Itoh Kohji, Satoh Yurie, Kadota Yoshito, Oheda Yukako, Kuwahara Jun, Shimmoto Michie, Sakuraba Hitoshi
Division of Medicinal Biotechnology, Institute of Medicinal Resources, Faculty of Pharmaceutical Sciences, The University of Tokushima, Tokushima 770-8505, Japan.
Neurochem Int. 2004 May;44(6):447-57. doi: 10.1016/j.neuint.2003.07.001.
Human neuroblastoma GOTO cell lines were established that stably express recombinant human lysosomal protective protein/cathepsin A (PPCA) cDNA by transfection. Intracellular cathepsin A (acid serine carboxypeptidase) activity increased four-fold compared with in those of the parent and mock-transfected cell lines. The immunoreactive 54 kDa precursor/zymogen and mature 32/20 kDa two-chain forms were produced in the cells. The amount of the latter form expressed in the GOTO cells was significantly larger than those in the PPCA-overexpressing CHO cell lines previously established. The intracellular proteins showed a typical lysosomal granular distribution and the glycosylated 54 kDa precursor was secreted into the culture medium without the addition of an alkalizing agent. The PPCA-overexpressing cell lines also retained the ability to differentiate bi-directionally as well as the parent cells; into neuronal cells on induction by dibutyryl cAMP in serum-free medium and into Schwannian cells on induction by bromodeoxyuridine. During the course of differentiation into neuronal and Schwannian cells, the intracellular cathepsin A activity further increased two and five times, respectively, which was associated with an increase in the expression of the 32/20 kDa two-chain form. The glycosylated precursor proteins were taken up via the mannose 6-phosphate receptors, and the cathepsin A, alpha-neuraminidase and beta-galactosidase (beta-Gal) activities deficient in the fibroblasts derived from a patient with PPCA deficiency (galactosialidosis) were restored. These results suggest that the bi-directional differentiation of GOTO cell lines stably expressing the recombinant human PPCA gene could be a model system for analyzing the functions of PPCA in peripheral neuronal cells and Schwannian cells as well as the recombinant PPCA could be a useful source for enzyme replacement therapy (ERT) for galactosialidosis patients.
通过转染建立了稳定表达重组人溶酶体保护蛋白/组织蛋白酶A（PPCA）cDNA的人神经母细胞瘤GOTO细胞系。与亲本细胞系和mock转染细胞系相比，细胞内组织蛋白酶A（酸性丝氨酸羧肽酶）活性增加了四倍。细胞中产生了免疫反应性54 kDa前体/酶原和成熟的32/20 kDa双链形式。在GOTO细胞中表达的后一种形式的量明显大于先前建立的过表达PPCA的CHO细胞系中的量。细胞内蛋白质呈现典型的溶酶体颗粒分布，并且在不添加碱化剂的情况下，糖基化的54 kDa前体分泌到培养基中。过表达PPCA的细胞系也保留了与亲本细胞一样的双向分化能力；在无血清培养基中由二丁酰cAMP诱导分化为神经元细胞，在由溴脱氧尿苷诱导下分化为雪旺氏细胞。在分化为神经元细胞和雪旺氏细胞的过程中，细胞内组织蛋白酶A活性分别进一步增加了两倍和五倍，这与32/20 kDa双链形式表达的增加有关。糖基化前体蛋白通过甘露糖6-磷酸受体被摄取，并且PPCA缺乏症（半乳糖唾液酸贮积症）患者来源的成纤维细胞中缺乏的组织蛋白酶A、α-神经氨酸酶和β-半乳糖苷酶（β-Gal）活性得以恢复。这些结果表明，稳定表达重组人PPCA基因的GOTO细胞系双向分化可能是分析PPCA在外周神经元细胞和雪旺氏细胞中的功能的模型系统，并且重组PPCA可能是半乳糖唾液酸贮积症患者酶替代疗法（ERT）的有用来源。
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