Identification of a multigene family encoding putative beta-glucan-binding proteins in Medicago truncatula

Branched 1,6-1,3-beta-glucans from Phytophthora sojae cell walls represent pathogen-associated molecular patterns (PAMPs) that have been shown to mediate the activation of plant defence reactions in many legumes. In soybean, a receptor protein complex containing a high affinity beta-glucan-binding protein (GBP) was identified and investigated in detail. In the model legume Medicago truncatula, used for functional genomic studies of various plant-microbe interactions, a high-affinity beta-glucan-binding site was characterized biochemically. However, to date, none of the genes encoding GBPs from M. truncatula have been described. Here, we report the identification of four full-length clones encoding putative beta-glucan-binding proteins from M. truncatula, MtGBP1, 2, 3, and 4, composing a multigene family encoding GBP-related proteins in this plant. Differences in expression patterns as well as in regulation on treatment with two different biotic elicitors are demonstrated for the members of the GBP family and for a selection of defence-related genes.     

Catalytic properties of the bifunctional soybean beta-glucan-binding protein, a member of family 81 glycoside hydrolases

The beta-glucan-binding protein (GBP) of soybean (Glycine max L.) has been shown to contain two different activities. As part of the plasma membrane-localized pathogen receptor complex, it binds a microbial cell wall elicitor, triggering the activation of defence responses. Additionally, the GBP is able to hydrolyze beta-1,3-glucans, as present in the cell walls of potential pathogens. The substrate specificity, the mode of action, and the stereochemistry of the catalysis have been elucidated. This defines for the first time the inverting mode of the catalytic mechanism of glycoside hydrolases belonging to family 81.     

The hepta-beta-glucoside elicitor-binding proteins from legumes represent a putative receptor family

The ability of legumes to recognize and respond to beta-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound beta-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-beta-glucoside conjugate (Kd = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (Kd = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex.     

Functional reconstitution of beta-glucan elicitor-binding activity upon incorporation into lipid vesicles.

In temperature-induced Triton X-114 phase separation experiments the beta-glucan elicitor-binding site from soybean (Glycine max L.) root membranes was identified as (a) hydrophobic membrane protein(s). The Zwittergent 3-12-solubilized beta-glucan-binding proteins were incorporated into lipid vesicles by the detergent-dilution procedure. Reconstituted binding proteins were functional in that binding of the hepta-beta-glucoside ligand was saturable, reversible and of high affinity (K(d)=6-7 nM). Competition studies using beta-glucans with different degrees of polymerization (DP 7-15; DP 15-25) showed effective displacement of the radioligand from the binding site whereas beta-glucan fragments with DP <7 were ineffective. The total amount of reconstituted binding activity was dependent on the acyl chain length of the phospholipids used for the reconstitution with a preference for decanoic (C10) and dodecanoic (C12) chains. Restored ligand binding was maximally 37% as compared to the former detergent-solubilized binding activity. The presence of a lipid environment stabilized the purified beta-glucan-binding proteins.     

High-affinity binding of fungal beta-glucan fragments to soybean (Glycine max L.) microsomal fractions and protoplasts

We have recently reported the existence of binding sites in soybean membranes for a beta-glucan fraction derived from the fungal pathogen Phytophthora megasperma f. sp. glycinea, which may play a role in the elicitor-mediated phytoalexin response of this plant [Schmidt, W. E. & Ebel, J. (1987) Proc. Natl Acad. Sci. USA 84, 4117-4121]. The specificity of beta-glucan binding to soybean membranes has now been investigated using a variety of competing polyglucans and oligoglucans of fungal origin. P. megasperma beta-glucan binding showed high apparent affinity for branched glucans with degrees of polymerization greater than 12. Binding affinity showed good correlation with elicitor activity as measured in a soybean cotyledon bioassay. Modification of the glucans at the reducing end with phenylalkylamine reagents had no effect on binding affinity. This characteristic was used to synthesize an oligoglucosyl tyramine derivative suitable for radioiodination. The 125I-glucan (15-30 Ci/mmol) provided higher sensitivity and lower detection limits for the binding assays while behaving in a manner identical to the [3H]glucan used previously. More accurate determinations of the Kd value for glucan binding indicated a higher affinity than previously shown (37 nM versus 200 nM). The 125I-glucan was used to provide the first reported evidence of specific binding of a fungal beta-glucan fraction in vivo to soybean protoplasts. The binding affinity to protoplasts proved identical to that found in microsomal fractions.     

