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

1.在参与神经退行性疾病的蛋白质中添加粘蛋白类型的碳水化合物研究了以四种α-T同工酶为酶来源，以在大肠杆菌中表达的α-突触核蛋白为底物，在体外将GalNAc加入到据报道在帕金森病中聚集形成不溶纤维的GalNAc-突触核蛋白。在四种同工酶中，只有两种同工酶将GalNAc转化为α-突触核蛋白。对培养的神经细胞施加内质网应激，以确定它是否可能导致神经元特异性GalNAc转移酶的转录诱导。2.神经组织发育和分化中粘蛋白-碳水化合物的作用当用RNAi方法抑制斑马鱼胚胎中神经元特异性GalNAc-转移酶同工酶的表达时，观察到后脑发育障碍。同样，在多能胚胎癌细胞P19中，这些同工酶的表达也受到抑制。3.GalNAc转运酶的结构与功能关系研究了GalNAc-T1凝集素样蛋白的功能。GalNAc-T1与包被GalNAc或Man的树脂结合，表明凝集素结构域具有糖结合活性。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筛选，寻找能够与该酶高亲和力结合的多肽，但到目前为止还没有得到任何多肽。

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.