Structure determination of Discoidin II from Dictyostelium discoideum and carbohydrate binding properties of the lectin domain

The social amoeba Dictyostelium discoideum adopts a cohesive stage upon starvation and then produces Discoidin I and II, two proteins able to bind galactose and N-acetyl-galactosamine. The N-terminal domain or discoidin domain (DS) is widely distributed in eukaryotes where it plays a role in extracellular matrix binding while the C-terminal domain displays sequence similarities to invertebrate lectins. We present the first X-ray structures of the wild-type and recombinant Discoidin II in unliganded state and in complex with monosaccharides. The protein forms a homotrimer which presents two binding surfaces situated on the opposite boundaries of the structure. The binding sites of the N-terminal domain contain PEG molecules that could mimics binding of natural ligand. The C-terminal lectin domain interactions with N-acetyl-D-galactosamine and methyl-beta-galactoside are described. The carbohydrate binding sites are located at the interface between monomers. Specificity for galacto configuration can be rationalized since the axial O4 hydroxyl group is involved in several hydrogen bonds with protein side chains. Titration microcalorimetry allowed characterization of affinity and demonstrated the enthalpy-driven character of the interaction. Those results highlight the structural differentiation of the DS domain involved in many cell-adhesion processes from the lectin activity of Dictyostelium discoidins.     

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.     

Mannan-binding lectins in the coelomic fluid of various species of Far Eastern echinoderms

A mannan-binding lectin activity was revealed in the coelomic fluid of the following echinoderm species inhabiting the coastal areas of the Sea of Japan, the holothurian Eupentacta fraudatrio, sea urchins Echinocardium cordatum, Strongylocentrotus nudus and S. intermedius, brittle star Amphipholis kochii, sea stars Asterina pectinifera, Lethasterias fusca, Lysastrosoma anthosticta, and Distolasterias nipon. It was shown that, concurrently with the general pattern of lectin interaction with branched bacterial mannans, there were also distinctions caused by the fine carbohydrate specificity of lectins. The obtained data preconditioned the further study of physical and chemical properties and structural features of the echinoderm MBL and the revelation of their role in the formation of the adaptive immune response and in other biological processes.     

Letting go: Deep computational modeling insights into pH-dependent calcium affinity

Calcium and other cofactors can feature as key additions to a molecular interface, to the extent that the cofactor is completely buried in the bound state. How can such an interaction be regulated then? The answer: By facilitating a switch through an allosteric network. Although a number of unbinding mechanisms are being characterized, an extensive computational study by Joswig et al. reveals a detailed model for the pattern recognition receptor langerin.     

Identification of a new adhesin‐like protein from Lactobacillus mucosae ME‐340 with specific affinity to the human blood group A and B antigens

Aims: To identify and characterize a new adhesin‐like protein of probiotics that show specific adhesion to human blood group A and B antigens. Methods and Results: Using the BIACORE assay, the adhesion of cell surface components obtained from four lactobacilli strains that adhered to blood group A and B antigens was tested. Their components showed a significant adhesion to A and B antigens when compared to the bovine serum albumin (BSA) control. The 1 mol l^?1 GHCl fraction extracted from Lactobacillus mucosae ME‐340 contained a 29‐kDa band (Lam29) using SDS–PAGE. The N‐terminal amino acid sequence and homology analysis showed that Lam29 was 90% similar to the substrate‐binding protein of the ATP‐binding cassette (ABC) transporter from Lactobacillus fermentum IFO 3956. The complete nucleotide sequence (858 bp) of Lam29 was determined and encoded a protein of 285 amino acid residues. Phylogenetic analysis and multiple sequence alignments indicated this protein may be related to the cysteine‐binding transporter. Conclusions: The adhesion of ME‐340 strain to blood group A and B antigens was mediated by Lam29 that is a putative component of ABC transporter as an adhesin‐like protein. Significance and Impact of the Study: Lactobacillus mucosae ME‐340 expressing Lam29 may be useful for competitive exclusion of pathogens via blood group antigen receptors in the human gastrointestinal mucosa and in the development of new probiotic foods.     

Interaction profile of galectin-5 with free saccharides and mammalian glycoproteins: probing its fine specificity and the effect of naturally clustered ligand presentation

