocytes and prevented DC-SIGN-mediated HIV-1 transmission to CD4+ target T lymphocytes. However, unlike many other antiviral carbohydrate-binding agents (CBAs) described so far, NICTABA did not block HIV-1 capture to DC-SIGN+ cells and it did not interfere with the binding of the human monoclonal antibody 2G12 to gp120. SPR studies with HIV-1 envelope glycoproteins showed that the affinity of NICTABA for gp120 and gp41 was in the low nanomolar range. The specific binding of NICTABA to gp120 could be prevented in the presence of a GlcNAc trimer, but not in the presence of mannose trimers. NICTABA displayed no antiviral activity against non-enveloped viruses. CONCLUSIONS: Since CBAs possess a high genetic barrier for the development of viral resistance and NICTABA shows a broad antiviral activity profile, this CBA may qualify as a potential antiviral candidate with a pleiotropic mode of action aimed at targeting the entry of enveloped viruses.

Comparative study of the phototoxicity of long-wavelength photosensitizers targeted by the MornigaG lectin.

Morniga G is a plant lectin selective for high density of tumor-associated carbohydrate T and Tn antigens on the surface of cells. The interaction of the protein with Tn induces its cell penetration. This property was used for targeting photosensitizers (consisting of the porphyrins TrMPyP and TPPS, the Al(III)-phthalocyanin AlPcS(4), and the chlorin e6) against leukemic Jurkat T cells after covalent coupling to the protein. The control of MornigaG/photosensitizer loading allowed the comparison of the toxicity of the different photosensitizer conjugates. Conjugate including a single AlPcS(4) per protein appeared promising, since it is poorly toxic when irradiated under white light, while it shows a strong phototoxicity (LD(50) = 4 nM) when irradiated in the therapeutic window, it preferentially kills cancerous lymphocytes, and the sugar binding specificity of the lectin part of the molecule remains unaltered.

Structural basis for sugar recognition, including the Tn carcinoma antigen, by the lectin SNA-II from Sambucus nigra.

Bark of elderberry (Sambucus nigra) contains a galactose (Gal)/N-acetylgalactosamine (GalNAc)-specific lectin (SNA-II) corresponding to slightly truncated B-chains of a genuine Type-II ribosome-inactivating protein (Type-II RIPs, SNA-V), found in the same species. The three-dimensional X-ray structure of SNA-II has been determined in two distinct crystal forms, hexagonal and tetragonal, at 1.90 A and 1.35 A, respectively. In both crystal forms, the SNA-II molecule folds into two linked beta-trefoil domains, with an overall conformation similar to that of the B-chains of ricin and other Type-II RIPs. Glycosylation is observed at four sites along the polypeptide chain, accounting for 14 saccharide units. The high-resolution structures of SNA-II in complex with Gal and five Gal-related saccharides (GalNAc, lactose, alpha1-methylgalactose, fucose, and the carcinoma-specific Tn antigen) were determined at 1.55 A resolution or better. Binding is observed in two saccharide-binding sites for most of the sugars: a conserved aspartate residue interacts simultaneously with the O3 and O4 atoms of saccharides. In one of the binding sites, additional interactions with the protein involve the O6 atom. Analytical gel filtration, small angle X-ray scattering studies and crystal packing analysis indicate that, although some oligomeric species are present, the monomeric species predominate in solution.

Related lectins from snowdrop and maize differ in their carbohydrate-binding specificity.

Searches in an EST database from maize revealed the expression of a protein related to the Galanthus nivalis (GNA) agglutinin, referred to as GNA(maize). Heterologous expression of GNA(maize) in Pichia pastoris allowed characterization of the first nucleocytoplasmic GNA homolog from plants. GNA(maize) is a tetrameric protein which shares 64% sequence similarity with GNA. Glycan microarray analyses revealed important differences in the specificity. Unlike GNA, which binds strongly to high-mannose N-glycans, the lectin from maize reacts almost exclusively with more complex glycans. Interestingly, GNA(maize) prefers complex glycans containing beta1-2 GlcNAc residues. The obvious difference in carbohydrate-binding properties is accompanied by a 100-fold reduced anti-HIV activity. Although the sequences of GNA and GNA(maize) are clearly related they show only 28% sequence identity. Our results indicate that gene divergence within the family of GNA-related lectins leads to changes in carbohydrate-binding specificity, as shown on N-glycan arrays.

Proteins with an Euonymus lectin-like domain are ubiquitous in Embryophyta.

