An initial event in the insect innate immune response: structural and biological studies of interactions between ?-1,3-glucan and the N-terminal domain of ?-1,3-glucan recognition protein.
ABSTRACT: In response to invading microorganisms, insect ?-1,3-glucan recognition protein (?GRP), a soluble receptor in the hemolymph, binds to the surfaces of bacteria and fungi and activates serine protease cascades that promote destruction of pathogens by means of melanization or expression of antimicrobial peptides. Here we report on the nuclear magnetic resonance (NMR) solution structure of the N-terminal domain of ?GRP (N-?GRP) from Indian meal moth (Plodia interpunctella), which is sufficient to activate the prophenoloxidase (proPO) pathway resulting in melanin formation. NMR and isothermal calorimetric titrations of N-?GRP with laminarihexaose, a glucose hexamer containing ?-1,3 links, suggest a weak binding of the ligand. However, addition of laminarin, a glucose polysaccharide (~6 kDa) containing ?-1,3 and ?-1,6 links that activates the proPO pathway, to N-?GRP results in the loss of NMR cross-peaks from the backbone (15)N-(1)H groups of the protein, suggesting the formation of a large complex. Analytical ultracentrifugation (AUC) studies of formation of the N-?GRP-laminarin complex show that ligand binding induces self-association of the protein-carbohydrate complex into a macro structure, likely containing six protein and three laminarin molecules (~102 kDa). The macro complex is quite stable, as it does not undergo dissociation upon dilution to submicromolar concentrations. The structural model thus derived from this study for the N-?GRP-laminarin complex in solution differs from the one in which a single N-?GRP molecule has been proposed to bind to a triple-helical form of laminarin on the basis of an X-ray crystallographic structure of the N-?GRP-laminarihexaose complex [Kanagawa, M., Satoh, T., Ikeda, A., Adachi, Y., Ohno, N., and Yamaguchi, Y. (2011) J. Biol. Chem. 286, 29158-29165]. AUC studies and phenoloxidase activation measurements conducted with the designed mutants of N-?GRP indicate that electrostatic interactions involving Asp45, Arg54, and Asp68 between the ligand-bound protein molecules contribute in part to the stability of the N-?GRP-laminarin macro complex and that a decreased stability is accompanied by a reduced level of activation of the proPO pathway. An increased level of ?-1,6 branching in laminarin also results in destabilization of the macro complex. These novel findings suggest that ligand-induced self-association of the ?GRP-?-1,3-glucan complex may form a platform on a microbial surface for recruitment of downstream proteases, as a means of amplification of the initial signal of pathogen recognition for the activation of the proPO pathway.
Project description:Pattern recognition receptors are known to participate in the activation of Prophenoloxidase system. In this study, a 1,3-?-D-glucan recognition protein was detected for the first time in Antheraea pernyi larvae (Ap-?GRP). Ap-?GRP was purified to 99.9% homogeneity from the hemolymph using traditional chromatographic methods. Ap-?GRP specifically bind 1,3-?-D-glucan and yeast, but not E. coli or M. luteus. The 1,3-?-D-glucan dependent phenoloxidase (PO) activity of the hemolymph inhibited by anti-Ap-?GRP antibody could be recovered by addition of purified Ap-?GRP. These results demonstrate that Ap-?GRP acts as a biosensor of 1,3-?-Dglucan to trigger the Prophenoloxidase system. A trace mount of 1,3-?-D-glucan or Ap-?GRP alone was unable to trigger the proPO system, but they both did. Ap-?GRP was specifically degraded following the activation of proPO with 1,3-?-Dglucan. These results indicate the variation in the amount of Ap-?GRP after specific immune challenge in A. pernyi hemolymph is an important regulation mechanism to immune response.
