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 -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.