Sugar-binding Properties Research Articles

Structural Basis for Multiple Sugar Recognition of Jacalin-related Human ZG16p Lectin

ZG16p is a soluble mammalian lectin, the first to be described with a Jacalin-related β-prism-fold. ZG16p has been reported to bind both to glycosaminoglycans and mannose. To determine the structural basis of the multiple sugar-binding properties, we conducted glycan microarray analyses of human ZG16p. We observed that ZG16p preferentially binds to α-mannose-terminating short glycans such as Ser/Thr-linked O-mannose, but not to high mannose-type N-glycans. Among sulfated glycosaminoglycan oligomers examined, chondroitin sulfate B and heparin oligosaccharides showed significant binding. Crystallographic studies of human ZG16p lectin in the presence of selected ligands revealed the mechanism of multiple sugar recognition. Manα1-3Man and Glcβ1-3Glc bound in different orientations: the nonreducing end of the former and the reducing end of the latter fitted in the canonical shallow mannose binding pocket. Solution NMR analysis using (15)N-labeled ZG16p defined the heparin-binding region, which is on an adjacent flat surface of the protein. On-array competitive binding assays suggest that it is possible for ZG16p to bind simultaneously to both types of ligands. Recognition of a broad spectrum of ligands by ZG16p may account for the multiple functions of this lectin in the formation of zymogen granules via glycosaminoglycan binding, and in the recognition of pathogens in the digestive system through α-mannose-related recognition.

Galectin-3 induces cell migration via a calcium-sensitive MAPK/ERK1/2 pathway.

The presence and level of circulating galectin-3 (Gal-3), a member of the galectin family, is associated with diverse diseases ranging from heart failure, immune disorders to cancer metastasis and serves as a biomarker of diagnosis and treatment response. However, the mechanisms by which exogenous Gal-3 affects pathobiology events remain elusive. In the current study, we found that exogenous Gal-3 slightly delays, while prolonging tyrosine phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in HeLa cells through a calcium-sensitive and PKC-dependent signaling pathway. The activation was dependent on the sugar-binding properties of Gal-3, since the antagonist lactose could inhibit it. The sugar-binding motif of Gal-3 was required for the activation of ERK1/2. The activation of ERK1/2 was necessary for the initiation and induction of cell migration associated with the phosphorylation of paxillin. All the results presented in this study suggest a novel calcium-sensitive and PKC-dependent pathway through which circulating Gal-3 promotes cell migration and activating the ERK1/2. Taken together, the data depicted here propose a biological function and a target for the diseases' associated circulating Gal-3.

Cadmium-Free Sugar-Chain-Immobilized Fluorescent Nanoparticles Containing Low-Toxicity ZnS-AgInS2 Cores for Probing Lectin and Cells

Sugar chains play a significant role in various biological processes through sugar chain-protein and sugar chain-sugar chain interactions. To date, various tools for analyzing sugar chains biofunctions have been developed. Fluorescent nanoparticles (FNPs) functionalized with carbohydrate, such as quantum dots (QDs), are an attractive imaging tool for analyzing carbohydrate biofunctions in vitro and in vivo. Most FNPs, however, consist of highly toxic elements such as cadmium, tellurium, selenium, and so on, causing problems in long-term bioimaging because of their cytotoxicity. In this study, we developed cadmium-free sugar-chain-immobilized fluorescent nanoparticles (SFNPs) using ZnS-AgInS2 (ZAIS) solid solution nanoparticles (NPs) of low or negligible toxicity as core components, and investigated their bioavailability and cytotoxicity. SFNPs were prepared by mixing our originally developed sugar-chain-ligand conjugates with ZAIS/ZnS core/shell NPs. In binding experiments with lectin, the obtained ZAIS/ZnS SFNPs interacted with an appropriate lectin to give specific aggregates, and their binding interaction was visually and/or spectroscopically detected. In addition, these SFNPs were successfully utilized for cytometry analysis and cellular imaging in which the cell was found to possess different sugar-binding properties. The results of the cytotoxicity assay indicated that SFNPs containing ZAIS/ZnS have much lower toxicity than those containing cadmium. These data strongly suggest that our designed SFNPs can be widely utilized in various biosensing applications involved in carbohydrates.

