Tunicate C-type Lectins as Acute Phase Reactants

被囊动物 C 型凝集素作为急性期反应物

• 批准号: 9406649
• 负责人: Gerardo Vasta
• 金额: --
• 依托单位: University of Maryland Biotechnology Institute
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9406649&HistoricalAwards=false
• 关键词: Tunicate; type; Lectins; Acute; Phase

9406649 Vasta As knowledge of the biochemical properties and gene sequences of animal lectins has accumulated in recent years, we have begun to understand not only their complex molecular and gene organization, but also their evolution and multiple biological roles. The goal of this research is to characterize the molecular and gene structures and binding properties of lectins from selected invertebrates in order to understand their evolution as self/non- self recognition molecules, and to elucidate the molecular mechanisms of their biological functions. With prior NSF support considerable progress has been made in isolation, purification, and biochemical characterization of four distinct L-fucosyl binding lectins, CPL-I, -II, -III, and -IV, from plasma and hemocytes of the protochordate Clavelina picta. Serological and primary structure evidence suggest that C. picta lectins are related to acute phase reactants from vertebrates, namely, human pro-clotting factor VIIIc (F VIIIc), C-reactive protein (CRP), and serum amyloid P (SAP). The C.picta lectin binding properties, including the fine specificity for L-fucose-containing oligosaccharides, affinity constants, divalent cation dependence and effect of toxic metals, have been extensively characterized, and the amino acid residues of the polypeptide chain involved in the interactions with the ligand have been identified by specific chemical modification. C. picta lectins bind preferentially to oligosaccharides that exhibit non- reducing terminal fucose but surprisingly, also bind phosphocholine, galactans, sialic and muramic acids, and proteoglycans. Preliminary studies to identify the "natural" ligands, both exogenous (environmental and colony-associated bacterial isolates) and endogenous (tunic sulfated galactan) have begun. Based on experimental results thus far, it appears that C. picta lectins are multifunctional recognition/effector mosaic molecules, involved not only in recognition and opsonization o f potentially pathogenic bacteria from the environment but also in wound repair upon injury of the body wall. Additional lectins have now been isolated from C. picta that have been characterized as belonging to the S-type. Until recently, S-type lectins were believed to be restricted to vertebrate species. This finding resulted not only in the isolation of S-type lectins in a variety of invertebrate species but also in the crystallization and resolution of the three dimensional structure of an animal lectin for the first time. To obtain the complete nucleotide sequence that will allow alignment of the extensive amino acid sequence obtained so far, a hemocyte cDNA library was constructed in the expression vector lunizap and through PCR techniques, a nucleic acid probe for CPL-III was developed. The hemocyte cDNA library was screened with this probe and positive recombinant phage clones were isolated. All the clones selected yield a product of similar size when used as templates for PCR with the same primers. These are currently being sequenced. Goals for the next three years are to conduct studies in two areas: 1) Protein and cDNA structure of C. picta lectins: By the end of the current funding period it is expected that substantial nucleotide sequence of CPL-III will be in hand, and the extent of homology to the acute phase reactants CRP, SAP, and F VIIIc will be determined. By the end of the new funding period equivalent information should be available for the other three C.picta lectins. 2) Biological role of C.picta lectins: Endogenous and exogenous "natural" ligands, i.e. tunic sulfated galactans and polysaccharides from selected environmental bacterial strains, will be sought and characterized. in vitro studies of the interactions of the characterized "natural" ligands or their relevant determinants on the C.picta hemocytes will be studied, and the inducibility of C.picta lectins by the putative natural ligands will be examined. %%% The goal of this research is to characterize the molecular structure, properties and biological functions of certain sugar- binding molecules ("lectins") from selected invertebrate animals to understand their evolution as self/non-self recognition molecules, and to clarify the molecular mechanisms of their biological functions which may be important in the defense mechanisms of a diversity of animals. ***

9406649瓦斯塔 近年来，随着对动物凝集素的生物化学性质和基因序列的了解的积累，我们不仅开始了解它们复杂的分子和基因组织，而且开始了解它们的进化和多种生物学作用。 本研究的目的是表征选定的无脊椎动物凝集素的分子和基因结构以及结合特性，以了解它们作为自我/非自我识别分子的进化，并阐明其生物学功能的分子机制。 与以前的NSF支持相当大的进展已经取得了分离，纯化和生化特性的四个不同的L-岩藻糖基结合凝集素，CPL-I，II，III和IV，从血浆和血细胞的原索动物Clavelina picta。 血清学和一级结构证据表明，C。picta凝集素与来自脊椎动物的急性期反应物，即人促凝血因子VIIIc（FVIIIc）、C-反应蛋白（CRP）和血清淀粉样蛋白P（SAP）相关。 已经广泛地表征了C.picta凝集素的结合性质，包括对含L-岩藻糖的寡糖的良好特异性、亲和常数、二价阳离子依赖性和有毒金属的影响，并且已经通过特异性化学修饰鉴定了参与与配体相互作用的多肽链的氨基酸残基。 C. Picta凝集素优先结合显示非还原性末端岩藻糖的寡糖，但令人惊讶的是，也结合磷酸胆碱、半乳聚糖、唾液酸和胞壁酸以及蛋白聚糖。 初步研究，以确定“天然”配体，外源性（环境和菌落相关的细菌分离株）和内源性（被膜硫酸半乳聚糖）已经开始。 根据目前的实验结果，C. picta凝集素是多功能的识别/效应嵌合分子，不仅参与识别和调理来自环境的潜在致病细菌，而且参与体壁损伤后的伤口修复。 现在已经从C. picta被认为属于S型。 直到最近，S型凝集素被认为仅限于脊椎动物物种。 这一发现不仅导致了在各种无脊椎动物物种中的S-型凝集素的分离，而且还导致了第一次结晶和解析动物凝集素的三维结构。 为了获得完整的核苷酸序列，这将允许迄今为止获得的大量氨基酸序列的比对，在表达载体lunizap中构建血细胞cDNA文库，并通过PCR技术，开发了CPL-III的核酸探针。 用该探针筛选血细胞cDNA文库，获得阳性重组噬菌体克隆。 当用相同的引物作为PCR模板时，所有选择的克隆产生相似大小的产物。 目前正在对它们进行排序。 今后三年的目标是在两个方面进行研究：1）C. picta凝集素：到目前的资助期结束时，预计将掌握CPL-III的大量核苷酸序列，并将确定与急性期反应物CRP、SAP和F VIIIc的同源性程度。 到新的资助期结束时，应可获得其他三种C.picta凝集素的等效信息。 2)C.picta凝集素的生物学作用： 内源性和外源性的“天然”配体，即被膜硫酸化半乳聚糖和多糖从选定的环境细菌菌株，将寻求和表征。 将研究所表征的“天然”配体或其相关决定簇在C.picta血细胞上的相互作用的体外研究，并将检查推定的天然配体对C.picta凝集素的诱导。 本研究的目的是表征来自选定的无脊椎动物的某些糖结合分子（“凝集素”）的分子结构、性质和生物学功能，以理解它们作为自我/非自我识别分子的进化，并阐明它们的生物学功能的分子机制，这可能在多种动物的防御机制中是重要的。 ***