title: An initial event in the insect innate immune response: structural and biological studies of interactions between β-1,3-glucan and the N-terminal domain of β-1,3-glucan recognition protein.
abstract: In response to invading microorganisms, insect β-1,3-glucan recognition protein (βGRP), 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 βGRP (N-βGRP) 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-βGRP with laminarihexaose, a glucose hexamer containing β-1,3 links, suggest a weak binding of the ligand. However, addition of laminarin, a glucose polysaccharide (~6 kDa) containing β-1,3 and β-1,6 links that activates the proPO pathway, to N-βGRP 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-βGRP-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-βGRP-laminarin complex in solution differs from the one in which a single N-βGRP 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-βGRP-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-βGRP indicate that electrostatic interactions involving Asp45, Arg54, and Asp68 between the ligand-bound protein molecules contribute in part to the stability of the N-βGRP-laminarin macro complex and that a decreased stability is accompanied by a reduced level of activation of the proPO pathway. An increased level of β-1,6 branching in laminarin also results in destabilization of the macro complex. These novel findings suggest that ligand-induced self-association of the βGRP-β-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.
authors.affiliation: Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
authors.forename: Huaien
authors.initials: H
authors.lastname: Dai
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Hiromasa
authors.forename: Daisuke
authors.initials: D
authors.lastname: Takahashi
authors.forename: David
authors.initials: D
authors.lastname: VanderVelde
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Fabrick
authors.forename: Michael R
authors.initials: MR
authors.lastname: Kanost
authors.forename: Ramaswamy
authors.initials: R
authors.lastname: Krishnamoorthi
mesh_terms: D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000276:immunology*; D005936:Glucans; D007113:Immunity, Innate; D019476:Insect Proteins / Q000737:chemistry / Q000276:immunology*; D007795:Laminaria / Q000276:immunology; D008958:Models, Molecular; D009036:Moths / Q000737:chemistry / Q000276:immunology* / Q000382:microbiology; D019906:Nuclear Magnetic Resonance, Biomolecular; D011134:Polysaccharides / Q000276:immunology; D017434:Protein Structure, Tertiary; D047071:beta-Glucans / Q000276:immunology*
chemical_list: D002352:Carrier Proteins; D005936:Glucans; D019476:Insect Proteins; D011134:Polysaccharides; C405332:beta-1,3-glucan recognition protein, insect; D047071:beta-Glucans; C008247:laminaran; C033363:beta-1,3-glucan