Interaction investigations of crustacean β‐GBP recognition toward pathogenic microbial cell membrane and stimulate upon prophenoloxidase activation

In invertebrates, crustaceans' immune system consists of pattern recognition receptors (PRRs) instead of immunoglobulin's, which involves in the microbial recognition and initiates the protein–ligand interaction between hosts and pathogens. In the present study, PRRs namely β‐1,3 glucan binding protein (β‐GBP) from mangrove crab Episesarma tetragonum and its interactions with the pathogens such as bacterial and fungal outer membrane proteins (OMP) were investigated through microbial aggregation and computational interaction studies. Molecular recognition and microbial aggregation results of Episesarma tetragonum β‐GBP showed the specific binding affinity toward the fungal β‐1,3 glucan molecule when compared to other bacterial ligands. Because of this microbial recognition, prophenoloxidase activity was enhanced and triggers the innate immunity inside the host animal. Our findings disclose the role of β‐GBP in molecular recognition, host–pathogen interaction through microbial aggregation, and docking analysis. In vitro results were concurred with the in silico docking, and molecular dynamics simulation analysis. This study would be helpful to understand the molecular mechanism of β‐GBP and update the current knowledge on the PRRs of crustaceans. Copyright © 2014 John Wiley & Sons, Ltd.     

Cell Wall Glucans of the Yeast and Mycelial Forms of Paracoccidioides brasiliensis

Glucans were isolated from the cell wall of the yeast (Y) and mycelial (M) forms of Paracoccidioides brasiliensis. The alkali-soluble glucan of the Y form had properties of alpha-1,3-glucan. The alkali-insoluble glucan of the M form was identified as a beta-glucan which contains a beta-(1 --> 3)-glycosidic linkage by infrared absorption spectrum, by effect of beta-1,3-glucanase, and by partial acid hydrolysis. The alkali-soluble glucans of the M form were a mixture of alpha- and beta-glucans and the ratio of alpha- to beta-glucan was variable, depending on the preparations.     

A glucanase-driven fractionation allows redefinition of Schizosaccharomyces pombe cell wall composition and structure: assignment of diglucan

Purified endoglucanases have been used to determine the composition of Schizosaccharomyces pombe cell wall. This structure has been traditionally studied after isolating its components (mannoproteins, alpha1,3-glucan, beta1,3-glucan, and a branched beta-glucan) with hot alkali. Instead, we sequentially removed the polysaccharides by digesting with endo-beta1,3-glucanase and with a novel endo-alpha1,3-glucanase (mutanase). After this gentle isolation we observed that a branched beta1,3-beta1,6-glucan is much more abundant than previously described. By scaling-up the new protocol we prepared large amounts of the highly branched glucan and determined its structural features. We have named this highly branched beta-glucan diglucan, reflecting its two types of beta linkages. We have also identified an insoluble endoglucanase-resistant type of 1,3-linked glucan present in S. pombe cell walls. We redefined the wall composition of S. pombe vegetative cells by this new method. Finally, to demonstrate its application, we determined the cell wall composition of known mutant strains.     

Enhanced defense against Pneumocystis carinii mediated by a novel dectin-1 receptor Fc fusion protein