Coupling Evolutionary Information with Functional Dynamics to Reengineer the Oligosaccharide Specificity of Cvn with Bp-Dock

Cyanovirin-N (CVN) is a cyanobacterial lectin with potent anti-HIV activity, mediated by binding to Man(1,2)Man with high affinity and specificity. These sugar-binding sites are located in two quasi-symmetric domains (A and B) of the CVN protein. We explore both the evolutionary and functional dynamics of CVN and reengineer CVN mutants with different binding affinity for the oligo-mannosides, through integrated computational and experimental strategies. We perform statistical analysis on CVN homologs to find correlated mutations between amino acids1 and in parallel; we also analyze the positions whose correlated fluctuations are critical for function through a dynamic flexibility index (dfi) metric.2 Integrating the evolutionary information with the dynamically coupled positions that are critical for function, we model various CVN mutants and obtain these variants in complex with di-mannose sugar with a fast and flexible docking method called BP-Dock.3 We later characterize these mutants experimentally with NMR or ITC. Overall, this study helps us to explore the sugar binding properties of CVN mutants obtained from evolutionary and functional analysis and thus provide better insights about the mechanism of glycan recognition.

Reengineering the Oligosaccharide Specificity of CVN with BP_Dock

Cyanovirin-N (CV-N) is a cyanobacterial lectin with potent anti-HIV activity, mediated by binding to Man(1,2)Man with high affinity and specificity. These sugar-binding sites are located in two quasi-symmetric domains (A and B) of the CV-N protein. We explore the sequence space of CV-N and reengineered CV-N mutants with different binding affinity for the oligo-mannosides, through integrated computational and experimental strategies. using the sequences of two engineered CV-N,1,2 we model various CV-N mutants, and obtain these variants in complex with di-mannose sugar with a fast and flexible docking method called BP-Dock. BP-Dock can integrate both backbone and side chain conformational changes in a protein through a multi-scale approach. It mimics the nature of binding induced events by perturbation of the binding site residues through small Brownian kicks as a first order approximation of an approaching ligand. Then it computes the response fluctuation profile of the chains using the perturbation response scanning method. The response fluctuation profiles are then used to generate binding induced multiple receptor conformations of CV-N mutants for ensemble docking. The results from docking simulation are then validated with NMR experiments. Overall, this study helps us to explore the sugar binding properties of CV-N mutants and provides a better insight about the binding site of CV-N.

Pre-feeding of a glycolipid binding protein LEC-8 from Caenorhabditis elegans revealed enhanced tolerance to Cry1Ac toxin in Helicoverpa armigera

Crystal toxins from Bacillus thuringiensis bind to glycolipids and glycoproteins using two different lectin domains in the toxin protein. Our previous observations suggested that the sequestration of crystal toxin depends on the functional interaction of a toxin lectin with glycolipids. Given the finding that competition of a galectin LEC-8 with Cry5B for binding to glycolipids resulting in reduced Bt toxicity in nematode, it is interesting to explore the role of LEC-8 in insects. Here, we reported that the LEC-8 can also be exploited by insect for their survival when they were fed with Bt toxin food. Bioassay with LEC-8 showed that pre-feeding of Helicoverpa armigera larvae reduced the Cry1Ac susceptibility. Both LEC-8 and Cry1Ac bind to the midgut glycolipid in a similar way. Further ELISA indicated that LEC-8 interacts with glycolipid from insect midgut, thus reduce Cry1Ac binding to glycolipid. This in turn enhances insect tolerance to Cry1Ac toxin. The sugar determinants of LEC-8 were studied by using haemagglutination (HA) and haemagglutination inhibition (HAI) assay. It was suggested that the terminal sugar of LEC-8 has multiple sugar binding property.

