title: Self-association of an insect β-1,3-glucan recognition protein upon binding laminarin stimulates prophenoloxidase activation as an innate immune response.
abstract: Insect β-glucan recognition protein (βGRP), a pathogen recognition receptor for innate immune responses, detects β-1,3-glucan on fungal surfaces via its N-terminal carbohydrate-binding domain (N-βGRP) 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 βGRP2 (N-βGRP2) with laminarin, a soluble form of β-1,3-glucan. We found that carbohydrate binding by N-βGRP2 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-βGRP2 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-βGRP molecules in the soluble complex. The pro-PO activation in naive plasma was strongly stimulated by addition of the insoluble aggregates of N-βGRP2. 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 βGRP with microbial polysaccharide promotes assembly of βGRP oligomers, which may form a platform needed to trigger the pro-PO pathway activation cascade.
authors.affiliation: From the Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas 66506.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Takahashi
authors.affiliation: From the Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas 66506.
authors.forename: Huaien
authors.initials: H
authors.lastname: Dai
authors.affiliation: From the Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas 66506.
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Hiromasa
authors.affiliation: From the Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas 66506.
authors.forename: Ramaswamy
authors.initials: R
authors.lastname: Krishnamoorthi
authors.affiliation: From the Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas 66506 kanost@ksu.edu.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Kanost
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D062610:Fungal Polysaccharides / Q000737:chemistry* / Q000276:immunology; D005786:Gene Expression Regulation / Q000276:immunology; D005936:Glucans / Q000737:chemistry* / Q000276:immunology; D007113:Immunity, Innate; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D018402:Manduca / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D015398:Signal Transduction
chemical_list: D002352:Carrier Proteins; D004792:Enzyme Precursors; D062610:Fungal Polysaccharides; D005936:Glucans; D019476:Insect Proteins; C405332:beta-1,3-glucan recognition protein, insect; C008247:laminaran; D014442:Monophenol Monooxygenase
keywords: Analytical Ultracentrifugation; Carbohydrate-binding Protein; Hemolymph; Innate Immunity; Insect Immunity; Mass Spectrometry (MS); Pattern Recognition Receptor (PRR); Protein Aggregation; Protein Assembly; Protein Cross-linking