Tunicate C-type Lectins as Acute Phase Reactants

9406649 VASTA随着近年来对动物凝集素的生化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。本研究的目的是对所选择的无脊椎动物凝集素的分子结构、基因结构和结合特性进行表征，以了解它们作为自我/非自我识别分子的进化，并阐明它们生物学功能的分子机制。在国家自然科学基金会的支持下，四种不同的L岩藻糖基结合凝集素CPL-I、-II、-III和-IV的分离、纯化和生化特性研究已经取得了相当大的进展。血清学和一级结构证据表明，苦瓜凝集素与脊椎动物的急性期反应物有关，即人凝血因子VIIIc(F VIIIc)、C反应蛋白(CRP)和血清淀粉样蛋白P(SAP)。苦瓜凝集素与L-岩藻糖低聚糖良好的结合特性、亲和常数、二价阳离子依赖性和有毒金属的作用已被广泛表征，并通过特定的化学修饰鉴定了参与与配基相互作用的多肽链上的氨基酸残基。C.Picta凝集素优先与显示非还原末端岩藻糖的低聚糖结合，但令人惊讶的是，还与磷胆碱、半乳糖、唾液酸和壁壁酸以及蛋白多糖结合。鉴定“天然”配体的初步研究已经开始，包括外源性(环境和菌落相关细菌分离株)和内源性(衣壳硫酸半乳聚糖)。根据目前的实验结果，Picta凝集素似乎是一种多功能的识别/效应嵌合体分子，不仅参与识别和调理来自环境的潜在致病菌，而且参与体壁损伤后的伤口修复。其他凝集素现已从已被鉴定为属于S类型的苦瓜中分离出来。直到最近，S类凝集素还被认为仅限于脊椎动物物种。这一发现不仅导致在各种无脊椎动物中分离出S类型的凝集素，而且首次结晶和解析了动物凝集素的三维结构。为了获得与迄今获得的广泛氨基酸序列进行比对的完整核苷酸序列，在表达载体LUNIZAP中构建了血细胞cDNA文库，并通过聚合酶链式反应技术开发了CPL-III的核酸探针。用该探针筛选血细胞c DNA文库，获得阳性重组噬菌体克隆。所有选择的克隆在用相同的引物作为模板进行聚合酶链式反应时都会产生大小相似的产物。目前正在对这些基因进行测序。未来三年的目标是在两个领域进行研究：1)苦瓜凝集素的蛋白质和cDNA结构：到目前的资助期结束时，预计将有大量CPL-III的核苷酸序列，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，应可获得其他三种苦瓜凝集素的同等信息。2)苦味葡萄球菌凝集素的生物学作用：将寻找和表征内源和外源“天然”配体，即来自选定环境细菌菌株的衣壳硫酸盐化半乳糖和多糖。我们将在体外研究具有特征的“天然”配体或它们的相关决定因素与微囊线虫血细胞的相互作用，并将检验可能的天然配体对微囊线虫凝集素的诱导作用。本研究的目的是确定某些糖结合分子(“凝集素”)的分子结构、性质和生物学功能，以了解它们作为自我/非自我识别分子的进化，并阐明它们生物学功能的分子机制，这可能在动物多样性的防御机制中起重要作用。***

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. ***