BACKGROUND: Cloning of the Euonymus lectin led to the discovery of a novel domain that also occurs in some stress-induced plant proteins. The distribution and the diversity of proteins with an Euonymus lectin (EUL) domain were investigated using detailed analysis of sequences in publicly accessible genome and transcriptome databases. RESULTS: Comprehensive in silico analyses indicate that the recently identified Euonymus europaeus lectin domain represents a conserved structural unit of a novel family of putative carbohydrate-binding proteins, which will further be referred to as the Euonymus lectin (EUL) family. The EUL domain is widespread among plants. Analysis of retrieved sequences revealed that some sequences consist of a single EUL domain linked to an unrelated N-terminal domain whereas others comprise two in tandem arrayed EUL domains. A new classification system for these lectins is proposed based on the overall domain architecture. Evolutionary relationships among the sequences with EUL domains are discussed. CONCLUSION: The identification of the EUL family provides the first evidence for the occurrence in terrestrial plants of a highly conserved plant specific domain. The widespread distribution of the EUL domain strikingly contrasts the more limited or even narrow distribution of most other lectin domains found in plants. The apparent omnipresence of the EUL domain is indicative for a universal role of this lectin domain in plants. Although there is unambiguous evidence that several EUL domains possess carbohydrate-binding activity further research is required to corroborate the carbohydrate-binding properties of different members of the EUL family.

Analysis of the sugar-binding specificity of mannose-binding-type Jacalin-related lectins by frontal affinity chromatography--an approach to functional classification.

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.

Phylogenetic and specificity studies of two-domain GNA-related lectins: generation of multispecificity through domain duplication and divergent evolution.

A re-investigation of the occurrence and taxonomic distribution of proteins built up of protomers consisting of two tandem arrayed domains equivalent to the GNA [Galanthus nivalis (snowdrop) agglutinin] revealed that these are widespread among monotyledonous plants. Phylogenetic analysis of the available sequences indicated that these proteins do not represent a monophylogenetic group but most probably result from multiple independent domain duplication/in tandem insertion events. To corroborate the relationship between inter-domain sequence divergence and the widening of specificity range, a detailed comparative analysis was made of the sequences and specificity of a set of two-domain GNA-related lectins. Glycan microarray analyses, frontal affinity chromatography and surface plasmon resonance measurements demonstrated that the two-domain GNA-related lectins acquired a marked diversity in carbohydrate-binding specificity that strikingly contrasts the canonical exclusive specificity of their single domain counterparts towards mannose. Moreover, it appears that most two-domain GNA-related lectins interact with both high mannose and complex N-glycans and that this dual specificity relies on the simultaneous presence of at least two different independently acting binding sites. The combined phylogenetic, specificity and structural data strongly suggest that plants used domain duplication followed by divergent evolution as a mechanism to generate multispecific lectins from a single mannose-binding domain. Taking into account that the shift in specificity of some binding sites from high mannose to complex type N-glycans implies that the two-domain GNA-related lectins are primarily directed against typical animal glycans, it is tempting to speculate that plants developed two-domain GNA-related lectins for defence purposes.

Recognition profile of Morus nigra agglutinin (Morniga G) expressed by monomeric ligands, simple clusters and mammalian polyvalent glycotopes.

The carbohydrate binding properties of a novel member of the subfamily of galactose-specific jacalin-related lectin isolated from the bark of black mulberry (Morus nigra) (Morniga G) was studied in detail by enzyme-linked lectinosorbent and inhibition assays using panels of monomeric saccharides, mammalian polyvalent glycotopes and polysaccharides. Among the natural glycans tested for lectin binding, Morniga G reacted best with glycoproteins (gps) presenting a high density of tumor-associated carbohydrate antigens Tn (GalNAcalpha1-Ser/Thr) and Talpha (Galbeta1-3GalNAcalpha1-). Their reactivities, on a nanogram basis, were up to 72.5, 3.9x10(3), 6.0x10(3), 8.8x10(3) and 2.9x10(4) times higher than that of Tn-containing glycopeptides (M.W.<3000 Da), monomeric T, Tn, GalNAc and Gal, respectively. It also reacted well with many multi-antennary N-glycans with II (Galbeta1-4GlcNAc) termini, ABH histo-blood group antigens and their precursors containing high densities of I/II and T/Tn glycotopes, and sialylated T/Tn. Among the mono-, di- and oligosaccharides tested, Thomsen-Friedenreich (T) disaccharide with aromatic aglycon [Galbeta1-3GalNAcalpha1-benzyl (Talpha1-benzyl)] and Tn glycopeptides were the best inhibitors. Molecular modeling and docking studies indicated the occurrence of a primary GalNAcalpha1- and Galbeta1-3GalNAc glycotope-binding site in Morniga G. Using a recently proposed system [Wu, A.M., 2003. Carbohydrate structural units in glycoproteins and polysaccharides as important ligands for Gal and GalNAc reactive lectins. J. Biomed. Sci. 10, 676-688], the binding properties of the combining sites of Morniga G can be defined as follows: (i) the monosaccharide specificity is GalNAc/Gal>>Man/Glc, GlcNAc and lFuc; (ii) the mammalian glycotope specificity is Talpha1-benzyl>T>Tn>GalNAcbeta1-3Gal (P), while B/E (Galalpha1-3/4Gal), I/II (Galbeta1-3/4GlcNAc), S (GalNAcbeta1-4Gal), F/A (GalNAcalpha1-3GalNAc/Gal) and L (Galbeta1-4Glc) are inactive; (iii) the most active ligand is T/Tn; (iv) simple clustered Tn or triantennary N-glycans with II termini (Tri-II) have limited impact; (v) high-density polyvalent glycotopes play a prominent role for enhancing Morniga G reactivity. These results provide evidence for the binding of this lectin to dense cell surface T/Tn glycoconjugates and facilitate future usage of this lectin in biotechnological and medical applications.

Plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle.

We describe the antiviral activity of plant lectins with specificity for different glycan structures against the severe acute respiratory syndrome coronavirus (SARS-CoV) and the feline infectious peritonitis virus (FIPV) in vitro. The SARS-CoV emerged in 2002 as an important cause of severe lower respiratory tract infection in humans, and FIPV infection causes a chronic and often fatal peritonitis in cats. A unique collection of 33 plant lectins with different specificities were evaluated. The plant lectins possessed marked antiviral properties against both coronaviruses with EC(50) values in the lower microgram/ml range (middle nanomolar range), being non-toxic (CC(50)) at 50-100 microg/ml. The strongest anti-coronavirus activity was found predominantly among the mannose-binding lectins. In addition, a number of galactose-, N-acetylgalactosamine-, glucose-, and N-acetylglucosamine-specific plant agglutinines exhibited anti-coronaviral activity. A significant correlation (with an r-value of 0.70) between the EC(50) values of the 10 mannose-specific plant lectins effective against the two coronaviruses was found. In contrast, little correlation was seen between the activity of other types of lectins. Two targets of possible antiviral intervention were identified in the replication cycle of SARS-CoV. The first target is located early in the replication cycle, most probably viral attachment, and the second target is located at the end of the infectious virus cycle.

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.

Expression of garlic leaf lectin under the control of the phloem-specific promoter Asus1 from Arabidopsis thaliana protects tobacco plants against the tobacco aphid (Myzus nicotianae).

BACKGROUND: To check for correlation between the insecticidal properties and the specificity of lectins, a comparative study was made of the insecticidal activities of two garlic lectins with different biological activities. RESULTS: The insecticidal activity of the garlic (Allium sativum L.) leaf lectin ASAL and bulb lectin ASAII towards the tobacco aphid Myzus nicotianae Blackman was studied using bioassays with transgenic tobacco (Nicotiana tabacum L. cv. Wisconsin 38). Bioassays were started with newborn nymphs of the tobacco aphid. Although during the first 7-8 days when nymphs developed to adults there were no apparent effects, part of the nymphal population was found to develop into winged (alate) forms. Later it became clear that transgenic plants expressing ASAL and ASAII had a significant effect on the reproduction capacity of the resulting adults, with a reduction of up to 40%. Different life table parameters such as prereproductive time, intrinsic rate of natural increase, generation time and doubling time were significantly affected (P < 0.05) in aphids grown on transgenic plant material expressing ASAL and ASAII. CONCLUSION: Bioassays with tobacco plants expressing ASAL and ASAII demonstrated a significant impact on the population growth of M. nicotianae. Therefore, both lectins can be considered as valuable candidate aphid control agents.

Crystal structure at 1.45-A resolution of the major allergen endo-beta-1,3-glucanase of banana as a molecular basis for the latex-fruit syndrome.

Resolution of the crystal structure of the banana fruit endo-beta-1,3-glucanase by synchrotron X-ray diffraction at 1.45-A resolution revealed that the enzyme possesses the eightfold beta/alpha architecture typical for family 17 glycoside hydrolases. The electronegatively charged catalytic central cleft harbors the two glutamate residues (Glu94 and Glu236) acting as hydrogen donor and nucleophile residue, respectively. Modeling using a beta-1,3 linked glucan trisaccharide as a substrate confirmed that the enzyme readily accommodates a beta-1,3-glycosidic linkage in the slightly curved catalytic groove between the glucose units in positions -2 and -1 because of the particular orientation of residue Tyr33 delimiting subsite -2. The location of Phe177 in the proximity of subsite +1 suggested that the banana glucanase might also cleave beta-1,6-branched glucans. Enzymatic assays using pustulan as a substrate demonstrated that the banana glucanase can also cleave beta-1,6-glucans as was predicted from docking experiments. Similar to many other plant endo-beta-1,3-glucanases, the banana glucanase exhibits allergenic properties because of the occurrence of well-conserved IgE-binding epitopes on the surface of the enzyme. These epitopes might trigger some cross-reactions toward IgE antibodies and thus account for the IgE-binding cross-reactivity frequently reported in patients with the latex-fruit syndrome.

