Grasshopper Agglutinin (Lectin): Molecular Biology of a Pathogen Surveillance Molecule

9304096 Hapner本研究涉及到存在于蚱蜢血淋巴中的凝集素（凝集素）的分子生物学和蛋白质结构。昆虫凝集素是一种防御反应分子，是无脊椎动物免疫机制的组成部分。它们作为监视分子，参与识别和中和非自身物质，包括变质碎片和致病微生物。这项工作的主要目的是：确定编码凝集素蛋白的cDNA的核苷酸序列；凝集素编码基因数量的测定及其表达和诱导；以及已知70 kD凝集素二聚体的物理化学特性的扩展，包括其氨基酸序列、分子均匀性、亚基同一性和单个碳水化合物结合结构域的碳水化合物识别特性，每个多肽链上有两个碳水化合物结合结构域。该研究的总体目标是将蚱蜢凝集素描述为一种具有广泛碳水化合物识别潜力的防御性监视分子。采用的方法包括从蚱蜢脂肪体中分离的凝集素编码CDNA的分子克隆和核苷酸序列测定，基因组DNA的Southern分析和从几种蝗虫组织中分离的mRNA的Northern分析，包括幼稚和免疫刺激。物理化学表征将包括糖苷和半乳糖Sepharose柱的亲和色谱，还原和非还原SDS聚丙烯酰胺凝胶电泳，等电聚焦和反相高效液相色谱。蛋白质碳水化合物将通过内糖苷酶处理来测定。将单个碳水化合物识别结构域从35kd单体亚基上蛋白水解切除，并通过亲和电泳研究其结合特性。70 Kd二聚体的亚基同一性或非同一性将通过纯化的35 Kd单体产生的CNBr的部分氨基酸序列或色氨酸片段来确认。这些结果将决定二聚体是代表一种基因产物还是两种基因产物，从而影响碳水化合物结合的多样性。昆虫凝集素是防御反应分子，是无脊椎动物免疫机制的一个组成部分。它们作为监视分子，参与识别和中和非自身物质，如变态后残留的碎片和致病微生物。本研究的目的是了解存在于蚱蜢的血淋巴（血液）中的凝集素（凝集素）的结构和功能。增加对无脊椎动物免疫机制及其潜在脆弱性的认识将有助于合理开发昆虫防治方法，也将改进有益无脊椎生物的管理和培养方法

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.***