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Abstract
Many therapeutic enzymes for lysosomal storage diseases require a high content of mannose-6-phosphate (M6P) glycan, which is important for cellular uptake and lysosomal targeting. We constructed glyco-engineered yeast harboring a high content of mannosylphosphorylated glycans, which can be converted to M6P glycans by uncapping of the outer mannose residue. In this study, the cell wall of this yeast was employed as a natural M6P glycan source for conjugation to therapeutic enzymes. The extracted cell wall mannoproteins were digested by pronase to generate short glycopeptides, which were further elaborated by uncapping and α(1,2)-mannosidase digestion steps. The resulting glycopeptides containing M6P glycans (M6PgPs) showed proper cellular uptake and lysosome targeting. The purified M6PgPs were successfully conjugated to a recombinant acid α-glucosidase (rGAA), used for the treatment of Pompe disease, by two-step reactions using two hetero-bifunctional crosslinkers. First, rGAA and M6PgPs were modified with crosslinkers containing azide and dibenzocyclooctyne, respectively. In the second reaction using copper-free click chemistry, the azide-functionalized rGAA was conjugated with dibenzocyclooctyne-functionalized M6PgPs without the loss of enzyme activity. The M6PgP-conjugated rGAA had a 16-fold higher content of M6P glycan than rGAA, which resulted in greatly increased cellular uptake and efficient digestion of glycogen accumulated in Pompe disease patient fibroblasts.
Highlights
Mannose-6-phosphate (M6P) glycan is a crucial signal for the trafficking of lysosomal enzymes to lysosomes
We thought that cell wall mannoproteins, a major component of yeast cell wall, would be a good source for M6P glycans because they can be obtained from the harvested cells18,20
The glycopeptides were efficiently purified on a graphitized carbon column due to the glycan moiety being much larger than the peptide part
Summary
Mannose-6-phosphate (M6P) glycan is a crucial signal for the trafficking of lysosomal enzymes to lysosomes. Lysosomal storage diseases are caused by a lysosomal enzyme deficiency, which results in abnormal accumulation of undigested molecules2,3 Because this induces cellular malfunctions leading to multiple tissue and organ failures, patients develop various clinical symptoms in childhood and often end up with early death. Yeast glyco-engineering approaches have been highlighted because they have enabled the production of therapeutic enzymes containing a high level of M6P glycans. Secretory expression levels of therapeutic enzymes from the glyco-engineered S. cerevisiae were very low Instead of employing it directly for enzyme production, we thought that this yeast could be used as a natural source of M6P glycans for conjugation to enzymes, which would be much more economical than M6P glycans obtained from lysosomal enzymes. The resulting M6PgP-conjugated rGAA showed greatly increased cellular uptake and lysosomal targeting
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