Pneumocystis carinii (PC) pneumonia is a leading opportunistic infection found among HIV-infected individuals worldwide. Although CD4(+) T cell deficiency clearly correlates with susceptibility to PC pneumonia, murine models of disease indicate that PC-directed Abs may prevent infection and/or inhibit growth of existing PC within the lungs. Recognition of PC by alveolar macrophages involves the beta-glucan receptor Dectin-1 and macrophage effector function against PC is enhanced by Abs derived from PC-vaccinated hosts. We developed a fusion protein consisting of the extracellular domain of Dectin-1 linked to the Fc portion of murine IgG1, which we hypothesized would enhance host recognition and opsonic phagocytosis of PC. The recombinant protein, Dectin-Fc, is dimeric and the Ag recognition site identifies beta-1,3 glucan linkages specifically and with high affinity (K(D) = 2.03 x 10(-7) M). Dectin-Fc enhances RAW264.7 macrophage recognition of the beta-glucan containing particulate zymosan in an FcgammaRII- and FcgammaRIII-dependent manner and preopsonization of PC organisms with Dectin-Fc increased alveolar and peritoneal macrophage-dependent killing of PC. SCID mice treated with a replication incompetent adenoviral vector expressing Dectin-Fc had attenuated growth of PC within the lungs, overall decreased PC lung burden, and diminished correlates of PC-related lung damage relative to SCID mice receiving a control vector. These findings demonstrate that targeting PC beta-glucan with Dectin-Fc enhances host recognition and clearance of PC in the absence of B and T cells, and suggest that FcgammaR-based targeting of PC, via cell wall carbohydrate recognition, may promote resistance against PC pneumonia in the immunodeficient host.     

Immunosuppressors in pathogenesis of potato-pathogen blight

The properties and effects of two plant resistance suppressors (1,3-beta-1,6-beta-glucan and a pentasaccharide of xyloglucan origin) involved in the pathosystem of potato (Solanum tuberosum) and the causal agent of blight (Phytophthora infestans (Mont) de Bary) were compared. The microbial 1,3-beta-1,6-beta-glucan suppressed the defense response over a narrow concentration range (10(-2) M), whereas the plant pentasaccharide had a broad range of effective concentrations (10(-12) to 10(-6) M). In the pathosystem of potato-causal agent of late blight, the beta-glucan caused a local and race-specific suppressor effect on the plant host defense response. In contrast, the pentasaccharide caused both local and systemic suppression of potato resistance, and the presence of terminal fucosyl residue in the xyloglucan oligosaccharine played a decisive role in its effect. The recognition of both suppressors by potato cell membrane sites is discussed.     

Enzymic activity of the K5-type yeast killer toxin and its characterization

K5-type yeast killer toxin secreted by P. anomala NCYC 434 cells has a broad killing spectrum. Competitive inhibiton of killer activity showed that glucans, mainly the beta-1,3 glucan, represent the primary toxin binding site within the cell wall of sensitive cells. Its hydrolytic activity on laminarin in an exo-like fashion revealed that the toxin exerts its killing effect by exo-beta-1,3-glucanase activity. Its specific activity on laminarin was 120 U/mg, and the Michaelis constants K(m) and V(max) for laminarin hydrolysis were 0.25 mg/ml and 370 micromol/min/mg. The toxin exerted its cytocidal effect after 2 h contact with the target cells. Production of the toxin by the cells was induced only when they were grown in culture media rich in beta-glucan sources, and the addition of glucose increased the specific production rate. The enzymic activity of the toxin was fully inhibited by Hg(+2), but increased with some other metal ions, most of all by Pb(+2).     

Molecular Interfaces of the Galactose-binding Protein Tectonin Domains in Host-Pathogen Interaction

β-Propeller proteins function in catalysis, protein-protein interaction, cell cycle regulation, and innate immunity. The galactose-binding protein (GBP) from the plasma of the horseshoe crab, Carcinoscorpius rotundicauda, is a β-propeller protein that functions in antimicrobial defense. Studies have shown that upon binding to Gram-negative bacterial lipopolysaccharide (LPS), GBP interacts with C-reactive protein (CRP) to form a pathogen-recognition complex, which helps to eliminate invading microbes. However, the molecular basis of interactions between GBP and LPS and how it interplays with CRP remain largely unknown. By homology modeling, we showed that GBP contains six β-propeller/Tectonin domains. Ligand docking indicated that Tectonin domains 6 to 1 likely contain the LPS binding sites. Protein-protein interaction studies demonstrated that Tectonin domain 4 interacts most strongly with CRP. Hydrogen-deuterium exchange mass spectrometry mapped distinct sites of GBP that interact with LPS and with CRP, consistent with in silico predictions. Furthermore, infection condition (lowered Ca²⁺ level) increases GBP-CRP affinity by 1000-fold. Resupplementing the system with a physiological level of Ca²⁺ did not reverse the protein-protein affinity to the basal state, suggesting that the infection-induced complex had undergone irreversible conformational change. We propose that GBP serves as a bridging molecule, participating in molecular interactions, GBP-LPS and GBP-CRP, to form a stable pathogen-recognition complex. The interaction interfaces in these two partners suggest that Tectonin domains can differentiate self/nonself, crucial to frontline defense against infection. In addition, GBP shares architectural and functional homologies to a human protein, hTectonin, suggesting its evolutionarily conservation for ∼500 million years, from horseshoe crab to human.     

