Carbohydrate Recognition Sites Research Articles

Targeting undruggable carbohydrate recognition sites through focused fragment library design

Carbohydrate-protein interactions are key for cell-cell and host-pathogen recognition and thus, emerged as viable therapeutic targets. However, their hydrophilic nature poses major limitations to the conventional development of drug-like inhibitors. To address this shortcoming, four fragment libraries were screened to identify metal-binding pharmacophores (MBPs) as novel scaffolds for inhibition of Ca2+-dependent carbohydrate-protein interactions. Here, we show the effect of MBPs on the clinically relevant lectins DC-SIGN, Langerin, LecA and LecB. Detailed structural and biochemical investigations revealed the specificity of MBPs for different Ca2+-dependent lectins. Exploring the structure-activity relationships of several fragments uncovered the functional groups in the MBPs suitable for modification to further improve lectin binding and selectivity. Selected inhibitors bound efficiently to DC-SIGN-expressing cells. Altogether, the discovery of MBPs as a promising class of Ca2+-dependent lectin inhibitors creates a foundation for fragment-based ligand design for future drug discovery campaigns.

Inflammasome NLRP3 activation induced by Convulxin, a C-type lectin-like isolated from Crotalus durissus terrificus snake venom

Convulxin (CVX), a C-type lectin-like protein isolated from the venom of the snake species, Crotalus durissus terrificus, stimulates platelet aggregation by acting as a collagen receptor agonist for glycoprotein VI found in the platelets. The effect of CVX on platelets has been studied, but its effect on human peripheral blood mononuclear cells (PBMCs) remains unclear. Given the significance of PBMCs in inflammation, this study explored the effect of CVX on PBMCs, specifically regarding NLRP3 inflammasome activation by assessing cell viability, ability to induce cell proliferation, reactive oxygen species (ROS) and nitric oxide production, interleukin (IL)-2 and IL-10 secretion, NLRP3 complex activation, and the role of C-type lectin-like receptors (CTLRs) in these. CVX was not toxic to PBMCs at the investigated concentrations and did not increase PBMC growth or IL-2 release; however, CVX induced IL-10 release and ROS generation via monocyte activation. It also activated the NLRP3 complex, resulting in IL-1β induction. Furthermore, the interaction between CVX and Dectin-2, a CTLR, induced IL-10 production. CVX interaction with CTLR has been demonstrated by laminarin therapy. Because of the involvement of residues near the Dectin-2 carbohydrate-recognition site, the generation of ROS resulted in inflammasome activation and IL-1β secretion. Overall, this work helps elucidate the function of CVX in immune system cells.

Lectin-Triggered Aggregation of Glyco-Gold Nanoprobes for Activity-based Sensing of Hydrogen Peroxide by the Naked Eye.

The purpose of this study was to develop a colorimetric assay for detecting hydrogen peroxide (H2 O2 ) through a combination of using an aryl boronate (AB) derivative and gold nanoparticles (AuNPs). The unique optical property of AuNPs is applied to design a detection probe. The aggregation of AuNPs could be directly observed as a color change by the naked eye. A mannoside-boronate-sulfide (MBS) ligand was designed that contains an arylboronate (AB), a mannoside, and a thiol group. The thiol group bonds covalently with the surface of AuNPs to obtain MBS@AuNPs. The mannoside moiety recognizes concanavalin A (Con A), a lectin with four carbohydrate recognition sites that can specifically recognize the non-reducing end of an α-D-mannoside or α-D-glucoside structure. The AB structure on MBS first reacts with H2 O2 and then inserts an oxygen atom in the B-H bond, which triggers intramolecular electron rearrangement to cleave the covalent bond, resulting in a MBSt mixture. The MBS or MBSt is then modified to citrate-coated AuNPs (c-AuNPs) to have MBS@AuNPs or MBSt@AuNPs. When the MBS@AuNPs are incubated with Con A, the Con A recognizes multiple mannosides on the surface of the MBS@AuNPs. Subsequently, the MBS@AuNPs aggregate and the solution's color changes from red to purple, but this color change does not occur in the case of MBSt@AuNPs. The phenomenon can be observed by the naked eye.