Project description:Insect ?-glucan recognition protein (?GRP), a pathogen recognition receptor for innate immune responses, detects ?-1,3-glucan on fungal surfaces via its N-terminal carbohydrate-binding domain (N-?GRP) and triggers serine protease cascades for the activation of prophenoloxidase (pro-PO) or Toll pathways. Using biophysical and biochemical methods, we characterized the interaction of the N-terminal domain from Manduca sexta ?GRP2 (N-?GRP2) with laminarin, a soluble form of ?-1,3-glucan. We found that carbohydrate binding by N-?GRP2 induces the formation of two types of protein-carbohydrate complexes, depending on the molar ratio of carbohydrate to protein ([C]/[P]). Precipitation, analytical ultracentrifugation, and chemical cross-linking experiments have shown that an insoluble aggregate forms when the molar ratio of carbohydrate to protein is low ([C]/[P] ? 1). In contrast, a soluble complex, containing at least five N-?GRP2 molecules forms at a higher molar ratio of carbohydrate/protein ([C]/[P] >5). A hypothesis that this complex is assembled partly due to protein-protein interactions was supported by chemical cross-linking experiments combined with LC-MS/MS spectrometry analysis, which permitted identification of a specific intermolecular cross-link site between N-?GRP molecules in the soluble complex. The pro-PO activation in naive plasma was strongly stimulated by addition of the insoluble aggregates of N-?GRP2. The soluble complex with laminarin formed in the plasma also stimulated pro-PO activation, but at a lower level. Taken together, these results provide experimental evidence for novel mechanisms in which associations of ?GRP with microbial polysaccharide promotes assembly of ?GRP oligomers, which may form a platform needed to trigger the pro-PO pathway activation cascade.
Project description:The innate ability to detect pathogens is achieved by pattern recognition receptors, which recognize non-self-components such as ?1,3-glucan. ?1,3-Glucans form a triple-helical structure stabilized by interchain hydrogen bonds. ?1,3-Glucan recognition protein (?GRP)/gram-negative bacteria-binding protein 3 (GNBP3), one of the pattern recognition receptors, binds to long, structured ?1,3-glucan to initiate innate immune response. However, binding details and how specificity is achieved in such receptors remain important unresolved issues. We solved the crystal structures of the N-terminal ?1,3-glucan recognition domain of ?GRP/GNBP3 (?GRP-N) in complex with the ?1,3-linked glucose hexamer, laminarihexaose. In the crystals, three structured laminarihexaoses simultaneously interact through six glucose residues (two from each chain) with one ?GRP-N. The spatial arrangement of the laminarihexaoses bound to ?GRP-N is almost identical to that of a ?1,3-glucan triple-helical structure. Therefore, our crystallographic structures together with site-directed mutagenesis data provide a structural basis for the unique recognition by such receptors of the triple-helical structure of ?1,3-glucan.
Project description:The beta-1,3-glucan recognition protein (betaGRP)/Gram-negative bacteria-binding protein 3 (GNBP3) is a crucial pattern-recognition receptor that specifically binds beta-1,3-glucan, a component of fungal cell walls. It evokes innate immunity against fungi through activation of the prophenoloxidase (proPO) cascade and Toll pathway in invertebrates. The betaGRP consists of an N-terminal beta-1,3-glucan-recognition domain and a C-terminal glucanase-like domain, with the former reported to be responsible for the proPO cascade activation. This report shows the solution structure of the N-terminal beta-1,3-glucan recognition domain of silkworm betaGRP. Although the N-terminal domain of betaGRP has a beta-sandwich fold, often seen in carbohydrate-binding modules, both NMR titration experiments and mutational analysis showed that betaGRP has a binding mechanism which is distinct from those observed in previously reported carbohydarate-binding domains. Our results suggest that betaGRP is a beta-1,3-glucan-recognition protein that specifically recognizes a triple-helical structure of beta-1,3-glucan.
