CDNA cloning, purification, properties, and function of a β-1,3-glucan recognition protein from a pyralid moth, Plodiainterpunctella

Abstract

Microorganisms possess distinctive biochemical or molecular patterns on their cell surfaces, such as those formed by the lipopolysaccharides, lipoteichoic acids, and/or peptidoglycans of bacteria and the β-1,3-glucans of fungi. Pattern recognition proteins that bind to these surface moieties have been implicated in the activation of the innate immune response in insects and other invertebrates. We report the purification and cloning of a cDNA for a 53-kDa β-1,3-glucan recognition protein (βGRP) from the Indianmeal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). βGRP cDNA contains an open reading frame that encodes 488 amino acids, of which the first 17 residues comprise the secretion signal peptide. The calculated molecular mass of the 471-residue mature protein is 53,311 Da. The protein consists of a carboxyl-terminal domain that is similar to other recognition proteins from invertebrates, β-1,3-glucanases from bacteria, and a β-1,3-glucanase from the sea urchin, Strongylocentrotus purpuratus. The amino-terminus of βGRP shares sequence similarity with other invertebrate recognition molecules and the β-1,3-glucanase from S. purpuratus. Affinity purification of a 53-kDa protein and subsequent sequencing of a peptide produced by tryptic cleavage confirmed the presence of the βGRP in P. interpunctella larval hemolymph. RT-PCR analysis indicates that βGRP is constitutively expressed in all life-stages, with no detectable induction following exposure of wandering larvae to microbial elicitors. Northern blot analysis indicates that the 1.8-kb βGRP transcript is transcribed within the fat body. Recombinant βGRP retains β-1,3-glucan-binding activity, binds to lipopolysaccharide and lipoteichoic acid in vitro, causes aggregation of microorganisms, and activates the prophenoloxidase cascade in the presence of soluble β-1,3-glucan. These data support the hypothesis that the 53-kDa βGRP functions to recognize pathogen surface molecules as nonself and subsequently activates insect innate immune responses.