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

1.将黏液型碳水化合物添加到与神经退行性疾病相关的蛋白质中研究以四种GalNAc- t同工酶为酶源，以大肠杆菌中表达的α-突触核蛋白为底物，在体外将GalNAc添加到α-突触核蛋白中，据报道，α-突触核蛋白在帕金森病中聚集形成不溶性原纤维。在四种同工酶中，只有两种同工酶将GalNAc转移到α-突触核蛋白上。对培养的神经细胞施加内质网应激，观察是否会引起神经元特异性galnac转移酶的转录诱导。然而，没有观察到诱导作用。利用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，观察后脑发育障碍。同样，这些同工酶的表达在多能胚胎癌P19细胞中被抑制。这些细胞的神经元分化也受到抑制。研究了GalNAc-T1凝集素样蛋白的结构-功能关系。GalNAc- t1与涂有GalNAc或Man的树脂结合，表明凝集素结构域的糖结合活性。此外，位点定向诱变研究表明，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。对神经元特异性同工酶进行底物筛选，寻找能与酶高亲和力结合的多肽，但目前尚未获得多肽。

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.