A tandem-repeat galectin-4 from Nile tilapia (Oreochromis niloticus) is involved in immune response to bacterial infection via mediating pathogen recognition and opsonization

Galectins are the family of carbohydrate-binding proteins that participate in host-pathogen interaction. In this study, a galectin-4 homolog (OnGal-4) from Nile tilapia (Oreochromis niloticus) was characterized. The open reading frame of OnGal-4 was 1194 bp, encoding a peptide of 397 amino including two CRD regions and two carbohydrate recognition sites. OnGal-4 mRNA was expressed in all examined tissues with the highest level in spleen. After Streptococcus agalactiae (S.agalactiae) challenge, the OnGal-4 expression was up-regulated in the spleen, head kidney, brain, and monocytes/macrophages (Mo/MΦ). The in vitro experiments showed that recombinant OnGal-4 (rOnGal-4) protein could bind and agglutinate S.agalactiae and A.hydrophila. Also, rOnGal-4 could induce cytokines expressions and increased bactericidal activity of Mo/MΦ. Further in vivo analysis indicated that OnGal-4 overexpression could protect O.niloticus from S.agalactiae infection through modulating inflammation response. Our study suggested that OnGal-4 could improve immune response against bacterial infection by mediating pathogen recognition and opsonization.

Fish Galectin8-Like Exerts Positive Regulation on Immune Response Against Bacterial Infection.

Galectin-8 is a member of the galectin family that is involved in immune response against pathogens. However, the roles of fish galectin-8 during pathogen infection require comprehensive studies. In this study, a galectin-8 homolog (OnGal8-like, OnGal8-L) was characterized from Nile tilapia (Oreochromis niloticus), and its roles in response to bacterial infection were analyzed. The OnGal8-L contains an open reading frame of 891 bp, encoding a peptide of 296 amino acids with two CRD regions of tandem-repeat galectin and two carbohydrate recognition sites. The OnGal8-L protein shares 46.42% identities with reported Oreochromis niloticus galectin-8 protein. Transcriptional expression analysis revealed that OnGal8-L was constitutively expressed in all examined tissues and was highly expressed in spleen. The transcript levels of OnGal8-L were up-regulated in the spleen, head kidney, and brain, following Streptococcus agalactiae (S. agalactiae) challenge. Further in vitro analysis indicated that the recombinant protein of OnGal8-L (rOnGal8L) could agglutinate erythrocyte, S. agalactiae, and A. hydrophila and bind S. agalactiae, A. hydrophila, and various PAMPs (lipopolysaccharides, lipoteichoic acid, poly I:C, peptidoglycan, galactose, mannose, and maltose). Also, rOnGal8L could regulate inflammatory-related gene expression, phagocytosis, and a respiratory burst of monocytes/macrophages. Moreover, in vivo analysis showed that OnGal8-L overexpression could protect O. niloticus from S. agalactiae infection through modulating serum antibacterial activity (AKP, ACP, and LZM), antioxidant capacity (CAT, POD, and SOD), and monocyte/macrophage proliferation and cytokine expression, as well as reducing bacterial burden and decreasing tissue damage. Our results collectively indicate that OnGal8-L plays important regulatory roles in immune response against bacterial infection.

Structures of human galectin-10/monosaccharide complexes demonstrate potential of monosaccharides as effectors in forming Charcot-Leyden crystals

The galectins are a family of β-galactoside-specific animal lectins, and have attracted much attention as novel regulators of the immune system. Galectin-10 is well-expressed in eosinophils, and spontaneously forms Charcot-Leyden crystals (CLCs), during prolonged eosinophilic inflammatory reactions, which are frequently observed in eosinophilic diseases. Although biochemical and structural characterizations of galectin-10 have been done, its biological role and molecular mechanism are still unclear, and few X-ray structures of galectin-10 in complex with monosaccharides/oligosaccharides have been reported. Here, X-ray structures of galectin-10 in complexes with seven monosaccharides are presented with biochemical analyses to detect interactions of galectin-10 with monosaccharides/oligosaccharides. Galectin-10 forms a homo-dimer in the face-to-face orientation, and the monosaccharides bind to the carbohydrate recognition site composed of amino acid residues from two galectin-10 molecules of dimers, suggesting that galectin-10 dimer likely captures the monosaccharides in solution and in vivo. d-Glucose, d-allose, d-arabinose, and D-N-acetylgalactosamine bind to the interfaces between galectin-10 dimers in crystals, and they affect the stability of molecular packing in crystals, leading to easy-dissolving of CLCs, and/or inhibiting the formation of CLCs. These monosaccharides may serve as effectors of G10 to form CLCs in vivo.