Activation of a tobacco glycine-rich protein gene by a fungal glucan preparation

A Phytophthora megasperma f.sp. glycinea cell wall glucan preparation was previously shown to protect tobacco plants against viral infection. Eleven plant defense-related genes were assayed for elevated mRNA accumulation levels in response to glucan treatment of tobacco plants. The expression of only one of these genes, a glycine-rich protein (GRP) gene, was induced by glucan application. Elevated GRP gene mRNA levels could be detected within 15 min of glucan treatment and reached maximum levels at 4 h post-treatment followed by a slow decline to 8 h. The maximum induction of the GRP gene was approximately ninefold above H₂O-treated control plants. Northern blot analysis showed that a single mRNA species of 1.4 kb was responding to the glucan treatment. GRP genes occur in tobacco as members of a multigene family, but only one specific GRP gene was induced by the glucan treatment. A genomic copy of this responding GRP gene was cloned and sequenced. This tobacco GRP gene is homologous to the petunia ptGRP1 gene and the French bean GRP1.8 gene, but is not closely related to the French bean GRP1.0 gene. GRP gene expression has previously been associated with disease resistance in plants, but it remains to be determined whether β-glucan activation of the tobacco GRP gene results in the observed resistance to virus.     

Further studies of the role of cyclic beta-glucans in symbiosis. An NdvC mutant of Bradyrhizobium japonicum synthesizes cyclodecakis-(1-->3)-beta-glucosyl

The cyclic beta-(1-->3),beta-(1-->6)-D-glucan synthesis locus of Bradyrhizobium japonicum is composed of at least two genes, ndvB and ndvC. Mutation in either gene affects glucan synthesis, as well as the ability of the bacterium to establish a successful symbiotic interaction with the legume host soybean (Glycine max). B. japonicum strain AB-14 (ndvB::Tn5) does not synthesize beta-glucans, and strain AB-1 (ndvC::Tn5) synthesizes a cyclic beta-glucan lacking beta-(1-->6)-glycosidic bonds. We determined that the structure of the glucan synthesized by strain AB-1 is cyclodecakis-(1-->3)-beta-D-glucosyl, a cyclic beta-(1-->3)-linked decasaccharide in which one of the residues is substituted in the 6 position with beta-laminaribiose. Cyclodecakis-(1-->3)-beta-D-glucosyl did not suppress the fungal beta-glucan-induced plant defense response in soybean cotyledons and had much lower affinity for the putative membrane receptor protein than cyclic beta-(1-->3),beta-(1-->6)-glucans produced by wild-type B. japonicum. This is consistent with the hypothesis presented previously that the wild-type cyclic beta-glucans may function as suppressors of a host defense response.     

A structural model for the mechanisms of elicitor release from fungal cell walls by plant beta-1,3-endoglucanase.

The release of elicitor-active carbohydrates from fungal cell walls by beta-1,3-endoglucanase contained in host tissues has been implicated as one of the earliest processes in the interaction between soybean (Glycine max) and the fungal pathogen Phytophthora megasperma f. sp. glycinea leading to host defense responses such as phytoalexin production. The present study was conducted to evaluate the primary structure of the glucanase-released elicitor (RE). Gel-filtration chromatography of carbohydrates released from mycelial walls by purified soybean beta-1,3-endoglucanase resolved them into the four fractions (elicitor-active RE-I, -II, and -III and elicitor-inactive RE-IV). Sugar composition analysis indicated that all of the fractions were composed almost entirely of glucose. 1H- and 13C-nuclear magnetic resonance analysis indicated the presence of both beta-1,3- and beta-1,6-linkages for the elicitor-active RE-I, -II, and -III fractions and only beta-1,3 linkage for the elicitor-inactive RE-IV fraction. Methylation analysis and degradation studies employing beta-1,3-endo- and beta-1,3-exoglucanase further suggested that the basic structure of elicitor-active RE consists of beta-1,6-linked glucan backbone chains of various lengths with frequent side branches composed of beta-1,3-linked one or two glucose moieties. From these structural analyses of RE, a structural model of how RE is originally present in fungal cell walls and released by host beta-1,3-endoglucanase is also proposed.     