Ring Structure and Aromatic Substituent Effects on the pK a of the Benzoxaborole Pharmacophore.

In this work, we present an investigation into the physical properties of a unique class of aromatic boronic acids, the benzoxaboroles. Using spectrophotometric methods, the ionization constants of a family of substituted benzoxaboroles are determined. Heterocyclic ring modifications are examined to determine their effects on the ionization of the boronic acid moiety. It is also shown that the substituent effects about the aromatic ring follow a Hammett relationship with the compounds' measured pK a values. Finally, these substituent effects are also shown to extend to the sugar binding properties of these compounds under physiologically relevant conditions. Combined, these data will inform medicinal chemists wishing to tailor the ionization and/or ability of this class of compound to bind diol-containing biomolecules.

Structural and Molecular Basis of Carbohydrate-Protein Interaction Systems as Potential Therapeutic Targets

The sugar chains covalently modifying proteins and lipids are recognized by a variety of proteins, thereby mediating a broad range of physiological and pathological events on cell surfaces as well as in cells. Hence, these carbohydrate-protein interaction systems could be potential therapeutic targets for various diseases, including viral infections, autoimmune diseases and neurodegenerative disorders. Cumulative crystallographic data of lectins complexed with their cognate carbohydrate ligands have elucidated the sugar recognition modes of these proteins, offering a structural basis for the design of drugs targeting carbohydrate-lectin interaction systems. In particular, structural and functional studies of animal L-type lectins, which possess a carbohydrate recognition domain with a structural resemblance to that of leguminous lectins such as concanavalin A, have demonstrated the molecular mechanisms underlying their distinct roles in sorting and trafficking of glycoproteins in cells, exemplifying the structure-based engineering that manipulates the sugar-binding properties of lectins. Furthermore, structural basis has been provided for the functional interplay between the L-type lectin ERGIC-53 and the EF-hand Ca²⁺-binding protein MCFD2 in the intracellular transport of the coagulation factors V and VIII. This article also deals with pathological carbohydrate-protein interactions involving ganglioside clusters on cell surfaces, particularly focusing on the interaction between amyloid β (Aβ) and GM1 ganglioside. This interaction triggers conformational transition and consequent aggregation of Aβ, and therefore, is considered to be a key step in Alzheimer's disease. The recently reported structural information of the Aβ-GM1 interaction is presented, underscoring the significance of assemblages of glycoconjugates as therapeutic targets.

Sugar-Binding Properties of the Two Lectin Domains of LEC-1 with Respect to the Gal.BETA.1-4Fuc Disaccharide Unit Present in Protostomia Glycoconjugates

Galβ1-4Fuc disaccharide unit was recently reported to be the endogenous structure recognized by the galectin LEC-6 isolated from the nematode Caenorhabditis elegans. LEC-1, which is another major galectin from this organism, is a tandem repeat-type galectin that contains two carbohydrate recognition domains, the N-terminal lectin domain (LEC-1Nh) and the C-terminal lectin domain (LEC-1Ch), and was also found to have an affinity for the Galβ1-4Fuc disaccharide unit. In the present study, we compared the binding strengths of LEC-1, LEC-1Nh, and LEC-1Ch to Galβ1-4Fuc, Galβ1-3Fuc, and Galβ1-4GlcNAc units as well as to LEC-6-ligand N-glycans by using frontal affinity chromatography (FAC) analysis. The two lectin domains of LEC-1 exhibited the highest affinity for Galβ1-4Fuc, though sugar-binding properties differed somewhat between LEC-1Nh and LEC-1Ch. Furthermore, these two domains had significantly lower affinities for the LEC-6-binding glycans. These results suggest that the endogenous recognition unit of LEC-1 is likely to be Galβ1-4Fuc, and that the endogenous ligands for LEC-1 are different from those for LEC-6.

