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In Vitro N-Glycan Mannosyl-Phosphorylation of a Therapeutic Enzyme by Using Recombinant Mnn14 Produced from Pichia pastoris
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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Korean Society for Microbiology and Biotechnology
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9705852/ https://www.ncbi.nlm.nih.gov/pubmed/33144549 http://dx.doi.org/10.4014/jmb.2010.10033 |
Sumario: | 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 76 amino acids spanning the transmembrane domain (46 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 (Man(8)GlcNAc(2)) as a substrate. In addition, rMnn14(77-935) was shown to mannosyl-phosphorylate high-mannose type Nglycans (Man(7-9)GlcNAc(2)) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bis-form which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-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. |
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