Akeboshi Hiromi, Kasahara Yoshiko, Tsuji Daisuke, Itoh Kohji, Sakuraba Hitoshi, Chiba Yasunori, Jigami Yoshifumi
Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566, Japan.
Glycobiology. 2009 Sep;19(9):1002-9. doi: 10.1093/glycob/cwp080. Epub 2009 Jun 8.
Effective enzyme replacement therapy for lysosomal storage diseases requires a recombinant enzyme with highly phosphorylated N-glycans. Recombinant human beta-hexosaminidase A is a potentially therapeutic enzyme for GM2-gangliosidosis. Recombinant HexA has been produced by using the methylotrophic yeast Ogataea minuta as a host, and the purified enzyme was tested for its replacement effect on cultured fibroblasts derived from GM2-gangliosidosis patients. Although the therapeutic effect was observed, in order to obtain the higher therapeutic effect with a little dose as possible, increased phosphorylation of recombinant beta-hexosaminidase A N-glycans is suggested to be prerequisite. In the budding yeast Saccharomyces cerevisiae, the overexpression of MNN4, which encodes a positive regulator of mannosylphosphate transferase, led to increased mannosylphosphate contents. In the present study, we cloned OmMNN4, a homologous gene to ScMNN4, based on the genomic sequence of O. minuta. We overexpressed the cloned gene under the control of the alcohol oxidase promoter in a beta-hexosaminidase A-producing yeast strain. Structural analysis of pyridylamine-labeled N-glycans by high-performance liquid chromatography revealed that the overexpression of MNN4 caused a 3-fold increase in phosphorylated N-glycans of recombinant beta-hexosaminidase A. The recombinant enzyme prepared from strains overexpressing OmMNN4 was more effectively incorporated into cultured fibroblasts and neural cells, and it more rapidly degraded the accumulated GM2-ganglioside as compared to the control enzyme. These results suggest that beta-hexosaminidase A produced in a strain that overexpresses OmMNN4 will act as an effective enzyme for use in replacement therapy of GM2-gangliosidosis.
针对溶酶体贮积病的有效酶替代疗法需要一种具有高度磷酸化N-聚糖的重组酶。重组人β-己糖胺酶A是治疗GM2-神经节苷脂贮积症的一种潜在治疗性酶。已使用甲基营养酵母奥默毕赤酵母作为宿主生产重组己糖胺酶A，并对纯化后的酶在源自GM2-神经节苷脂贮积症患者的培养成纤维细胞上的替代效果进行了测试。尽管观察到了治疗效果，但为了尽可能以小剂量获得更高的治疗效果，重组β-己糖胺酶A的N-聚糖磷酸化增加被认为是先决条件。在芽殖酵母酿酒酵母中，编码甘露糖磷酸转移酶正调节因子的MNN4的过表达导致甘露糖磷酸含量增加。在本研究中，我们基于奥默毕赤酵母的基因组序列克隆了与ScMNN4同源的基因OmMNN4。我们在产β-己糖胺酶A的酵母菌株中，在醇氧化酶启动子的控制下过表达了克隆基因。通过高效液相色谱对吡啶胺标记的N-聚糖进行结构分析表明，MNN4的过表达使重组β-己糖胺酶A的磷酸化N-聚糖增加了3倍。与对照酶相比，从过表达OmMNN4的菌株制备的重组酶更有效地被整合到培养的成纤维细胞和神经细胞中，并且能更快地降解积累的GM2-神经节苷脂。这些结果表明，在过表达OmMNN4的菌株中产生的β-己糖胺酶A将作为一种有效的酶用于GM2-神经节苷脂贮积症的替代疗法。