Cytotoxic Effects of Native and Recombinant Frutalin, a Plant Galactose‐Binding Lectin, on HeLa Cervical Cancer Cells

Frutalin is the α-D-galactose-binding lectin isolated from breadfruit seeds. Frutalin was obtained from two different sources: native frutalin was purified from its natural origin, and recombinant frutalin was produced and purified from Pichia pastoris. This work aimed to study and compare the effect of native and recombinant frutalin on HeLa cervical cancer cells proliferation and apoptosis. Furthermore, the interaction between frutalin and the HeLa cells was investigated by confocal microscopy. Despite having different carbohydrate-binding affinities, native and recombinant frutalin showed an identical magnitude of cytotoxicity on HeLa cells growth (IC50~100 μg/mL) and equally induced cell apoptosis. The interaction studies showed that both lectins were rapidly internalised and targeted to HeLa cell's nucleus. Altogether, these results indicate that frutalin action is not dependent on its sugar-binding properties. This study provides important information about the bioactivity of frutalin and contributes to the understanding of the plant lectins cytotoxic activity.

Role of the Lectin VIP36 in Post-ER Quality Control of Human α1-Antitrypsin

The leguminous-type (L-type) lectin VIP36 localizes to the Golgi apparatus and cycles early in the secretory pathway. In vitro, VIP36 binds high-mannose glycans with a pH optimum of 6.5, a value similar to the luminal pH of the Golgi apparatus. Although the sugar-binding properties of VIP36 in vitro have been characterized in detail, the function of VIP36 in the intact cell remains unclear as no convincing glycoprotein cargo has been identified. Here, we used yellow fluorescent protein (YFP) fragment complementation to identify luminal interaction partners of VIP36. By screening a human liver cDNA library, we identified the glycoprotein alpha1-antitrypsin (alpha1-AT) as a cargo of VIP36. The VIP36/alpha1-AT complex localized to Golgi and endoplasmic reticulum (ER). In the living cell, VIP36 bound exclusively to the high-mannose form of alpha1-AT. The binding was increased when complex glycosylation was prevented by kifunensine and abolished when the glycosylation sites of alpha1-AT were inactivated by mutagenesis. Silencing VIP36 accelerated alpha1-AT transport, arguing against a role of VIP36 in anterograde traffic. The complex formed by VIP36 and alpha1-AT in the Golgi recycled back to the ER. The combined data are most consistent with a function of VIP36 in post-ER quality control of alpha1-AT.

Real-time monitoring and kinetic parameter estimation of the affinity interaction of jArtinM and rArtinM with peroxidase glycoprotein by the electrogravimetric technique

ArtinM is a D-mannose binding lectin that has been arousing increasing interest because of its biomedical properties, especially those involving the stimulation of Th1 immune response, which confers protection against intracellular pathogens. The potential pharmaceutical applications of ArtinM have motivated the production of its recombinant form (rArtinM) so that it is important to compare the sugar-binding properties of jArtinM and rArtinM in order to take better advantage of the potential applications of the recombinant lectin. In this work, a biosensor framework based on a Quartz Crystal Microbalance was established with the purpose of making a comparative study of the activity of native and recombinant ArtinM protein. The QCM transducer was strategically functionalized to use a simple model of protein binding kinetics. This approach allowed for the determination of the binding/dissociation kinetics rate and affinity equilibrium constant of both forms of ArtinM with horseradish peroxidase glycoprotein (HRP), a N-glycosylated protein that contains the trimannoside Manα1–3[Manα1–6]Man, which is a known ligand for jArtinM ( Jeyaprakash et al., 2004). Monitoring of the real-time binding of rArtinM shows that it was able to bind HRP, leading to an analytical curve similar to that of jArtinM, with statistically equivalent kinetic rates and affinity equilibrium constants for both forms of ArtinM. The lower reactivity of rArtinM with HRP than jArtinM was considered to be due to a difference in the number of Carbohydrate Recognition Domains (CRDs) per molecule of each lectin form rather than to a difference in the energy of binding per CRD of each lectin form.

