Analysis of the function of human brain carboxypeptidase B and its diagnostic significance in dementia of Alzheimer's type

1.人脑羧肽酶B(HBCPB)CPB-BP结合口袋的生化和电子显微分析：为了了解HBCPB及其特异性结合蛋白CPB-BP(1和2)的作用机制，我们利用这两种蛋白的一级结构产生的抗体进行了表位定位分析。这些单抗(6个克隆)表现出足够的特异性和敏感性，结合CPB-BP(1和2)及其调节的分析使这些抗体除了特异性和敏感性外，还可以根据三维结构的差异进行分类。这一成果为下一步实验方案中结合口袋和SBDD的结构分析奠定了基础。人血清中CPB-BP及其相关蛋白的寻找与分析。为了便于HTP分析，结合MALDI-TOF-MS进行了表面等离子激元相互作用分析。使用与CPB的C端表位相对应的多肽配体，对去除了主要丰富蛋白的人外周血清样品进行了分析。除了先前通过免疫沉淀确定的成功鉴定的分子外，还鉴定了一个新的分子，通过它可以识别与候选分子的相互作用。病理标本的免疫组织化学分析。所有AD脑样本和正常老年人的部分脑样本都在海马区显示CPB-BP2免疫反应。

1. Biochemical and in silico analyses of the CPB-BP binding pocket of human brain carboxypeptidase B (HBCPB) : In an attempt to understand the mechanism of action of HBCPB and its specific binding protein, CPB-BP (1 & 2), we have started an epitope mapping analyses by using antibodies generated based upon primary structures of the both proteins. These monoclonal antibodies (6 clones) exhibit sufficient specificity and sensitivity, and analyses of binding to CPB-BP (1 & 2) and its regulation have enabled classification of these in terms of difference in three-dimensional structures, apart from specificity and sensitivity. This achievement embodies a basis for structural analysis of the binding pocket and SBDD in the next experimental program.2. A search and analysis for CPB-BP and related proteins in human serum. To facilitate HTP analyses for this purpose, the surface plasmon interaction analysis in combination with MALDI- TOF-MS was utilized. Using a peptide ligand corresponding to the C-terminal epitope of CPB, human peripheral serum samples, from which major abundant proteins were removed, were analyzed. In addition to the successful identification molecules previously determined by immunoprecipitation, a new molecule was identified, with which an interaction to the candidate molecule is recognized.3. Immunohistochemical analyses of pathological specimen. All AD brain samples and a portion of brain samples from the normal aged both exhibit CPB-BP2 immunoreactivity in the hippocampus.