β-淀粉样蛋白前体N端神经节苷脂GM1结合位点定位分析  

Ganglioside-GM1 binding site on the N-terminus of amyloid β-protein precurs or

作　　者：丁吉新[1] 阮燕[1] 朱忠军[1] 张岱[1] 

机构地区：[1]北京大学精神卫生研究所,北京100083

出　　处：《北京大学学报（医学版）》2002年第5期594-598,共5页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金 ( 3 9870 2 76;3 9840 0 10 );国家重点基础研究发展规划项目 (G19990 64 0 0 7)资助

摘　　要：目的 :确定 β 淀粉样蛋白前体 (amyloidβ proteinprecursor ,APP)分子N端的神经节苷脂GM1结合位点。 方法 :利用重组DNA技术获得不同长度Mty4 APP融合蛋白以及合成APP多肽 ,用于分析和确定APP N端GM1的活性区 ;用免疫沉淀Western印迹方法探讨不同神经节苷脂与APP N端融合蛋白的结合能力。结果 :Western印迹法证实APP18 81、APP18 65和APP18 58片段可与GM1分子结合 ,而APP18 51片段不与GM1分子相互作用。多肽抑制实验证明 ,多肽APP52～ 81明显阻断了融合蛋白APP18 81与GM1的结合。不同神经节苷脂与APP N端融合蛋白的结合能力实验表明 ,融合蛋白APP18 81与GM1特异性结合 ,而不与GD1a ,GT1b ,GALCER相互作用。结论 :APP分子N端存在着一个GM1的活性区域 ,位于 5 2～ 81氨基酸残基区间。此区域与GM1存在着特异性相互作用 ,有赖于GM1糖链的特殊构象 ,而与神经节苷脂上唾液酸的数目无关 ,也与神经酰胺母链无关。GM1在APP N端活性区域的发现和定位 ,对探讨GM1代谢异常与 βObjective: To identify the binding site of ganglioside GM1 on amyloid β -protein precursor (APP) in vitro for exploring the relationship between GM 1 and amyloid β-protein (Aβ) generation on Alzheimer disease. Methods: Four plasmids, APP 18-81, 18-65, 18-58, 18-51 , were constructed by gene recombination . The frag ments of N-terminal APP (APP-NTFs) were expressed in Escherichia coli and then p urified. The purified products were examined for GM1 binding abilities by Wester n blotting. Immunoprecipitation Western blotting was used to identify the bindin g specialities between different gangliosides and APP 18-81 . The synthetic peptid es spanning residues 52-81 of APP blocked GM1 binding to APP 18-81 i n order to confirm the specific GM1-binding region within APP-NTFs. Results: Western blo tting showed that GM1 could bind to the APP 18-81 , APP 18-65 and APP 18-58 fragment s, but could not bind to APP 18-51 . The synthetic peptide APP 52-81 could block GM 1 binding to APP 18-81 , however, the synthetic peptide APP 52-65 , AP P 66-81 could n ot block GM1 interacting with APP 18-81 . The binding test of different gan gliosid es and APP 18-81 showed there was a specific interaction between N-termin us of AP P and GM1, which depended on the sugar moiety of GM1, rather than on neither the ceramide part nor the number of sialic acids. Conclusion: The binding site of GM1 on APP locat es on 52-81 amino acid residues of the APP molecule. These results suggest th at GM1 might affect the physiological function of APP, change APP span-membrane p rocess and interfere with APP trafficking and internalization by anchoring this molecule on the membrane, which may provide much more substrates for γ-secreta se and enhance Aβ generation on the cells.

关 键 词：阿尔茨海默病 Β-淀粉样蛋白前体 神经节苷脂GM1 结合位点 

分 类 号：R749.16[医药卫生—神经病学与精神病学]