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

9304096哈普纳这项研究涉及蝗虫血淋巴中凝集素(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。它们充当监视分子，参与识别和中和包括变质碎片和病原微生物在内的非我物质。这项工作的主要目标是：确定编码凝集素蛋白的cDNA的核苷酸序列；确定编码凝集素的基因的数量及其表达和诱导；以及扩展70kD凝集素二聚体的已知物理化学特性，包括其氨基酸序列、分子同源性、亚基同一性和单个糖结合结构域的糖识别特性，其中每个多肽链有两个。这项研究的总体目标是将蝗虫凝集素描述为一种具有广泛的碳水化合物识别潜力的防御性监视分子。采用的方法包括从蝗虫脂肪体中克隆编码凝集素的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天然和免疫刺激的mRNA的Northern分析。物理化学特性将包括糖苷和半乳糖苷Sepharose柱上的亲和层析、还原和非还原的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法、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切糖苷酶处理来测定。用蛋白水解法从35kD的单体亚基中分离出单个碳水化合物识别结构域，并用亲和电泳法研究它们的结合特性。70kD二聚体的亚基同一性或非同一性将通过CNBR的部分氨基酸序列或从纯化的35kD单体产生的胰酶片段来确认。这些结果应该确定二聚体是否代表一个或两个基因产物，从而对碳水化合物结合多样性产生影响。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。它们充当监视分子，参与识别和中和非我物质，如变态后留下的碎片和病原微生物。本研究的目的是了解蝗虫血淋巴(血)中存在的蛋白质凝集素(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了解，将有助于合理开发昆虫控制方法，并将改进有益无脊椎动物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.***