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Relationship between brain-specific addition of mucin-carbohydrates and neurodegenerative diseases

脑特异性添加粘蛋白碳水化合物与神经退行性疾病之间的关系

基本信息

批准号：
15590077
负责人：
KUROSAKA Akira
金额：
$ 2.24万
依托单位：
Kyoto Sangyo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

1.Addition of mucin-type carbohydrates to a protein involved in neurodegenerative diseasesIn vitro addition of GalNAc to α-synuclein, which is reported to aggregate to form insoluble fibrils in Parkinson's disease, was investigated using four GalNAc-T isozymes as enzyme source, and α-synuclein expressed in E.coli as substrate. Of four isozymes, only two isozymes transferred GalNAc to α-synuclein. Endoplasmic reticulum stress was given to cultured neural cells to see if it may cause transcriptional induction of neuron-specific GalNAc-transferases. No induction was, however, observed.2.Function of mucin-carbohydrates during development and differentiation of nervous tissuesWhen expression of neuron-specific GalNAc-transferase isozymes was depressed using an RNAi method in zebrafish embryos, disorder of hindbrain development was observed. Similarly, expression of these isozymes was suppressed in pluripotent embryonic carcinoma P19 cells. Neuronal differentiation of these cells was inhibited as well.3.Structure-function relationship of a GalNAc-transferaseFunction of the lectin-like of GalNAc-T1 was investigated. GalNAc-T1 bound to resins coated with either GalNAc or Man, suggesting the sugar binding activity of the lectin domain. Moreover, study with site-directed mutagenesis indicated that one of the tryptophan residues in the Gal/GalNAc-T motif is involved in the recognition of both donor and acceptor substrates.4.Substrate screening of neuron-specific isozymesPhage-display screening was carried out to find peptides that can bind to the enzyme with high affinity, but no peptides have been obtained so far.

1.以大肠杆菌表达的α-突触核蛋白为底物，以4种GalNAc-T同工酶为酶源，研究了GalNAc与α-突触核蛋白的体外加成作用。在4种同工酶中，只有2种同工酶将GalNAc转移到α-突触核蛋白。将内质网应激给予培养的神经细胞以观察其是否可引起神经元特异性GalNAc转移酶的转录诱导。2.粘蛋白-碳水化合物在神经组织发育和分化过程中的作用利用RNAi方法抑制斑马鱼胚胎神经元特异性GalNAc-转移酶同工酶的表达，观察到后脑发育障碍。类似地，这些同工酶的表达在多能胚胎癌P19细胞中被抑制。3. GalNAc-转移酶的结构与功能关系研究了GalNAc-T1的凝集素样功能。GalNAc-T1结合到用GalNAc或Man包被的树脂上，表明凝集素结构域的糖结合活性。此外，定点突变的研究表明，Gal/GalNAc-T基序中的一个色氨酸残基参与了对供体和受体底物的识别。4.神经元特异性同工酶的底物筛选通过噬菌体展示技术筛选能与该酶高亲和力结合的肽段，但目前还没有筛选到。