Purification and cDNA cloning of galactosylceramidase 1

半乳糖神经酰胺酶1的纯化和cDNA克隆

基本信息

批准号：
02454246
负责人：
KOBAYASHI Takuro
金额：
$ 4.22万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454246/
关键词：
Krabbe disease Galactosylceramidase I galactosylceramidase I

项目摘要

Krabbe disease (GLD) is one of the geneti leukodystrophies, in which galactosylc eramidase I is deficient. For 10 years, we have studied the pathogenesis of this particular disorder and found that the hydrolysis of galactosylceramide is catalyzed by 2 acid hydrolases, namely galactosylceramidase I and 11, and only the former enzyme is deficient in GLD. This finding could answer the question why galactosylsphingosine but not galactosylceramide accumulates in the tissue of GLD patient. Because the accumulated galactosylsphingosine is cytotoxic, it is suggested that myelin-forming cells, oflgodendroglia and Schwann cells, are dead and demyelination occurs.The aim of this project is to characterize the molecular properties of the defici ent enzyme in GLD. The purification of the enzyme has been very difficult, and no in vestigators could succeed to purify it. In 1990, we have purified the enzyme up to I 0000 folds from the crude sample of human placenta, using several chromatographic tec hniques. The final product contained 2 bands of 58kDa and 2OkDa, as cheeked with SDS -PAGE. After blotting the 2 bands to an appropriatemembrane and the corresponding proteins were digested by endopeptidase. But the amino acid sequence of the 58kDa band revealed the homology to IgG, as checked by compute research. We could not detect amino acid in the 2OkDa band protein. Therefore, in 1991, we changed the starting material to porcine kidney which contained relatively high specific acitivit of the enzyme. After purification to about 20000 folds, the final product contained a main band of 54 kDa and several faint bands. The amino acid sequence of the main protein was that which has never been reported. We are now trying to clone the CDNA of the protein, and the results will soon be able to be obtained.

Krabbe疾病（GLD）是Geneti白细胞营养不良的一种，其中半乳糖基质酶I缺乏。十年来，我们研究了这种特定疾病的发病机理，发现半乳糖酰胺的水解是由2种酸水解酶催化的，即半乳糖苷酶I和11，只有前一个酶在GLD中缺乏。这一发现可以回答一个问题，为什么半乳糖基肾上腺素（但不能在GLD患者的组织中积累半乳糖酰胺）。由于累积的半乳糖基肾上腺素是细胞毒性的，因此建议形成髓磷脂的细胞，Oflgodendroglia和Schwann细胞已死亡，脱髓鞘发生。酶的纯化非常困难，遗迹者无法成功将其净化。在1990年，我们使用几种色谱Tec Hniques将酶从人体胎盘的粗糙样品中纯化至I 0000倍。最终产品包含2个带有58kDa和2okda的频段，带有SDS -Page的脸颊。将2个谱带吸收到适当的膜，并被内肽酶消化相应的蛋白质。但是，如Compute研究所检查的那样，58KDA带的氨基酸序列揭示了与IgG的同源性。我们无法在2OKDA带蛋白中检测到氨基酸。因此，在1991年，我们将起始材料更改为猪肾脏，猪肾脏包含酶的特异性相对较高。在纯化约20000倍之后，最终产品包含一个54 kDa和几个微弱带的主带。主要蛋白的氨基酸序列是从未报道过的氨基酸序列。我们现在正在尝试克隆蛋白质的cDNA，结果很快就能获得。