Project description:The prophenoloxidase (proPO) system is activated upon recognition of pathogens by pattern recognition proteins (PRPs), including a lipopolysaccharide- and ?-1,3-glucan-binding protein (LGBP). However, shrimp LGBPs that are involved in the proPO system have yet to be clarified. Here, we focus on characterizing the role of a Penaeus monodon LGBP (PmLGBP) in the proPO system. We found that PmLGBP transcripts are expressed primarily in the hemocytes and are increased at 24 h after pathogenic bacterium Vibrio harveyi challenge. The binding studies carried out using ELISA indicated that recombinant (r)PmLGBP binds to ?-1,3-glucan and LPS with a dissociation constant of 6.86 × 10(-7) M and 3.55 × 10(-7) M, respectively. Furthermore, we found that rPmLGBP could enhance the phenoloxidase (PO) activity of hemocyte suspensions in the presence of LPS or ?-1,3-glucan. Using dsRNA interference-mediated gene silencing assay, we further demonstrated that knockdown of PmLGBP in shrimp in vivo significantly decreased the PmLGBP transcript level but had no effect on the expression of the other immune genes tested, including shrimp antimicrobial peptides (AMPs). However, suppression of proPO expression down-regulated PmLGBP, proPO-activating enzyme (PmPPAE2), and AMPs (penaeidin and crustin). Such PmLGBP down-regulated shrimp showed significantly decreased total PO activity. We conclude that PmLGBP functions as a pattern recognition protein for LPS and ?-1,3-glucan in the shrimp proPO activating system.
Project description:Endo-1,3-?-glucanases derived from marine mollusks have attracted much attention in recent years because of their unique transglycosylation activity. In this study, a novel endo-1,3-?-glucanase from the scallop Chlamys farreri, named Lcf, was biochemically characterized. Unlike in earlier studies on marine mollusk endo-1,3-?-glucanases, Lcf was expressed in vitro first. Enzymatic analysis demonstrated that Lcf preferred to hydrolyze laminarihexaose than to hydrolyze laminarin. Furthermore, Lcf was capable of catalyzing transglycosylation reactions with different kinds of glycosyl acceptors. More interestingly, the transglycosylation specificity of Lcf was different from that of other marine mollusk endo-1,3-?-glucanases, although they share a high sequence identity. This study enhanced our understanding of the diverse enzymatic specificities of marine mollusk endo-1,3-?-glucanases, which facilitated development of a unique endo-1,3-?-glucanase tool in the synthesis of novel glycosides.
Project description:Detection of pathogenic invaders is the essential first step of a successful defense response in multicellular organisms. In this study, we have identified a new member of the ?-1,3-glucanase-related protein superfamily from the tobacco hornworm Manduca sexta. This protein, designated microbe binding protein (MBP), is 61% identical in sequence to Bombyx mori Gram-negative bacteria binding protein, but only 34-36% identical to M. sexta ?-1,3-glucan recognition protein-1 and 2. Its mRNA levels were strongly up-regulated in hemocytes and fat body of immune challenged larvae, along with an increase in concentration of the plasma protein. We expressed M. sexta MBP in a baculovirus-insect cell system. The purified protein associated with intact bacteria and fungi. It specifically bound to lipoteichoic acid, lipopolysaccharide, diaminopimelic acid-type peptidoglycans (DAP-PGs) from Escherichia coli and Bacillus subtilis, but less so to laminarin or Lys-type PG from Staphylococcus aureus. The complex binding pattern was influenced by other plasma factors and additional microbial surface molecules. After different amounts of MBP had been incubated with larval plasma on ice, a concentration-dependent increase in phenoloxidase (PO) activity occurred in the absence of any microbial elicitor. The activity increase was also observed in the mixture of plasma and a bacterial or fungal cell wall component. The prophenoloxidase (proPO) activation became more prominent when DAP-PGs, Micrococcus luteus Lys-PG, or lipoteichoic acid was included in the mixture of MBP and plasma. Statistic analysis suggested that a synergistic enhancement of proPO activation was caused by an interaction between MBP and these elicitors, but not S. aureus Lys-PG, lipopolysaccharide, curdlan, or laminarin. These data indicate that M. sexta MBP is a component of the surveillance mechanism and, by working together with other pattern recognition molecules and serine proteinases, triggers the proPO activation system.
