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Study on the selection and traffic of lysosomal enzyme

溶酶体酶的选择与运输研究

基本信息

批准号：
12680704
负责人：
NISHIKAWA Atsushi
金额：
$ 2.24万
依托单位：
Tokyo University of Agriculture and Technology (2001)Okayama University of Science (2000)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680704/
关键词：
Iysosome N-linked Dnase I manuose-6-phosphate Oligosacchary Itames ferase DNaseI

项目摘要

In many cell types, newly synthesized acid hydrolases acquire mannose 6-phosphate residues which serve as high affinity ligands for binding to Man-6-P receptors and targeting to lysosomes. A key step in this pathway is the ability of the enzyme UDP-GlcNAc : Lysosomal enzyme N-acetylglucosaminyl phosphotransferase to selective recognize and phosphorylate acid hydrolases. We have previously demonstrated that phosphotransferase recognizes conformational dependent protein determinants present on the acid hydrolases and absent in most nonlysosomal glycoproteins. It was recently reported that the secretory glycoprotein DNase I contains Man-6-P residues and we found that the human, bovine and mouse forms of this enzymes are phosphorylated to a low extent when expressed in COS cells. The poor phosphorylation of human and bovine DNase I is due to the lack of two critical lysines (position 27 and 74) present in the mouse enzyme whereas the poor phosphorylation of mouse Dnase I appears to be due to the presence of "inhibitory" amino acid (Val23) that block the phosphotransferase recognition domain and the disappear of two critical amino acids (Tyr54 and Serl90).Then, we also report the substrate specificity of GPT for the oligosaccharides using variety of fluorescence labeled high mannose type oligosaccharides. As a point to be noticed, GPT can transfer the GlcNAc-phosphate to the GlcMan9GlcNAc2 that still remains glucose.

在许多细胞类型中，新合成的酸性水解酶获得甘露糖6-磷酸残基，其用作结合Man-6-P受体和靶向溶酶体的高亲和力配体。该途径中的关键步骤是酶UDP-GlcNAc：溶酶体酶N-乙酰葡糖胺基磷酸转移酶选择性识别和磷酸化酸水解酶的能力。我们以前已经证明，磷酸转移酶识别构象依赖的蛋白质决定簇上的酸水解酶和大多数非溶酶体糖蛋白缺席。最近报道分泌糖蛋白DNase I含有Man-6-P残基，并且我们发现当在COS细胞中表达时，人、牛和小鼠形式的这种酶被低程度地磷酸化。人和牛的DNA酶I磷酸化能力差是由于缺少两个关键的赖氨酸小鼠Dnase I的磷酸化差似乎是由于存在阻断磷酸转移酶识别结构域的“抑制性”氨基酸（Val 23）和两个关键氨基酸（Val 23）的消失。（Tyr 54和Ser 190）。然后，我们还报道了使用多种荧光标记的高甘露糖型寡糖的GPT对寡糖的底物特异性。值得注意的是，GPT可以将GlcNAc-磷酸转移到仍保留葡萄糖的GlcMan 9 GlcNAc 2。