An initial event in 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

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Show simple item record Dai, Huaien Hiromasa, Yasuaki Takahashi, Daisuke VanderVelde, David Fabrick, Jeffrey A. Kanost, Michael R. Krishnamoorthi, Ramaswamy 2013-03-21T13:34:04Z 2013-03-21T13:34:04Z 2013-03-21
dc.description.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 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 15N-1H groups of the protein, suggesting the formation of a large complex. Analytical ultra centrifugation (AUC) studies of formation of N-βGRP:laminarin complex show that ligand-binding induces sel-fassociation 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 sub-micromolar concentrations. The structural model thus derived from the present studies for 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 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 carried out 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 N-βGRP:laminarin macro complex and that a decreased stability is accompanied by a reduced activation of the proPO pathway. Increased β-1,6 branching in laminarin also results in destabilization of the macro complex. These novel findings suggest that ligand-induced self-association of 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. en_US
dc.language.iso en_US en_US
dc.relation.uri en_US
dc.rights This document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in Biochemistry, copyright © American Chemical Society after peer review. To access the final edited and published work see en_US
dc.subject Pathogen recognition en_US
dc.subject Innate immunity en_US
dc.subject β-1,3-glucan en_US
dc.subject βGRP en_US
dc.title An initial event in 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 en_US
dc.type Article (author version) en_US 2013 en_US
dc.citation.doi doi:10.1021/bi301440p en_US
dc.citation.epage 170 en_US
dc.citation.issue 1 en_US
dc.citation.jtitle Biochemistry en_US
dc.citation.spage 161 en_US
dc.citation.volume 52 en_US
dc.contributor.authoreid hiromasa en_US
dc.contributor.authoreid kanost en_US
dc.contributor.authoreid krish en_US
dc.contributor.authoreid dskmokks en_US

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