L-Fucose prevention of renal ischaemia/reperfusion injury in Mice.

In a recent study, we identified a fucosylated damage‐associated ligand exposed by ischemia on renal tubule epithelial cells, which after recognition by collectin‐11 (CL‐11 or collectin kidney 1 (CL‐K1)), initiates complement activation and acute kidney injury. We exploited the ability to increase the local tissue concentration of free l‐fucose following systemic administration, in order to block ligand binding by local CL‐11 and prevent complement activation. We achieved a thirty‐five‐fold increase in the intrarenal concentration of l‐fucose following an IP bolus given before the ischemia induction procedure ‐ a concentration found to significantly block in vitro binding of CL‐11 on hypoxia‐stressed renal tubule cells. At this l‐fucose dose, complement activation and acute post‐ischemic kidney injury are prevented, with additional protection achieved by a second bolus after the induction procedure. CL‐11−/− mice gained no additional protection from l‐fucose administration, indicating that the mechanism of l‐fucose therapy was largely CL‐11‐dependent. The hypothesis is that a high dose of l‐fucose delivered to the kidney obstructs the carbohydrate recognition site on CL‐11 thereby reducing complement‐mediated damage following ischemic insult. Further work will examine the utility in preventing post‐ischemic injury during renal transplantation, where acute kidney injury is known to correlate with poor graft survival.

Exploration of carbohydrate binding behavior and anti-proliferative activities of Arisaema tortuosum lectin

BackgroundLectins have come a long way from being identified as proteins that agglutinate cells to promising therapeutic agents in modern medicine. Through their specific binding property, they have proven to be anti-cancer, anti-insect, anti-viral agents without affecting the non-target cells. The Arisaema tortuosum lectin (ATL) is a known anti-insect and anti-cancer candidate, also has interesting physical properties. In the present work, its carbohydrate binding behavior is investigated in detail, along with its anti-proliferative property.ResultsThe microcalorimetry of ATL with a complex glycoprotein asialofetuin demonstrated trivalency contributed by multiple binding sites and enthalpically driven spontaneous association. The complex sugar specificity of ATL towards multiple sugars was also demonstrated in glycan array analysis in which the trimannosyl pentasaccharide core N-glycan [Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ] was the highest binding motif. The high binding glycans for ATL were high mannans, complex N-glycans, core fucosylated N-glycans and glycans with terminal lactosamine units attached to pentasaccharide core. ATL induced cell death in IMR-32 cells was observed as time dependent loss in cell number, formation of apoptotic bodies and DNA damage. As a first report of molecular cloning of ATL, the in silico analysis of its cDNA revealed ATL to be a β-sheet rich heterotetramer. A homology model of ATL showed beta prism architecture in each monomer with 85% residues in favoured region of Ramachandran plot.ConclusionsDetailed exploration of carbohydrate binding behavior indicated ATL specificity towards complex glycans, while no binding to simple sugars, including mannose. Sequence analysis of ATL cDNA revealed that during the tandem evolutionary events, domain duplication and mutations lead to the loss of mannose specificity, acquiring of new sugar specificity towards complex sugars. It also resulted in the formation of a two-domain single chain polypeptide with both domains having different binding sites due to mutations within the consensus carbohydrate recognition sites [QXDXNXVXY]. This unique sugar specificity can account for its significant biological properties. Overall finding of present work signifies anti-cancer, anti-insect and anti-viral potential of ATL making it an interesting molecule for future research and/or theragnostic applications.