Cell-surface glycans are functional docking sites for tissue lectins such as the members of the galectin family. This interaction triggers a wide variety of responses; hence, there is a keen interest in defining its structural features. Toward this aim, we have used enzyme-linked lectinosorbent (ELLSA) and inhibition assays with the prototype rat galectin-5 and panels of free saccharides and glycoconjugates. Among 45 natural glycans tested for lectin binding, galectin-5 reacted best with glycoproteins (gps) presenting a high density of Galbeta1-3/4GlcNAc (I/II) and multiantennary N-glycans with II termini. Their reactivities, on a nanogram basis, were up to 4.3 x 10(2), 3.2 x 10(2), 2.5 x 10(2), and 1.7 x 10(4) times higher than monomeric Galbeta1-3/4GlcNAc (I/II), triantennary-II (Tri-II), and Gal, respectively. Galectin-5 also bound well to several blood group type B (Galalpha1-3Gal)- and A (GalNAcalpha1-3Gal)-containing gps. It reacted weakly or not at all with tumor-associated Tn (GalNAcalpha1-Ser/Thr) and sialylated gps. Among the mono-, di-, and oligosaccharides and mammalian glycoconjugates tested, blood group B-active II (Galalpha1-3Gal beta1-4GlcNAc), B-active IIbeta1-3L (Galalpha1-3Galbeta1-4GlcNAc beta1-3Galbeta1-4Glc), and Tri-II were the best. It is concluded that (1) Galbeta1-3/4GlcNAc and other Galbeta1-related oligosaccharides with alpha1-3 extensions are essential for binding, their polyvalent form in cellular glycoconjugates being a key recognition force for galectin-5; (2) the combining site of galectin-5 appears to be of a shallow-groove type sufficiently large to accommodate a substituted beta-galactoside, especially with alpha-anomeric extension at the non-reducing end (e.g., human blood group B-active II and B-active IIbeta1-3L); (3) the preference within beta-anomeric positioning is Galbeta1-4 > or = Galbeta1-3 > Galbeta1-6; and (4) hydrophobic interactions in the vicinity of the core galactose unit can enhance binding. These results are important for the systematic comparison of ligand selection in this family of adhesion/growth-regulatory effectors with potential for medical applications.     

Clec14a is specifically expressed in endothelial cells and mediates cell to cell adhesion

Clec14a is a member of the thrombomodulin (TM) family, but its function has not yet been determined. Here, we report that Clec14a is a plasma membrane protein of endothelial cells (ECs) expressed specifically in the vasculature of mice. Deletion mutant analysis revealed that Clec14a mediates cell–cell adhesion through its C-type lectin-like domain. Knockdown of Clec14a in ECs suppressed cell migratory activity and filopodial protrusion, and delayed formation of tube-like structures. These findings demonstrate that Clec14a is a novel EC-specific protein that appears to play a role in cell–cell adhesion and angiogenesis.     

Pea lectin unfolding reveals a unique molten globule fragment chain

Pea lectin (PSL) is a dimeric protein in which each subunit comprises two intertwined, post-translationally processed polypeptide chains -a long β-fragment and a short α-fragment. Using guanidine hydrochloride-induced denaturation, we have investigated and characterized the species obtained in the unfolding equilibrium of PSL by steady-state and time-resolved fluorescence, phosphorescence, and selective chemical modification. During unfolding, the fragment chains become separated, and the unfolding pattern reveals a β-fragment as intermediate that has the molten globule characteristics. As examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, the fragment intermediate shows ∼ 20 fold increase in ANS fluorescence, and a large increase in ANS lifetime (12.8 ns). The tryptophan environment of the molten globule β-fragment has been probed by selective modification with N-bromosuccinimide (NBS), which shows that two tryptophans, possibly Trp 53 and Trp 152 are oxidized while the other Trp 128 remains resistant to oxidation. The different types of tryptophan environment for the intermediate are supported by phosphorescence studies at 77 K, which gives a (0,0) band at 410 nm. These results seem to indicate that the larger fragment chain of PSL can independently behave as a monomeric or single domain protein that undergoes unfolding through intermediate state(s), and may provide important insight into the folding problem of oligomeric proteins in general and lectins in particular.     

Implications of oligomeric forms of POD-1 and POD-2 proteins isolated from cell walls of the biocontrol agent Pythium oligandrum in relation to their ability to induce defense reactions in tomato

The cell wall protein fraction (CWP) isolated from the biocontrol agent Pythium oligandrum induces defense reactions in tomato. CWP contains two novel elicitin-like proteins, POD-1 and POD-2, both with seven cysteines. To determine the essential structure in the defense-eliciting components of CWP, five fractions (F1, F2, F3, F4 and F5) were fractionated from CWP using cation chromatography and their components and disulfide bond compositions were analyzed. The expression levels of three defense-related genes (PR-6, LeCAS and PR-2b) were determined in tomato roots treated with each of the five fractions. Of the five fractions, F4 containing a heterohexamer of POD-1 and POD-2, and F5 containing a homohexamer of POD-1, both with disulfide bonds formed between all cysteine residues, induced the expression of three genes. F4 treatment also induced the accumulation of ethylene in tomato. The predicted three-dimensional structures of POD-1 and POD-2, and the results of SEC and MALDI-TOF MS analyses suggest that F4 consists of three POD-1 and POD-2 disulfide-bonded heterodimers that interleave into a hexameric ring through noncovalent association. These results suggest that this structure, which F5 also appears to form, is essential for stimulating defense responses in tomato.