Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn14_77-935 with deletion of N-terminal 77 amino acids spanning the transmembrane domain (47 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn14_77-935 were determined through enzyme assays with a high-mannose type N-glycan (Man₈GlcNAc₂) as a substrate. In addition, rMnn14_77-935 was shown to mannosyl-phosphorylate high-mannose type N-glycans (Man_7-9GlcNAc₂) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bisform which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitroN-glycan mannosyl-phosphorylation reaction using rMnn14_77-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.

中文翻译：

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使用毕赤酵母产生的重组 Mnn14 对治疗酶进行体外 N-聚糖甘露糖磷酸化。

溶酶体贮积病的酶替代疗法通常需要含有 6-磷酸甘露糖 (M6P) 聚糖的重组酶，用于细胞摄取和溶酶体靶向。此处首次建立了一种利用源自酿酒酵母的重组 Mnn14 蛋白对高甘露糖型N-聚糖进行体外甘露糖磷酸化的策略。在毕赤酵母中表达的一系列 N 末端或 C 末端缺失的重组 Mnn14 蛋白中，rMnn14 _77-935缺失跨越跨膜结构域（47 个氨基酸）和部分茎区（30 个氨基酸）的 N 末端 77 个氨基酸，显示出最高水平的甘露糖磷酸化活性。_{rMnn14 77-935}的最佳反应条件是通过以高甘露糖型N-聚糖 (Man ₈ GlcNAc ₂ ) 作为底物的酶测定法确定的。此外，rMnn14 _77-935显示甘露糖磷酸化高甘露糖型N-聚糖 (Man _7-9 GlcNAc ₂) 对重组人溶酶体α-葡萄糖苷酶 (rhGAA) 具有非常高的效率。此外，大多数生成的甘露糖磷酸化聚糖是双型的，可以转化为具有优异溶酶体靶向能力的双磷酸化 M6P 聚糖。使用 rMnn14 _77-935的体外N-聚糖甘露糖磷酸化反应将提供一种灵活而直接的方法来增加 M6P 聚糖含量，从而产生“Biobetter”治疗酶。