Mannoside and 1,2-mannobioside β-cyclodextrin-scaffolded NO-photodonors for targeting antibiotic resistant bacteria

Two β-cyclodextrin derivatives randomly appended on the primary face with both the nitric oxide (NO) photodonor 4-nitro-3-(trifluoromethyl)aniline and a mannose or α(1→2)mannobioside residue are reported to construct targeted NO photoreleasing nanocarriers. 2D ROESY and PGSE NMR suggested supramolecular homodimerization in water by inclusion of the nitroaniline group into the facing macrocycle cavities. Isothermal titration calorimetry on their concanavalin A lectin binding showed an exothermic binding event to the lectin and an endothermic process during the dilution of the conjugates. Both α(1→2)mannobioside and the nitroaniline moieties significantly enhanced the binding to the lectin. These effects might arise from a better fit within the carbohydrate-recognition site in the former case and a multivalent effect caused by homodimerization in the latter. Direct detection of NO by amperometric technique shows that both β-cyclodextrin derivatives release this radical upon excitation with visible light with higher efficiency than the unfunctionalized NO photodonor.

Interplay of a secreted protein with type IVb pilus for efficient enterotoxigenic Escherichia coli colonization

Initial attachment and subsequent colonization of the intestinal epithelium comprise critical events allowing enteric pathogens to survive and express their pathogenesis. In enterotoxigenic Escherichia coli (ETEC), these are mediated by a long proteinaceous fiber termed type IVb pilus (T4bP). We have reported that the colonization factor antigen/III (CFA/III), an operon-encoded T4bP of ETEC, possesses a minor pilin, CofB, that carries an H-type lectin domain at its tip. Although CofB is critical for pilus assembly by forming a trimeric initiator complex, its importance for bacterial attachment remains undefined. Here, we show that T4bP is not sufficient for bacterial attachment, which also requires a secreted protein CofJ, encoded within the same CFA/III operon. The crystal structure of CofB complexed with a peptide encompassing the binding region of CofJ showed that CofJ interacts with CofB by anchoring its flexible N-terminal extension to be embedded deeply into the expected carbohydrate recognition site of the CofB H-type lectin domain. By combining this structure and physicochemical data in solution, we built a plausible model of the CofJ-CFA/III pilus complex, which suggested that CofJ acts as a molecular bridge by binding both T4bP and the host cell membrane. The Fab fragments of a polyclonal antibody against CofJ significantly inhibited bacterial attachment by preventing the adherence of secreted CofJ proteins. These findings signify the interplay between T4bP and a secreted protein for attaching to and colonizing the host cell surface, potentially constituting a therapeutic target against ETEC infection.

Mannosylcalix[n]arenes as multivalent ligands for DC-SIGN

DC-SIGN is a receptor protruded from the membrane of immature dendritic cells (DCs) that participates in the activation of the immune response through the recognition of pathogen-associated molecular patterns (PAMPs). On the other hand, HIV exploits the interaction between high-mannose structures of its envelope glycoprotein gp120 and DC-SIGN to be transported towards and infect T-cells. DC-SIGN is involved in the recognition process in the form of a tetramer and the multiple exposition of carbohydrate recognition sites (CRSs) is amplified by the formation on the DCs membrane of patches of tetramers. DC-SIGN is then considered an interesting target to fight the virus and multivalent systems exposing multiple copies of ligating units for its CRSs are becoming valuable tools to reach this goal. We herein prepared four mannosylated calix[n]arenes (1a-d) and tested them by Surface Plasmon Resonance (SPR) competition assays as inhibitors of the binding between DC-SIGN and a mannosylated BSA used as model of HIV gp120. IC50s in the μM range were found evidencing in particular for compound 1a that, although rather moderate, a multivalent effect is taking place in the inhibition activity of this cluster. A relative potency (rp/n) around 4, respect to the monovalent methyl α-mannoside and normalized for the number of monosaccharide on the scaffold, was observed. This result, compared with previously reported data relative to dendrimers with the same valency, indicates the calixarene as a promising scaffold to build efficient inhibitors for DC-SIGN and, in perspective, for HIV.