Dual Specificity of Langerin to Sulfated and Mannosylated Glycans via a Single C-type Carbohydrate Recognition Domain

Langerin is categorized as a C-type lectin selectively expressed in Langerhans cells, playing roles in the first line of defense against pathogens and in Birbeck granule formation. Although these functions are thought to be exerted through glycan-binding activity of the C-type carbohydrate recognition domain, sugar-binding properties of Langerin have not been fully elucidated in relation to its biological functions. Here, we investigated the glycan-binding specificity of Langerin using comprehensive glycoconjugate microarray, quantitative frontal affinity chromatography, and conventional cell biological analyses. Langerin showed outstanding affinity to galactose-6-sulfated oligosaccharides, including keratan sulfate, while it preserved binding activity to mannose, as a common feature of the C-type lectins with an EPN motif. By a mutagenesis study, Lys-299 and Lys-313 were found to form extended binding sites for sulfated glycans. Consistent with the former observation, the sulfated Langerin ligands were found to be expressed in brain and spleen, where the transcript of keratan sulfate 6-O-sulfotransferase is expressed. Moreover, such sulfated ligands were up-regulated in glioblastoma relative to normal brain tissues, and Langerin-expressing cells were localized in malignant brain tissues. Langerin also recognized pathogenic fungi, such as Candida and Malassezia, expressing heavily mannosylated glycans. These observations provide strong evidence that Langerin mediates diverse functions on Langerhans cells through dual recognition of sulfated as well as mannosylated glycans by its uniquely evolved C-type carbohydrate-recognition domain.

Conjugation Process in Spirogyra varians Monitored with FITC-lectins(Zygnemataceae, Chlorophyta)

In Spirogyra, two compatible filaments align parallel to each other making numerous papillae, which connect the opposite cells during conjugation and serve as a passage for a male cytoplasm to move into the female cell (Hoek et al. 1995; Sze 1998; Kim et al. 2007). Conjugation process involves a complex series of events but it always begins with the contact between two papillae protruded from each opposite cell. Therefore, recognition factors are expected along their surfaces. Although there are few studies on the environmental factors inducing conjugation in the zygnematacean species (Grote 1977; Simons et al. 1984; Stabenau and Saeftel 1989; Kato and Ooshima 1997), the key factors which trigger the conjugation and the mechanism of recognition between the compatible filaments are still unknown (Kim et al. 2007). Cell surface carbohydrates are highly suitable for encoding biological information because of their complexity and structural diversity, and hence they have been reported as primary markers for cell-cell recognition events in many organisms (Sharon and Lis 1993, Kim and Kim 1999). Such recognition systems depend on the complementary binding between carbohydrate moieties of a glycoconjugate on one cell with a lectin protein on another cell (Sharon and Lis 1989; Chrispeels and Raikhel 1991; Kim and Kim 1999; Kim et al. 2005). Because of their sugar-binding properties, fluoresceinlabeled lectins have been used as a powerful tool to analyze the characteristics and the distribution of cell surface carbohydrates in many organisms (e.g. Wassarman 1987; Sharon and Lis 1989; Karlsson 1991; Kim and Fritz 1993a, b; Kim et al. 1996; Kim and Kim 1999). Cheli and De Vecchi (1989) showed that the extracellular mucilage of Spirogyra filaments disappeared during conjugation and some glycoproteinaceous materials were accumulated in the cell wall of the developing conjugation papilla, suggesting that the chemical composition in cell surface changed during the process. Our previous study on the conjugation process of Zygnema cruciatum also showed that the surface carbohydrate moiety changes during the conjugation and newly appeared glyAlgae Volume 24(1): 39-45, 2009

