The enzyme activity sites and the conjugation sites for dimers formation in hexosaminidase S (α α) and B (ββ)

氨基己糖苷酶 S (α α) 和 B (ββ) 中形成二聚体的酶活性位点和结合位点

• 批准号: 10670750
• 负责人: TANAKA Akemi
• 金额: $ 1.98万
• 依托单位: Osaka City University Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670750/
• 关键词: β-HEXOSMINIDASE Βsubunit; GM2-GANGLIOSIDOSIS; GENE EXPRESSION; IN VITRO MUTAGENESIS; DIMER FORMATION; GATALYTIC SITE; BINDING SITE

Three distinct mutant alleles of β-hexosaminidase β-subunit gene in Japanese patients with infantile Sandhoff disease (GM2-gangliosidosis type O) have been found for their molecular basis of their severe phenotype. They were the allele with a tripe mutation (P417L, K121R and S255R), a double (P417L and K121R) mutation, and an intronic mutation away from the intron 10/exon11 junction (-17, a→g). S255R is novel without prior description in the literature. Both P417L and the intronal muatation lVS 10, 17 a→g caused splicing abnormalities, although they did not abolish the consensus sequence for splicing. An expression study of the normally spliced cDNA with the double and the triple mutations gave 78% and 28% of normal activity, respectively. This finding suggested that S255R further reduces the catalytic activity of the already below-the-threshold amount of normally spliced mRNA and accounts for the severe phenotype.Number of missence mutations in β-hexosaminidase β-subunit gene that cause GM2-gangliosidosis type O with various phenotypes have been reported. To determine the active site for the enzyme activity or the binding site for the dimerization, we constructed four kinds of mutant cDNAs with l207V, S255R, Y456S, or C533Y by in vitro mutagenesis. The human cultured fibroblasts which do not produce α-subunit protein but produce mal β-subunit protein were transformed with those mutant cDNAs and analysed hexosaminidase B activity. When the β-subunit protein derived from mutant cDNA could bind with normal β-subunit protein, the activity of hexosaminidase B would decrease by a dominant negative like effect.

在日本婴儿Sandhoff病（gm2 -神经节脂质沉积症O型）患者中发现了三个不同的β-己糖氨酸酶β-亚基基因突变等位基因，它们是其严重表型的分子基础。这些等位基因分别为三型突变（P417L、K121R和S255R）、双突变（P417L和K121R）和远离内含子10/外显子11连接处的内含子突变（-17,a→g）。S255R是新颖的，在文献中没有事先描述。P417L和内部突变lVS 10,17 a→g都导致剪接异常，尽管它们没有取消剪接的共识序列。正常剪接的双突变和三突变cDNA的表达研究分别获得78%和28%的正常活性。这一发现表明，S255R进一步降低了已经低于阈值的正常剪接mRNA的催化活性，并解释了严重的表型。据报道，β-己糖氨酸酶β-亚基基因缺失突变导致多种表型的gm2 -神经节脂质沉积症O型。为了确定酶活性的活性位点或二聚化的结合位点，我们通过体外诱变构建了l207V、S255R、Y456S和C533Y四种突变cdna。用这些突变cdna转化不产生α-亚基蛋白但产生少量α-亚基蛋白的人培养成纤维细胞，分析其己糖氨酸酶B活性。当由突变体cDNA衍生的β亚基蛋白与正常β亚基蛋白结合时，己糖胺酶B的活性以显性负样效应降低。

项目成果

M.Fujimaru,A.Tanaka et al.: "Two mutations remote from an exon/intron junction in the β-hexosaminidase β subunit gene affect 3'-splice site selection and cause Sandhoff disease." Human Genetics. 103・4. 462-469 (1998)

M.Fujimaru、A.Tanaka 等人：“β-己糖胺酶 β 亚基基因中远离外显子/内含子连接的两个突变影响 3 剪接位点选择并导致桑德霍夫病 103・4。” -469 (1998)

A. Tanaka, M. Fujimaru et al.: "Novel mutations including the second most common in Japan in the β-hexosaminidase α subunit gene, and a simple screening of Japanese patients with Tay-Sachs diseae"Journal of Human Genetics. 44・1. 91-95 (1999)

A. Tanaka、M. Fujimaru 等人：“新型突变，包括日本第二常见的 β-己糖胺酶 α 亚基基因，以及对日本泰萨克斯病患者的简单筛查”《人类遗传学杂志》44。 1.91-95 (1999)