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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...

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Detalles Bibliográficos
Autores principales: Kang, Ji-Yeon, Choi, Hong-Yeol, Kim, Dong-Il, Kwon, Ohsuk, Oh, Doo-Byoung
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Korean Society for Microbiology and Biotechnology 2021
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
Descripción
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.