Proteomic approaches to study structure, functions and toxicity of legume seeds lectins. Perspectives for the assessment of food quality and safety

Lectins are a structurally diverse class of (glyco)proteins which bind mono- and oligosaccharides with high specificity and in a reversible way. For many years, the unique sugar binding properties of plant lectins have been exploited for the development of biochemical tools for glycoprotein isolation and characterisation, and the use of lectins as a glycoprofiling tool has became much more sophisticated with the advent of lectin microarrays, in which a panel of lectins are immobilized on a single chip for glycomic analysis. Among the numerous lectins studied so far, those from legumes represent the largest family. They can be present at relatively high amounts depending on genetic as well as environmental factors, and are accumulated especially in the seeds. For this reason, some lectins as the phytohemagglutinin from the common bean Phaseolus vulgaris constitute a possible risk, since consumption of raw or incorrectly processed beans has been shown to cause outbreaks of gastroenteritis, nausea and diarrhoea. On the other hand, for these anti-nutritional properties, bean extracts enriched in lectins or in lectin-related amylase inhibitors are also finding a growing use as active ingredients of "weight-blockers" in dietetic preparations for obesity treatment. Current methods to determine the lectin levels in foods are based on immunoenzymatic or toxicity tests, which are largely aspecific. Very recently, the availability of proteomic methodologies has allowed to start development and validation of sensitive and specific assays for detecting trace amounts of harmful lectins in either raw or processed foods. In this review, the main aspects of current and perspective applications of mass spectrometry and proteomic technologies to the structural characterisation of legumes are presented, with focus on issues related to detection, identification, and quantification of phytohemagglutinins relevant for their biochemical, immunological and toxicological aspects.

Sugar-complex structures of the C-half domain of the galactose-binding lectin EW29 from the earthwormLumbricus terrestris

R-type lectins are one of the most prominent types of lectin; they exist ubiquitously in nature and mainly bind to the galactose unit of sugar chains. The galactose-binding lectin EW29 from the earthworm Lumbricus terrestris belongs to the R-type lectin family as represented by the plant lectin ricin. It shows haemagglutination activity and is composed of a single peptide chain that includes two homologous domains: N-terminal and C-terminal domains. A truncated mutant of EW29 comprising the C-terminal domain (rC-half) has haemagglutination activity by itself. In order to clarify how rC-half recognizes ligands and shows haemagglutination activity, X-ray crystal structures of rC-half in complex with D-lactose and N-acetyl-D-galactosamine have been determined. The structure of rC-half is similar to that of the ricin B chain and consists of a beta-trefoil fold; the fold is further divided into three similar subdomains referred to as subdomains alpha, beta and gamma, which are gathered around the pseudo-threefold axis. The structures of sugar complexes demonstrated that subdomains alpha and gamma of rC-half bind terminal galactosyl and N-acetylgalactosaminyl glycans. The sugar-binding properties are common to both ligands in both subdomains and are quite similar to those of ricin B chain-lactose complexes. These results indicate that the C-terminal domain of EW29 uses these two galactose-binding sites for its function as a single-domain-type haemagglutinin.

A C-type lectin of Caenorhabditis elegans: Its sugar-binding property revealed by glycoconjugate microarray analysis

C-type lectins are a family of proteins with an affinity to carbohydrates in the presence of Ca 2+. In the genome of Caenorhabditis elegans, almost 300 genes encoding proteins containing C-type lectin-like domains (CTLDs) have been assigned. However, none of their products has ever been shown to have carbohydrate-binding activity. In the present study, we selected 6 potential C-type lectin genes and prepared corresponding recombinant proteins. One of them encoded by clec-79 was found to have sugar-binding activity by using a newly developed glycoconjugate microarray based on evanescent-field excited fluorescence. CLEC-79 exhibited affinity to sugars containing galactose at the non-reducing terminal, especially to the Galβ1-3GalNAc structure, in the presence of Ca 2+. Combined with structural information of the glycans of C. elegans, these results suggest that CLEC-79 preferentially binds to O-glycans in vivo.

