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

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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.