Combined replacement effects of human modified β-hexosaminidase B and GM2 activator protein on GM2 gangliosidoses fibroblasts.
Combined replacement effects of human modified β-hexosaminidase B and GM2 activator protein on GM2 gangliosidoses fibroblasts.
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DOI：
10.1016/j.bbrep.2016.04.012
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发表时间：
2016-09
影响因子：
2.7
通讯作者：
Itoh K
中科院分区：
文献类型：
--
作者：
Kitakaze K;Tasaki C;Tajima Y;Hirokawa T;Tsuji D;Sakuraba H;Itoh K
GM2 神经节苷脂病是由 HEXA、HEXB 和 GM2A 基因突变引起的常染色体隐性溶酶体贮积病 (LSD)，这些基因分别编码人溶酶体 β-己糖胺酶 (Hex) α 和 β 亚基以及 GM2 激活蛋白 (GM2A)。这些疾病与有神经症状的患者大脑中 GM2 神经节苷脂 (GM2) 的过度积累有关。在这里，我们建立了过表达人GM2A的CHO细胞系，并从条件培养基中纯化了GM2A，该条件培养基被来自GM2A缺陷患者的成纤维细胞摄取，当添加到培养基中时，具有减少成纤维细胞中积累的GM2的治疗效果。我们还首次证明，重组GM2A可以增强具有GM2降解活性的人修饰HexB（modB）的替代作用，该修饰由含有α亚基部分氨基酸序列的同源二聚体改变的β亚基组成，包括与GM2A结合所需的GSEP环，对减少来自Tay-Sachs病患者的成纤维细胞中积累的GM2，一种HexA（αβ）异二聚体）缺陷，由 HEXA 突变引起。根据 X 射线晶体结构，我们预测 GM2A 与位于修饰的 HexB β 亚基中的 GSEP 环的结合方式与天然 HexA α 亚基中的结合方式相同。这些发现表明，涉及人类修饰的 HexB 和 GM2A 的联合替代疗法对于 GM2 神经节苷脂病是有效的。通过过表达 GM2A 的 CHO 细胞系纯化重组人 GM2A 蛋白。通过 GM2A 替代减少 GM2A 缺陷成纤维细胞中积累的 GM2。修饰的 HexB 和 GM2A 对 HexA 缺陷成纤维细胞的联合作用。改进的 HexB 和 GM2A 之间分子相互作用的计算机预测。
GM2 gangliosidoses are autosomal recessive lysosomal storage diseases (LSDs) caused by mutations in the HEXA, HEXB and GM2A genes, which encode the human lysosomal β-hexosaminidase (Hex) α- and β-subunits, and GM2 activator protein (GM2A), respectively. These diseases are associated with excessive accumulation of GM2 ganglioside (GM2) in the brains of patients with neurological symptoms. Here we established a CHO cell line overexpressing human GM2A, and purified GM2A from the conditioned medium, which was taken up by fibroblasts derived from a patient with GM2A deficiency, and had the therapeutic effects of reducing the GM2 accumulated in fibroblasts when added to the culture medium. We also demonstrated for the first time that recombinant GM2A could enhance the replacement effect of human modified HexB (modB) with GM2-degrading activity, which is composed of homodimeric altered β-subunits containing a partial amino acid sequence of the α-subunit, including the GSEP loop necessary for binding to GM2A, on reduction of the GM2 accumulated in fibroblasts derived from a patient with Tay-Sachs disease, a HexA (αβ heterodimer) deficiency, caused by HEXA mutations. We predicted the same manner of binding of GM2A to the GSEP loop located in the modified HexB β-subunit to that in the native HexA α-subunit on the basis of the x-ray crystal structures. These findings suggest the effectiveness of combinational replacement therapy involving the human modified HexB and GM2A for GM2 gangliosidoses. Purification of recombinant human GM2A proteins by CHO cell line overexpressing GM2A. Reduction of GM2 accumulated in GM2A deficiency fibroblasts by GM2A replacement. Combined effects of modified HexB and GM2A for HexA deficiency fibroblasts. In silico prediction of molecular interaction between modified HexB and GM2A.