Structural studies and nociceptive activity of a native lectin from Platypodium elegans seeds (nPELa)

A native lectin (nPELa), purified from seeds of the species Platypodium elegans, Dalbergieae tribe, was crystallized and structurally characterized by X-ray diffraction crystallography and bioinformatics tools. The obtained crystals diffracted to 1.6Å resolution, and nPELa structure were solved through molecular substitution. In addition, nPELa has a metal binding site and a conserved carbohydrate recognition domain (CRD) similar to other Dalbergieae tribe lectins, such as PAL (Pterocarpus angolensis) and CTL (Centrolobium tomentosum). Molecular docking analysis indicated high affinity of this lectin for different mannosides, mainly trimannosides, formed by α-1,3 or α-1,6 glycosidic bond, as evidenced by the obtained scores. In addition, molecular dynamics simulations were performed to demonstrate the structural behavior of nPELa in aqueous solution. In solution, nPELa was highly stable, and structural modifications in its carbohydrate recognition site allowed interaction between the lectin and the different ligands. Different modifications were observed during simulations for each one of the glycans, which included different hydrogen bonds and hydrophobic interactions through changes in the relevant residues. In addition, nPELa was evaluated for its nociceptive activity in mice and was reported to be the first lectin of the Dalbergieae tribe to show CRD-dependent hypernociceptive activity.

Sauromatum guttatum lectin: Spectral studies, lectin-carbohydrate interaction, molecular cloning and in silico analysis

Non-immune carbohydrate binding proteins are broadly defined as lectins. Having been reported from all kingdoms of life, phytolectins are the most widely studied group of lectins. Sauromatum guttatum agglutinin (SGA) was isolated from the plant tubers and characterized for structural variations due to solvent perturbation using polarimetry, fluorescence and light scattering. For the β-sheet rich SGA, a pH and temperature induced molten globule like intermediate was identified. In isothermal titration microcalorimetry, SGA demonstrated cooperative binding to a complex glycoprotein in enthalpically driven mechanism. Fine sugar specificity exploration identified core pentasaccharide as the most common and highest binding motif with complex N-glycans and fucosylated core N-glycans as additional motifs. Molecular cloning of SGA which has previously been demonstrated to have anti-cancer and anti-insect activities is being reported for the first time. Full length cDNA sequence was obtained with RACE-PCR based upon the conserved carbohydrate recognition site [QXDXNXVXY] present in all GNA-related lectins. Quaternary structure was proposed by homology modeling and an attempt was made to explain the structure-function relationship by in silico analysis.

The Atomic Structure of the Phage Tuc2009 Baseplate Tripod Suggests that Host Recognition Involves Two Different Carbohydrate Binding Modules.

ABSTRACTThe Gram-positive bacterium Lactococcus lactis, used for the production of cheeses and other fermented dairy products, falls victim frequently to fortuitous infection by tailed phages. The accompanying risk of dairy fermentation failures in industrial facilities has prompted in-depth investigations of these phages. Lactococcal phage Tuc2009 possesses extensive genomic homology to phage TP901-1. However, striking differences in the baseplate-encoding genes stimulated our interest in solving the structure of this host’s adhesion device. We report here the X-ray structures of phage Tuc2009 receptor binding protein (RBP) and of a “tripod” assembly of three baseplate components, BppU, BppA, and BppL (the RBP). These structures made it possible to generate a realistic atomic model of the complete Tuc2009 baseplate that consists of an 84-protein complex: 18 BppU, 12 BppA, and 54 BppL proteins. The RBP head domain possesses a different fold than those of phages p2, TP901-1, and 1358, while the so-called “stem” and “neck” domains share structural features with their equivalents in phage TP901-1. The BppA module interacts strongly with the BppU N-terminal domain. Unlike other characterized lactococcal phages, Tuc2009 baseplate harbors two different carbohydrate recognition sites: one in the bona fide RBP head domain and the other in BppA. These findings represent a major step forward in deciphering the molecular mechanism by which Tuc2009 recognizes its saccharidic receptor(s) on its host.

Mammalian Cell Surface Display as a Novel Method for Developing Engineered Lectins with Novel Characteristics.

