9304096 Hapner This research involves the molecular biology and protein structure of the agglutinin (lectin) present in the hemolymph of the grasshopper, Melanoplus differentialis. Insect agglutinins are defense response molecules that are a component of invertebrate immune mechanisms. They act as surveillance molecules and take part in the recognition and neutralization of non-self substances including metamorphic debris and pathogenic microorganisms. Primary objectives of the work are: determination of the nucleotide sequence of the cDNA encoding the agglutinin protein; determination of the number of genes coding for the agglutinin and their expression and induction; and the extension of known physicochemical characteristics of the 70 kD agglutinin dimer, including its amino acid sequence, molecular homogeneity, subunit identity, and carbohydrate recognition properties of individual carbohydrate-binding domains, of which there are two per polypeptide chain. The overall aim of the research is to characterize grasshopper agglutinin as a defensive surveillance molecule exhibiting broad carbohydrate recognition potential. Methods to be employed include molecular cloning and nucleotide sequence determination of agglutinin-encoding CDNA from grasshopper fat body, Southern analysis of genomic DNA and Northern analysis of mRNA isolated from several grasshopper tissues, both naive and immuno-stimulated. Physicochemical characterization will include affinity chromatography on glucosidic and galactosidic Sepharose columns, reducing and nonreducing SDS polyacrylamide gel electrophoresis, isoelectric focusing, and reverse phase high performance liquid chromatography. Protein carbohydrate will be determined by treatment with endoglycosidases. Individual carbohydrate recognition domains are to be proteolytically excised from the 35 Kd monomer subunit and their binding characteristics studied by affinity electrophoresis. Subunit identity or nonidentity of the 70 Kd dimer will be confirmed by partial amino acid sequence of CNBr or tryptic fragments generated from the purified 35 Kd monomer. These results should determine whether or not the dimer represents one gene product or two, with consequent implications for carbohydrate binding diversity. %%% Insect agglutinins are defense response molecules that are a component of invertebrate immune mechanisms. They act as surveillance molecules and take part in the recognition and neutralization of non-self substances such as debris left after metamorphosis and pathogenic microorganisms. The goal of this research is to understand the structure and function of the protein agglutinin (lectin) present in the hemolymph (blood) of the grasshopper. Increased knowledge of invertebrate immune mechanisms and their potential vulnerability will contribute to the rational development of insect control methods, and will also enhance methods for management and culturing of beneficial invertebrate organisms.***