Glucanolytic Actinomycetes Antagonistic to Phytophthora fragariae var. rubi, the Causal Agent of Raspberry Root Rot

A collection of about 200 actinomycete strains was screened for the ability to grow on fragmented Phytophthora mycelium and to produce metabolites that inhibit Phytophthora growth. Thirteen strains were selected, and all produced (beta)-1,3-, (beta)-1,4-, and (beta)-1,6-glucanases. These enzymes could hydrolyze glucans from Phytophthora cell walls and cause lysis of Phytophthora cells. These enzymes also degraded other glucan substrates, such as cellulose, laminarin, pustulan, and yeast cell walls. Eleven strains significantly reduced the root rot index when inoculated on raspberry plantlets.     

Conserved repeat motifs and glucan binding by glucansucrases of oral streptococci and Leuconostoc mesenteroides

Glucansucrases of oral streptococci and Leuconostoc mesenteroides have a common pattern of structural organization and characteristically contain a domain with a series of tandem amino acid repeats in which certain residues are highly conserved, particularly aromatic amino acids and glycine. In some glucosyltransferases (GTFs) the repeat region has been identified as a glucan binding domain (GBD). Such GBDs are also found in several glucan binding proteins (GBP) of oral streptococci that do not have glucansucrase activity. Alignment of the amino acid sequences of 20 glucansucrases and GBP showed the widespread conservation of the 33-residue A repeat first identified in GtfI of Streptococcus downei. Site-directed mutagenesis of individual highly conserved residues in recombinant GBD of GtfI demonstrated the importance of the first tryptophan and the tyrosine-phenylalanine pair in the binding of dextran, as well as the essential contribution of a basic residue (arginine or lysine). A microplate binding assay was developed to measure the binding affinity of recombinant GBDs. GBD of GtfI was shown to be capable of binding glucans with predominantly alpha-1,3 or alpha-1,6 links, as well as alternating alpha-1,3 and alpha-1,6 links (alternan). Western blot experiments using biotinylated dextran or alternan as probes demonstrated a difference between the binding of streptococcal GTF and GBP and that of Leuconostoc glucansucrases. Experimental data and bioinformatics analysis showed that the A repeat motif is distinct from the 20-residue CW motif, which also has conserved aromatic amino acids and glycine and which occurs in the choline-binding proteins of Streptococcus pneumoniae and other organisms.     

Beta-glucosidase excretion in Trichoderma strains with different cell wall bound beta-1,3-glucanase activities

Two different strains of Trichoderma pseudokoningii (SE1 A8 and SE1 D81) and Trichoderma viride QM 9123 release into the medium different proportions of the total beta-glucosidase activity produced. This observation correlates with the degree of beta-1,3-glucanase binding to the cell wall found for each strain. DEAE-Sephadex ion-exchange chromatography revealed three peaks of beta-1,3-glucanase activity. These three enzymes (enzyme I, enzyme II, and enzyme III) differ in their extent of binding to the cell walls, their activity on isolated cell walls and Trichoderma beta-glucan, and their affinity for beta-glucan. Of these enzymes, enzyme II shows the largest variation in relative importance among the three strains and is located predominantly within the mural compartment. Enzyme II has the highest activity on and affinity for Trichoderma beta-glucan. Enzyme II is also the most active in releasing beta-glucosidase from cell walls of strain SE1 A8 (the strain excreting a high proportion of its beta-glucosidase into the culture fluid) as well as from strain SE1 D81 (little beta-glucosidase activity in the culture fluid). It is concluded that the action of beta-1,3-glucanase II on cell wall beta-glucan may be responsible for the in vivo release of cell wall bound beta-glucosidase into the culture fluid.