Leguminous lectins have a conserved carbohydrate recognition site comprising four loops (A–D). Here, we randomly mutated the sequence and length of loops C and D of peanut agglutinin (PNA) and expressed the proteins on the surface of mouse green fluorescent protein (GFP)-reporter cells. Flow cytometry, limiting dilution, and cDNA cloning were used to screen for several mutated PNAs with distinct properties. The mutated PNA clones obtained using NeuAcα2-6(Galβ1-3)GalNAc as a ligand showed preference for NeuAcα2-6(Galβ1-3)GalNAc rather than non-sialylated Galβ1-3GlcNAc, whereas wild-type PNA binds to Galβ1-3GlcNAc but not sialylated Galβ1-3GalNAc. Sequence analyses revealed that for all of the glycan-reactive mutated PNA clones, (i) loop C was eight amino acids in length, (ii) loop D was identical to that of wild-type PNA, (iii) residue 127 was asparagine, (iv) residue 125 was tryptophan, and (v) residue 130 was hydrophobic tyrosine, phenylalanine, or histidine. The sugar-binding ability of wild-type PNA was increased nine-fold when Tyr125 was mutated to tryptophan, and that of mutated clone C was increased more than 30-fold after His130 was changed to tyrosine. These results provide an insight into the relationship between the amino acid sequences of the carbohydrate recognition site and sugar-binding abilities of leguminous lectins.

Identification of a fourth mannose 6-phosphate binding site in the cation-independent mannose 6-phosphate receptor.

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) plays an essential role in lysosome biogenesis by targeting ∼ 60 different phosphomannosyl-containing acid hydrolases to the lysosome. This type I membrane glycoprotein has a large extracellular region comprised of 15 homologous domains. Two mannose 6-phosphate (M6P) binding sites have been mapped to domains 3 and 9, whereas domain 5 binds preferentially to the phosphodiester, M6P-N-acetylglucosamine (GlcNAc). A structure-based sequence alignment predicts that the C-terminal domain 15 contains three out of the four conserved residues identified as essential for carbohydrate recognition by domains 3, 5 and 9 of the CI-MPR, but lacks two cysteine residues that are predicted to form a disulfide bond. To determine whether domain 15 of the CI-MPR has lectin activity and to probe its carbohydrate-binding specificity, truncated forms of the CI-MPR were tested for binding to acid hydrolases with defined N-glycans in surface plasmon resonance analyses, and used to interrogate a phosphorylated glycan microarray. The results show that a construct encoding domains 14-15 binds both M6P and M6P-GlcNAc with similar affinity (Kd = 13 and 17 μM, respectively). Site-directed mutagenesis studies demonstrate the essential role of the conserved Tyr residue in domain 15 for phosphomannosyl binding. A structural model of domain 15 was generated that predicted an Arg residue to be in the binding pocket and mutagenesis studies confirmed its important role in carbohydrate binding. Together, these results show that the CI-MPR contains a fourth carbohydrate-recognition site capable of binding both phosphomonoesters and phosphodiesters.

Computational and experimental prediction of human C-type lectin receptor druggability.

Mammalian C-type lectin receptors (CTLRS) are involved in many aspects of immune cell regulation such as pathogen recognition, clearance of apoptotic bodies, and lymphocyte homing. Despite a great interest in modulating CTLR recognition of carbohydrates, the number of specific molecular probes is limited. To this end, we predicted the druggability of a panel of 22 CTLRs using DoGSiteScorer. The computed druggability scores of most structures were low, characterizing this family as either challenging or even undruggable. To further explore these findings, we employed a fluorine-based nuclear magnetic resonance screening of fragment mixtures against DC-SIGN, a receptor of pharmacological interest. To our surprise, we found many fragment hits associated with the carbohydrate recognition site (hit rate = 13.5%). A surface plasmon resonance-based follow-up assay confirmed 18 of these fragments (47%) and equilibrium dissociation constants were determined. Encouraged by these findings we expanded our experimental druggability prediction to Langerin and MCL and found medium to high hit rates as well, being 15.7 and 10.0%, respectively. Our results highlight limitations of current in silico approaches to druggability assessment, in particular, with regard to carbohydrate-binding proteins. In sum, our data indicate that small molecule ligands for a larger panel of CTLRs can be developed.