Solution and Crystal Structures of a Sugar Binding Site Mutant of Cyanovirin-N: No Evidence of Domain Swapping

The cyanobacterial lectin Cyanovirin-N (CV-N) exhibits antiviral activity against HIV at a low nanomolar concentration by interacting with high-mannose oligosaccharides on the virus surface envelope glycoprotein gp120. Atomic structures of wild-type CV-N revealed a monomer in solution and a domain-swapped dimer in the crystal, with the monomer comprising two independent carbohydrate binding sites that individually bind with micromolar affinity to di- and trimannoses. In the mutant CVN(mutDB), the binding site on domain B was abolished and the protein was found to be completely inactive against HIV. We determined the solution NMR and crystal structures of this variant and characterized its sugar binding properties. In solution and the crystal, CVN(mutDB) is a monomer and no domain-swapping was observed. The protein binds to Man-3 and Man-9 with similar dissociation constants ( approximately 4 muM). This confirms that the nanomolar activity of wild-type CV-N is related to the multisite nature of the protein carbohydrate interaction.

Caenorhabditis elegans galectins LEC-1–LEC-11: Structural features and sugar-binding properties

Galectins form a large family of β-galactoside-binding proteins in metazoa and fungi. This report presents a comparative study of the functions of potential galectin genes found in the genome database of Caenorhabditis elegans. We isolated full-length cDNAs of eight potential galectin genes ( lec-2– 5 and 8– 11) from a λZAP cDNA library. Among them, lec-2–5 were found to encode 31–35-kDa polypeptides containing two carbohydrate-recognition domains similar to the previously characterized lec-1, whereas lec-8–11 were found to encode 16–27-kDa polypeptides containing a single carbohydrate-recognition domain and a C-terminal tail of unknown function. Recombinant proteins corresponding to lec-1–4, -6, and 8– 10 were expressed in Escherichia coli, and their sugar-binding properties were assessed. Analysis using affinity adsorbents with various β-galactosides, i.e., N-acetyllactosamine (Galβ1-4GlcNAc), lacto- N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc), and asialofetuin, demonstrated that LEC-1–4, -6, and -10 have a significant affinity for β-galactosides, while the others have a relatively lower affinity. These results indicate that the integrity of key amino acid residues responsible for recognition of lactose (Galβ1-4Glc) or N-acetyllactosamine in vertebrate galectins is also required in C. elegans galectins. However, analysis of their fine oligosaccharide-binding properties by frontal affinity chromatography suggests their divergence towards more specialized functions.

Isolation and characterization of four type 2 ribosome inactivating pulchellin isoforms from Abrus pulchellus seeds

Abrus pulchellus seeds contain at least seven closely related and highly toxic type 2 ribosome-inactivating pulchellins, each consisting of a toxic A-chain linked to a sugar binding B-chain. In the present study, four pulchellin isoforms (termed P I, P II, P III and P IV) were isolated by affinity, ion exchange and chromatofocusing chromatographies, and investigated with respect to toxicity and sugar binding specificity. Half maximal inhibitory concentration and median lethal dose values indicate that P I and P II have similar toxicities and that both are more toxic to cultured HeLa cells and mice than P III and P IV. Interestingly, the secondary structural characteristics and sugar binding properties of the respective pairs of isoforms correlate well with the two toxicity levels, in that P I/P II and P III/P IV form two specific subgroups. From the deduced amino acids sequences of the four isoforms, it is clear that the highest similarity within each subgroup is found to occur within domain 2 of the B-chains, suggesting that the disparity in toxicity levels might be attributed to subtle differences in B-chain-mediated cell surface interactions that precede and determine toxin uptake pathways.