Tunicate Lectins as Acute Phase Reactants

这项研究计划的总体目标是表征 充分利用了分子和基因结构以及 从选定的模式无脊椎动物凝集素，以了解 它们作为识别分子的进化，并阐明了 其生物学功能的分子机制。 期间 在过去三年中，在NSF的支持下，四种不同的L-岩藻糖基结合 凝集素已经从原脊索动物的血浆中分离出来， C.picta，以及生物化学性质和结合特异性， 除了L-岩藻糖之外，还具有其它特性。 C. picta 凝集素结合磷酸胆碱、半乳聚糖、唾液酸和胞壁 acids. 此外，通过使用单克隆抗体和 针对纯化的蛋白质开发的常规抗血清， 合成肽，它表明，这些凝集素是交叉- 与哺乳动物急性期反应物反应，特别是与 C-反应蛋白（CRP）、血清淀粉样蛋白（SAP）和凝血功能 因子VIII（F VIII）。 部分氨基酸序列已被 确定了两种凝集素，和一种肽形式之一， 凝集素（CPL-I）已被表征为表现出高度同源性 人F VIII。 一种来自血细胞的含有poly（A）+的RNA已被发现， 分离、合成双链cDNA、构建文库 使用表达载体λ gt 11，和12个阳性的 已经分离出重组噬菌体克隆。 下一个目标 三年是：1）完成氨基酸和核苷酸 两种凝集素的序列，以确定同源性的程度， 急性时相蛋白，CRP，SAP和F VIII; 2）发展 为了鉴定和分离基因组克隆， 描述他们的基因结构; 3）进行 研究以确定和表征C. picta 凝集素及其推定的天然配体（外源或 内源性），特别是与 C. picta菌落和被膜硫酸化半乳聚糖。 该实验室先前的研究表明， 原脊索动物，被囊动物，有碳水化合物结合蛋白 （凝集素）在他们的血液中，类似于某些蛋白质发现， 高等脊椎动物的血液，即所谓的“急性期 蛋白质”，被认为是原始防御的组成部分 反应 继续这些分子水平的研究， 被囊动物凝集素将增加我们的生理知识， 这些有趣的海洋生物，扩大我们对 宿主防御机制的起源。

The overall goals of this research program are to characterize fully the molecular and gene structures and binding properties of lectins from selected model invertebrates in order to understand their evolution as recognition molecules and elucidate the molecular mechanisms of their biological functions. During the past three years, with NSF support, four distinct L-fucosyl-binding lectins have been isolated from the plasma of the protochordate, C.picta, and the biochemical properties and binding specificities, in addition to L-fucose, have been characterized. C. picta lectins bind phosphocholine, galactan, and sialic and muramic acids. Furthermore, by the use of monoclonal antibodies and conventional antisera developed against the purified proteins and synthetic peptides, it was shown that those lectins are cross- reactive with mammalian acute phase reactants, in particular with c-reactive protein (CRP), serum amyloid P (SAP) and coagulation factor VIII (F VIII). Partial amino acid sequences have been determined for two of the lectins, and a peptide form one of the lectins (CPL-I) has been characterized that exhibits high homology to human F VIII. A poly(A)+ containing RNA from hemocytes has been isolated, double stranded cDNA synthesized, a library constructed using the expression vector, lambda gt11, and twelve positive recombinant phage clones have been isolated. Goals for the next three years are: 1) to complete the amino acid and nucleotide sequences of both lectins to determine the extent of homology to the acute phase proteins, CRP, SAP, and F VIII; 2) to develop probes required to identify and isolate genomic clones in order to characterize the structure of their genes; and 3) to carry out studies to identify and characterize the interactions of C. picta lectins with their putative natural ligands (exogenous or endogenous), in particular the bacterial community associated with C. picta colonies and the tunic sulphated galactans. Prior research from this laboratory has shown that the protochordates, tunicates, have carbohydrate-binding proteins (lectins) in their blood that resemble certain proteins found in the blood of higher vertebrates, the so-called "acute phase proteins", that are thought to be components of a primitive defense response. The continuation of these molecular-level studies of tunicate lectins will increase our knowledge of the physiology of these interesting marine organisms and expand our understanding of the origins of host defense mechanisms.