Inefficiency in GM2 ganglioside elimination by human lysosomal β-hexosaminidase β-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice
Inefficiency in GM2 ganglioside elimination by human lysosomal β-hexosaminidase β-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice
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DOI：
10.1248/bpb.29.1564
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发表时间：
2006-08-01
影响因子：
2
通讯作者：
Itoh, Kohji
中科院分区：
文献类型：
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作者：
Itakura, Tomohiro;Kuroki, Aya;Itoh, Kohji
Sandhoff病(SD)是一种常染色体隐性遗传性GM2神经节苷脂增多症，由溶酶体β-氨基己糖苷酶(Hex)β亚单位基因缺陷引起，与神经体症状相关。在编码小鼠β-亚基的Hexb基因等位基因突变所致的Sandhoff病模型小鼠(SD小鼠)上，观察了Hex亚单位基因转导的治疗效果。在这里，我们证明了当只转导HEXB基因时，GM2神经节苷脂(GM2)在SD小鼠成纤维细胞系(FSD)中积聚的消除不会发生。相反，对中性底物，包括GA2(asialo-GM2)和在其非还原末端携带末端N-乙酰氨基葡萄糖残基的寡糖(G1cNAc-寡糖)的HexB(β-β同源二聚体)活性显著增加。天然聚丙烯酰胺凝胶电泳法(Native-PAGE)后用抗人六(a，6)二聚体血清免疫印迹显示，人HEXB基因产物很难与小鼠α-亚基结合形成嵌合的六聚体。然而，共导入编码人a亚基和HEXB基因的HEXA通过产生人HEXA而对GM2的降解产生显著的纠正作用。这些结果表明，重组人HexA可以与小鼠GM2激活蛋白发生种间结合，从而降解积聚在FSD细胞中的GM2。因此，利用SD小鼠可以评价重组人HEXB同工酶而不是人HEXB基因产物的治疗效果。
Sandhoff disease (SD) is an autosomal recessive GM2 gangliosidosis caused by the defect of lysosomal beta-hexosaminidase (Hex) beta-subunit gene associated with neurosomatic manifestations. Therapeutic effects of Hex subunit gene transduction have been examined on Sandhoff disease model mice (SD mice) produced by the allelic disruption of Hexb gene encoding the murine beta-subunit. We demonstrate here that elimination of GM2 ganglioside (GM2) accumulated in the fibroblastic cell line derived from SD mice (FSD) did not occur when the HEXB gene only was transfected. In contrast, a significant increase in the HexB (beta beta homodimer) activity toward neutral substrates, including GA2 (asialo-GM2) and oligosaccharides carrying the terminal N-acetylglucosamine residues at their non-reducing ends (G1cNAc-oligosaccharides) was observed. Immunoblotting with anti-human HexA (a,6 heterodimer) serum after native polyacrylamide gel electrophoresis (Native-PAGE) revealed that the human HEXB gene product could hardly form the chimeric HexA through associating with the murine a-subunit. However, co-introduction of the HEXA encoding the human a-subunit and HEXB genes caused significant corrective effect on the GM2 degradation by producing the human HexA. These results indicate that the recombinant human HexA could interspeciesly associate with the murine GM2 activator protein to degrade GM2 accumulated in the FSD cells. Thus, therapeutic effects of the recombinant human HexA isozyme but not human HEXB gene product could be evaluated by using the SD mice.