Ligand Induced Galectin-3 Protein Self-association

Many functions of galectin-3 entail binding of its carbohydrate recognition site to glycans of a glycoprotein, resulting in cross-linking thought to be mediated by its N-terminal noncarbohydrate-binding domain. Here we studied interaction of galectin-3 with the model glycoprotein asialofetuin (ASF), using a fluorescence anisotropy assay to measure the concentration of free galectin carbohydrate recognition sites in solution. Surprisingly, in the presence of ASF, this remained low even at high galectin-3 concentrations, showing that many more galectin-3 molecules were engaged than expected due to the about nine known glycan-based binding sites per ASF molecule. This suggests that after ASF-induced nucleation, galectin-3 associates with itself by the carbohydrate recognition site binding to another galectin-3 molecule, possibly forming oligomers. We named this type-C self-association to distinguish it from the previously proposed models (type-N) where galectin-3 molecules bind to each other through the N-terminal domain, and all carbohydrate recognition sites are available for binding glycans. Both types of self-association can result in precipitates, as measured here by turbidimetry and dynamic light scattering. Type-C self-association and precipitation occurred even with a galectin-3 mutant (R186S) that bound poorly to ASF but required much higher concentration (∼50 μM) as compared with wild type (∼1 μM). ASF also induced weaker type-C self-association of galectin-3 lacking its N-terminal domains, but as expected, no precipitation. Neither a monovalent nor a divalent N-acetyl-D-lactosamine-containing glycan induced type-C self-association, even if the latter gave precipitates with high concentrations of galectin-3 (>∼50 μM) in agreement with published results and perhaps due to type-N self-association.

Structural Basis of the Anti-HIV Activity of the Cyanobacterial Oscillatoria Agardhii Agglutinin

The cyanobacterial Oscillatory Agardhii agglutinin (OAA) is a recently discovered HIV-inactivating lectin that interacts with high-mannose sugars. Nuclear magnetic resonance (NMR) binding studies between OAA and α3,α6-mannopentaose (Manα(1-3)[Manα(1-3)[Manα(1-6)]Manα(1-6)]Man), the branched core unit of Man-9, revealed two binding sites at opposite ends of the protein, exhibiting essentially identical affinities. Atomic details of the specific protein-sugar contacts in the recognition loops of OAA were delineated in the high-resolution crystal structures of free and glycan-complexed protein. No major changes in the overall protein structure are induced by carbohydrate binding, with essentially identical apo- and sugar-bound conformations in binding site 1. A single peptide bond flip at W77-G78 is seen in binding site 2. Our combined NMR and crystallographic results provide structural insights into the mechanism by which OAA specifically recognizes the branched Man-9 core, distinctly different from the recognition of the D1 and D3 arms at the nonreducing end of high-mannose carbohydrates by other antiviral lectins.

Crystal structure of a β-prism II lectin from Remusatia vivipara

The crystal structure of a β-prism II (BP2) fold lectin from Remusatia vivipara, a plant of traditional medicinal value, has been determined at a resolution of 2.4Å. This lectin (RVL, Remusatia vivipara lectin) is a dimer with each protomer having two distinct BP2 domains without a linker between them. It belongs to the "monocot mannose-binding" lectin family, which consists of proteins of high sequence and structural similarity. Though the overall tertiary structure is similar to that of lectins from snowdrop bulbs and garlic, crucial differences in the mannose-binding regions and oligomerization were observed. Unlike most of the other structurally known proteins in this family, only one of the three carbohydrate recognition sites (CRSs) per BP2 domain is found to be conserved. RVL does not recognize simple mannose moieties. RVL binds to only N-linked complex glycans like those present on the gp120 envelope glycoprotein of HIV and mannosylated blood proteins like fetuin, but not to simple mannose moieties. The molecular basis for these features and their possible functional implications to understand the different levels of carbohydrate affinities in this structural family have been investigated through structure analysis, modeling and binding studies. Apart from being the first structure of a lectin to be reported from the Araceae/Arum family, this protein also displays a novel mode of oligomerization among BP2 lectins.