Purification, cDNA cloning, and biological significance of a beta-1,3-glucan recognition protein and its amino- and carboxyl-terminal domains from the Indianmeal moth, Plodia interpunctella

Invertebrates rely on innate immunity for protection against a variety of infectious, parasitic, or opportunistic organisms. Defense systems of arthropods involve both cellular and humoral facets and are activated by a wide variety of pattern recognition receptors. Many of these pattern recognition proteins have overlapping specificities for microbial surface molecules and may function to activate various immune pathways. I 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. 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. Affinity purification of a 53-kDa protein from larval hemolymph and subsequent sequencing of a peptide produced by tryptic cleavage confirmed the presence of the βGRP in P. interpunctella. RT-PCR analysis indicates that βGRP is constitutively expressed in all life-stages, with no detectable induction following exposure of larvae to microbial elicitors. Northern blot analysis indicates that the 1.8-kb βGRP mRNA 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. A recombinant polypeptide corresponding to the amino-terminal domain of the P. interpunctella βGRP bound to β-1,3-glucans, lipopolysaccharide, and lipoteichoic acid found on microorganisms and activated the prophenoloxidase activating system, an important immune response pathway in arthropods. The carboxyl-terminal domain has less affinity for these compounds and did not activate the prophenoloxidase activating system. P. interpunctella βGRP also participates in formation of a prophenoloxidase-activation complex that consists of several proteins with known roles in insect immunity. These data support the hypothesis that the 53-kDa P. interpunctella βGRP functions as a pattern recognition protein for the recognition of microbes and that its two domains bind microbial polysaccharides with differing specificity. The amino-terminal domain, which is unique to this class of pattern recognition receptors from invertebrates, is critical for recognition of “nonself” and subsequent activation of insect innate immune responses.