In this study we developed an efficient method to prepare glycoengineered β-N-acetylhexosaminidase containing multiple mannose-6-phosphates (M6Ps) by combining genetic code expansion with bioorthogonal ligation techniques. We found that multiple M6P-conjugated enzymes were produced with a high efficiency by using combined techniques. Importantly, glycoengineered enzymes entered lysosomes of patient-derived primary cells, which lack endogenous lysosomal β-N-acetylhexosaminidase, more readily than commercialized human β-hexosaminidase. Moreover, glycoengineered enzymes successfully removed GM2-ganglioside stored in lysosomes of diseased cells, indicating that its activity is restored in diseased cells. We also synthesized and applied a lysosome-targeting fluorogenic substrate to monitor endogenous and supplemental glycoengineered β-N-acetylhexosaminidase activities in lysosomes. The results of this study indicate that the present strategy, which relies on genetic code expansion and bioorthogonal ligation techniques, is highly attractive to generate multi-M6P-containing lysosomal enzymes that can be used to study lysosomal storage disorders associated with lysosomal enzyme deficiencies.

中文翻译：

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具有多个甘露糖6磷酸的糖工程化的酶被内化到患病的细胞中以恢复其在溶酶体中的活性。

在这项研究中，我们开发了一种有效的方法，通过结合遗传密码扩展和生物正交连接技术，制备包含多种甘露糖6-磷酸酯（M6Ps）的糖基化β-N-乙酰基己糖胺酶。我们发现通过使用组合技术可以高效生产多种M6P共轭酶。重要的是，糖基化酶进入了患者来源的原代细胞的溶酶体，该溶酶体缺乏内源性溶酶体β-N-乙酰基己糖胺酶，比商品化的人β-己糖胺酶更容易。此外，糖基化酶成功去除了患病细胞溶酶体中储存的GM2-神经节苷脂，表明其活性在患病细胞中得以恢复。我们还合成并应用了靶向溶酶体的荧光底物，以监测溶酶体中的内源性和补充性糖工程化的β-N-乙酰基己糖胺酶活性。这项研究的结果表明，依赖于遗传密码扩展和生物正交连接技术的本策略对于产生可用于研究与溶酶体酶缺乏症相关的溶酶体贮积病的含M6P的溶酶体酶具有很高的吸引力。