Development of fine micro analytical method of drug recognition function of sugar chains

采用毛细管电泳（CE）和质谱（MS）相结合的方法研究糖蛋白结构对药物结合亲和力的影响。以α1-酸性糖蛋白（AGP）为模型糖蛋白，评价其糖型和遗传变异与模型基础药物的结合亲和力。(a)仅位于AGP分子上药物结合位点附近的双触角糖链对AGP与模型基础药物（双酰胺和维拉帕米）的对端选择性结合没有显著影响。(b) AGP有三种主要的基因变异，a型变异、f1型变异和s型变异。与F1^*S变异相比，a变异在与模型药物对映体结合时表现出更高的对映选择性，而这两种遗传变异在与维拉帕米对映体结合时表现出几乎相同的对映选择性。(c)采用无鞘ESI法的CE/MS系统在AGP的胰蛋白酶消化分析中可提高一个数量级的检出率。为了研究糖蛋白的微观异质性对蛋白质功能和二级结构的影响，采用核磁共振、分子轨道法和CE光谱对合成的na通道蛋白和朊蛋白的多肽片段进行了结构化学研究。合成的片段对应于负责蛋白质二级结构和/或功能的主要肽部分。发现了以下内容。(a)由于溶剂的性质和成分，模型肽片段的二级结构随环境的疏水性而变化。(b)同时含有疏水片段和疏水片段的聚糖，根据聚糖的性质、序列和分支结构的不同，会产生具有不同微介电常数的局部疏水空间。提示疏水局部环境的产生会引起蛋白质二级结构的改变，从而调控其功能和淀粉样纤维的形成。少

A novel analytical system using capillary electrophoresis (CE) and mass spectroscopy (MS) was developed to investigate the structural effect of glycoproteins upon drug binding affinity. α1-acid glycoprotein (AGP) was used as a model glycoprotein, and the binding affinities of AGP glycoforms and genetic variants to model basic drugs were evaluated.(a) The biantennary glycochains which locate only near the drug binding site on AGP molecule does not give any significant effect upon the enantioselective binding of AGP to model basic drugs (disopyramide and verapamil).(b) AGP has three major genetic variants, A-variant, F1-variant and S-variant. A-variant shows higher enantioselectivity in the binding with the enantiomers of model drugs than F1^*S variants, while these genetic variants show almost the same enantioselectivity in the binding to verapamil enantiomers.(c) CE/MS system using sheathless ESI method allows one-order higher detectability in the analyses of tryptic digests of AGP var … More iants than conventional CE method using UV detection.In order to investigate the effect of microheterogeneity of glycoproteins upon the function and the secondary structure of the proteins, the structural chemistry of several synthesized peptide fragments of Na-channel protein and prion protein were studied by using NMR, molecular orbital method and CE spectroscopy. The synthesized fragments correspond to the major peptide portion responsible for the secondary structure and/or the function of the proteins. The followings were found.(a) The secondary structure of model peptide fragments changes depending on the hydrophobicity of environment due to the nature and the component of solvents.(b) Glycans containing both hydrophobic moiety and hydrophobic moiety create local hydrophobic space with different micro-permittivity depending on nature, sequence and the branching structure of glycans. It is suggested that the creation of hydrophobic local environment would cause the change of secondary structure of protein, resulting in the regulation of function and amyloid fibril formation. Less