Project description:BH0236 from Bacillus halodurans is a multimodular ?-1,3-glucanase comprising an N-terminal family 81 glycoside hydrolase catalytic module, an internal family 6 carbohydrate-binding module (CBM) that binds the nonreducing end of ?-1,3-glucan chains, and an uncharacterized C-terminal module classified into CBM family 56. Here, we determined that this latter CBM, BhCBM56, bound the soluble ?-1,3-glucan laminarin with a dissociation constant (Kd ) of ?26 ?m and displayed higher affinity for insoluble ?-1,3-glucans with Kd values of ?2-10 ?m but lacked affinity for ?-1,3-glucooligosaccharides. The X-ray crystal structure of BhCBM56 and NMR-derived chemical shift mapping of the binding site revealed a ?-sandwich fold, with the face of one ?-sheet possessing the ?-1,3-glucan-binding surface. On the basis of the functional and structural properties of BhCBM56, we propose that it binds a quaternary polysaccharide structure, most likely the triple helix adopted by polymerized ?-1,3-glucans. Consistent with the BhCBM56 and BhCBM6/56 binding profiles, deletion of the CBM56 from BH0236 decreased activity of the enzyme on the insoluble ?-1,3-glucan curdlan but not on soluble laminarin; additional deletion of the CBM6 also did not affect laminarin degradation but further decreased curdlan hydrolysis. The pseudo-atomic solution structure of BH0236 determined by small-angle X-ray scattering revealed structural insights into the nature of avid binding by the BhCBM6/56 pair and how the orientation of the active site in the catalytic module factors into recognition and degradation of ?-1,3-glucans. Our findings reinforce the notion that catalytic modules and their cognate CBMs have complementary specificities, including targeting of polysaccharide quaternary structure.
Project description:Recognition of pathogens by insect pattern recognition receptors is critical to mount effective immune responses. In this study, we reported a new member (?GRP3) of the ?-1, 3-glucan recognition protein (?GRP) family from the tobacco hornworm Manduca sexta. Unlike other members of the M. sexta ?GRP family proteins, which contain an N-terminal small glucan binding domain and a C-terminal large glucanase-like domain, ?GRP3 is 40-45 residues shorter at the N-terminus and lacks the small glucan binding domain. The glucanase-like domain of ?GRP3 is most similar to that of M. sexta microbe binding protein (MBP) with 78% identity. ?GRP3 transcript was mainly expressed in the fat body, and both its mRNA and protein levels were not induced by microorganisms in larvae. Recombinant ?GRP3 purified from Drosophila S2 cells could bind to several Gram-negative and Gram-positive bacteria and yeast, as well as to laminarin (?-1, 3-glucan), mannan, lipopolysaccharide (LPS), lipoteichoic acid (LTA), and meso-diaminopimelic acid (DAP)-type peptidoglycan (PG), but did not bind to Lysine-type PG. Binding of ?GRP3 to laminarin could be competed well by free laminarin, mannan, LPS and LTA, but almost not competed by free PGs. Recombinant ?GRP3 could agglutinate Bacillus cereus and Escherichia coli in a calcium-dependent manner and showed antibacterial (bacteriostatic) activity against B. cereus, novel functions that have not been reported for the ?GRP family proteins before. M. sexta ?GRP3 may serve as an immune surveillance receptor with multiple functions.
Project description:Silkworm ?-1,3-glucan recognition protein (?GRP) tightly and specifically associates with ?-1,3-glucan. We report here an affinity purification system named the 'GRP system', which uses the association between the ?-1,3-glucan recognition domain of ?GRP (GRP-tag), as an affinity tag, and curdlan beads. Curdlan is a water-insoluble ?-1,3-glucan reagent, the low cost of which (about 100 JPY/g) allows the economical preparation of beads. Curdlan beads can be readily prepared by solubilization in an alkaline solution, followed by neutralization, sonication and centrifugation. We applied the GRP system to preparation of several proteins and revealed that the expression levels of the GRP-tagged proteins in soluble fractions were two or three times higher than those of the glutathione S-transferase (GST)-tagged proteins. The purity of the GRP-tagged proteins on the curdlan beads was comparable to that of the GST-tagged proteins on glutathione beads. The chemical stability of the GRP system was more robust than conventional affinity systems under various conditions, including low pH (4-6). Biochemical and structural analyses revealed that proteins produced using the GRP system were structurally and functionally active. Thus, the GRP system is suitable for both the large- and small-scale preparation of recombinant proteins for functional and structural analyses.