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Title: CDNA CLONING, PURIFICATION, PROPERTIES, AND FUNCTION OF A BETA-1, E-GLUCAN RECOGNITION PROTEIN FROM A PYRALID MOTH, PLODIA INTERPUNCTELLA

Author
item FABRICK, JEFFREY - 5430-05-30
item Baker, James
item KANOST, MICHAEL - KANSAS STATE UNIV

Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/6/2003
Publication Date: 5/20/2003
Citation: FABRICK,J.A., BAKER,J.E., KANOST,M.R., CDNA CLONING, PURIFICATION, PROPERTIES, AND FUNCTION OF A BETA-1, E-GLUCAN RECOGNITION PROTEIN FROM A PYRALID MOTH, PLODIA INTERPUNCTELLA, INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 33:579-594. 2003.

Interpretive Summary: Multicellular organisms have evolved with the ability to protect themselves from pathogens. In many organisms, this protection is provided through the action of an immune system. An effective immune response is however dependent on the initial recognition of foreign pathogens as nonself. A recognition strategy utilized by the innate immune system of both vertebrates and invertebrates is based on the detection of conserved molecular patterns unique to microbes. We have conducted a study to characterize one such recognition protein from the insect pest the Indianmeal moth. A comprehensive understanding of the immune system of such insect pests may provide logical avenues for which effective insect control may be acquired via ecologically sound biological control methods.

Technical 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 beta-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 beta-1,3-glucan recognition protein (beta GRP) from the Indianmeal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). Beta GRP 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, beta-1,3-glucanases from bacteria, and a beta-1,3-glucanase from the sea urchin, Strongylocentrotus purpuratus. The amino-terminus of beta GRP shares sequence similarity with other invertebrate recognition molecules and the beta-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 beta GRP in P. interpunctella larval hemolymph. RT-PCR analysis indicates that beta GRP 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 beta GRP transcript is transcribed within the fat body. Recombinant beta GRP produced in bacteria retains beta-1,3-glucan-binding activity, binds to lipopolysaccharide and lipoteichoic acid in vitro, possesses the ability to cause aggregation of microorganisms, and activates the prophenoloxidase cascade in insects in the presence of soluble beta-1,3-glucan. These data support the hypothesis that the 53-kDa beta GRP functions to recognize pathogen surface molecules as nonself and subsequently activates insect innate immune responses.