Localization and in vitro binding studies suggest that the cytoplasmic/nuclear tobacco lectin can interact in situ with high-mannose and complex N-glycans.

The possible in vivo interaction of the Nicotiana tabacum agglutinin (Nictaba) with endogenous glycoproteins was corroborated using a combination of confocal/electron microscopy of an EGFP-Nictaba fusion protein expressed in tobacco Bright Yellow-2 (BY-2) cells and biochemical analyses. In vitro binding studies demonstrated that the expressed EGFP-Nictaba possesses carbohydrate-binding activity. Microscopic analyses confirmed the previously reported cytoplasmic/nuclear location of Nictaba in jasmonate-treated tobacco leaves and provided evidence for the involvement of a nuclear localization signal-dependent transport mechanism. In addition, it became evident that the lectin is not uniformly distributed over the nucleus and the cytoplasm of BY-2 cells. Far Western blot analysis of extracts from whole BY-2 cells and purified nuclei revealed that Nictaba interacts in a glycan inhibitable way with numerous proteins including many nuclear proteins. Enzymatic deglycosylation with PNGase F indicated that the observed interaction depends on the presence of N-glycans. Glycan array screening, which showed that Nictaba exhibits a strong affinity for high-mannose and complex N-glycans, provided a reasonable explanation for this observation. The cytoplasmic/nuclear localization of a plant lectin that has a high affinity for high-mannose and complex N-glycans and specifically interacts with conspecific glycoproteins suggests that N-glycosylated proteins might be more important in the cytoplasm and nucleus than is currently believed.

Resolution of the structure of the allergenic and antifungal banana fruit thaumatin-like protein at 1.7-A.

The structure of a thaumatin-like protein from banana (Musa acuminata) fruit, an allergen with antifungal properties, was solved at 1.7-A-resolution, by X-ray crystallography. Though the banana protein exhibits a very similar overall fold as thaumatin it markedly differs from the sweet-tasting protein by the presence of a surface exposed electronegative cleft. Due to the presence of this electronegative cleft, the banana thaumatin-like protein (Ban-TLP) acquires a strong (local) electronegative character that eventually explains the observed antifungal activity. Our structural analysis also revealed the presence of conserved residues of exposed epitopic determinants that are presumably responsible for the allergenic properties of banana fruit towards susceptible individuals, and provided evidence that the Ban-TLP shares some structurally highly conserved IgE-binding epitopes with thaumatin-like proteins from fruits or pollen from other plants. In addition, some overlap was detected between the predicted IgE-binding epitopes of the Ban-TLP and IgE-binding epitopes previously identified in the mountain cedar Jun a 3 TLP aeroallergen. The presence of these common epitopes offers a molecular basis for the cross-reactivity between aeroallergens and fruit allergens.

Deterrent activity of plant lectins on cowpea weevil Callosobruchus maculatus (F.) oviposition.

A set of 14 plant lectins was screened in a binary choice bioassay for inhibitory activity on cowpea weevil Callosobruchus maculatus (F.) oviposition. Coating of chickpea seeds (Cicer arietinum L.) with a 0.05% (w/v) solution of plant lectins caused a significant reduction in egg laying. Control experiments with heat inactivated lectin and BSA indicated that the observed deterrent effects are specific and require carbohydrate-binding activity. However, no clear correlation could be established between deterrent activity and sugar-binding specificity/molecular structure of the lectins. Increasing the insect density reduced the inhibitory effect of the lectins confirming that female insects are capable of adjusting their oviposition rates as a function of host availability.

Structural analysis of the jacalin-related lectin MornigaM from the black mulberry (Morus nigra) in complex with mannose.

The structures of MornigaM and the MornigaM-mannose complex have been determined at 1.8 A and 2.0 A resolution, respectively. Both structures adopt the typical beta-prism motif found in other jacalin-related lectins and their tetrameric assembly closely resembles that of jacalin. The carbohydrate-binding cavity of MornigaM readily binds mannose. No major structural rearrangements can be observed in MornigaM upon binding of mannose. These results allow corroboration of the structure-function relationships within the small group of Moraceae lectins.