Tunicate C-type Lectins as Acute Phase Reactants

随着近年来对动物凝集素生化特性和基因序列知识的积累，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来自无脊椎动物的凝集素的分子和基因结构及其结合特性，以了解其作为自我/非自我识别分子的进化过程，并阐明其生物学功能的分子机制。在先前NSF的支持下，从原脊索动物clvelina picta的血浆和血细胞中分离、纯化和生化表征四种不同的L- focusyl结合凝集素（cpli、- ii、- iii和- iv）取得了相当大的进展。血清学和初级结构证据表明，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）。picta凝集素的结合特性，包括对含L-焦点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，已经被广泛地表征，并且通过特定的化学修饰鉴定了与配体相互作用的多肽链的氨基酸残基。C. picta凝集素优先结合表现出非还原性末端聚焦的低聚糖，但令人惊讶的是，它也结合磷酸胆碱、半乳糖、唾液酸和乳酸菌酸以及蛋白聚糖。鉴定“天然”配体的初步研究已经开始，包括外源性（环境和菌落相关的细菌分离）和内源性（外衣硫酸半乳酰胺）。从目前的实验结果来看，C. picta凝集素是一种多功能的识别/效应镶嵌分子，不仅参与对环境中潜在致病菌的识别和调理，还参与体壁损伤后的伤口修复。现在已经从C. picta中分离出了其他凝集素，这些凝集素被定性为属于s型。直到最近，s型凝集素被认为仅限于脊椎动物物种。这一发现不仅在多种无脊椎动物中分离到了s型凝集素，而且首次实现了动物凝集素的结晶和三维结构的解析。为了获得完整的核苷酸序列，以便对迄今为止获得的广泛的氨基酸序列进行比对，我们在表达载体lunizap上构建了血细胞cDNA文库，并通过PCR技术开发了CPL-III的核酸探针。利用该探针对白细胞cDNA文库进行筛选，分离出阳性重组噬菌体克隆。当用相同的引物作为PCR模板时，所选择的所有克隆产生的产物大小相似。目前正在对它们进行测序。未来三年的目标是在两个方面进行研究：1)C. picta凝集素的蛋白质和cDNA结构：到本资助期结束时，预计将获得ccp - iii的大量核苷酸序列，并确定与急性期反应物CRP， SAP和fviic的同源程度。到新的供资期结束时，其他三次C.picta选举的同等资料应可获得。2) picta凝集素的生物学作用：寻找和表征内源性和外源性“天然”配体，即从选定的环境菌株中提取的囊膜硫酸半乳和多糖。将在体外研究所表征的“天然”配体或其相关决定因素对picta血细胞的相互作用，并研究这些假定的天然配体对picta凝集素的诱导性。本研究的目的是表征某些来自无脊椎动物的糖结合分子（“凝集素”）的分子结构，性质和生物学功能，以了解其作为自我/非自我识别分子的进化，并阐明其生物学功能的分子机制，这可能对多种动物的防御机制具有重要意义。* * *

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. ***