In vitro N-glycan mannosyl-phosphorylation of a therapeutic enzyme by using recombinant Mnn14 produced from Pichia pastoris

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Title
In vitro N-glycan mannosyl-phosphorylation of a therapeutic enzyme by using recombinant Mnn14 produced from Pichia pastoris
Author(s)
Ji-Yeon Kang; H Y Choi; D I Kim; Ohsuk KwonDoo-Byoung Oh
Bibliographic Citation
Journal of Microbiology and Biotechnology, vol. 31, no. 1, pp. 163-170
Publication Year
2021
Abstract
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, rMnn1477-935 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 rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type Nglycans (Man7-9GlcNAc2) 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 rMnn1477-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.
Keyword
Mannosyl-phosphorylation, Mannose-6-phosphate, Mnn14, Enzyme replacement therapyLysosomal storage disease
ISSN
1017-7825
DOI
http://dx.doi.org/10.4014/jmb.2010.10033
Type
Article
Appears in Collections:
Division of Bio Technology Innovation > SME Support Center > 1. Journal Articles
Division of Research on National Challenges > Environmental diseases research center > 1. Journal Articles
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