Division of Cardiac Repair and Regeneration, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan.
Mol Biol Rep. 2011 Jun;38(5):3145-52. doi: 10.1007/s11033-010-9985-5. Epub 2010 Feb 4.
Fabry disease is an inherited lysosomal disorder caused by a deficiency of alpha-galactosidase A (α-gal A). The systemic accumulation of substrate, mainly globotriaosylceramide (Gb3), results in organ failure. Although Gb3 accumulation has been observed in an α-gal A-deficient mouse model, important clinical manifestations were not seen. The pursuit of effective treatment for Fabry disease through gene therapy, for example, has been hampered by the lack of a relevant large animal model to assess the efficacy and safety of novel therapies. Towards assembling the tools to generate an alternative animal model, we have sequenced and characterized the porcine ortholog of the α-gal A gene. When compared to the human α-gal A, the porcine α-gal A showed a high level of homology in the coding regions and located at chromosome Xq22. Cell lysate and supernatants from Fabry patient-derived fibroblasts transduced with a lentiviral vector (LV) carrying the porcine α-gal A cDNA (LV/porcine α-gal A), showed high levels of α-gal A activity and its enzymological stability was similar to that of human α-gal A. Uptake of secreted porcine α-gal A was observed into non-transduced cells and was partially inhibited by soluble mannose-6-phosphate. Furthermore, Gb3 accumulation was reduced in Fabry patient-derived fibroblasts transduced with the LV/porcine α-gal A. In conclusion, we elucidated and characterized the porcine α-gal A gene and enzyme. Similarity in enzymatic profile and chromosomal location between α-gal A of porcine and human origins may be of great advantage for the development of a large animal model for Fabry disease.
法布瑞氏病是一种遗传性溶酶体贮积病，由α-半乳糖苷酶 A（α-gal A）缺乏引起。底物的全身积累，主要是神经节苷脂 Gb3，导致器官衰竭。尽管在缺乏α-gal A 的小鼠模型中观察到 Gb3 积累，但并未出现重要的临床表现。通过基因治疗来寻找治疗法布瑞氏病的有效方法，例如，由于缺乏相关的大型动物模型来评估新型疗法的疗效和安全性，一直受到阻碍。为了组装生成替代动物模型的工具，我们对猪α-gal A 基因进行了测序和特征分析。与人类α-gal A 相比，猪α-gal A 在编码区域具有高度同源性，位于 Xq22 染色体上。用携带猪α-gal A cDNA 的慢病毒载体（LV/猪α-gal A）转导的法布瑞氏病患者来源的成纤维细胞的细胞裂解物和上清液显示出高水平的α-gal A 活性，其酶学稳定性与人类α-gal A 相似。观察到分泌的猪α-gal A 被非转导细胞摄取，并被可溶性甘露糖-6-磷酸部分抑制。此外，转导 LV/猪α-gal A 的法布瑞氏病患者来源的成纤维细胞中 Gb3 积累减少。总之，我们阐明并鉴定了猪α-gal A 基因和酶。猪和人来源的α-gal A 在酶谱和染色体位置上的相似性可能对法布瑞氏病大型动物模型的开发具有重要优势。
