earticle

영어

Lysosomal storage diseases (LSDs), a group of inherent diseases, are caused by hydrolase deficiency, which results in the accumulation of undigested metabolites in the lysosome. For the treatment of LSDs, enzyme replacement therapies with recombinant enzymes have been used successfully. Most of the therapeutic enzymes, except for the ones for Gaucher disease, require mannose-6-phosphate (M6P) glycans that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Therefore, M-6-P content is a key factor for efficient MPR binding and lysosomal delivery, which is essential for clearance of the accumulated molecules. Several glyco-engineering strategies for increasing M6P glycan content have been designed to produce “Biobetter” enzymes for LSDs [1]. We constructed the glyco-engineered yeast harboring high content of mannosylphosphorylated glycans, which can be converted M6P glycans by uncapping of the outer mannose residue. The short glycopeptides containing M6P glycans (M6PgPs) were prepared from cell wall mannoproteins of the glyco-engineered yeast by using hot-citrate buffer extraction and pronase digestion. The purified M6PgPs were successfully conjugated to a therapeutic enzyme, recombinant acid a-glucosidase (rGAA) used for treatment of Pompe disease, by two-step reactions using two hetero-bifunctional corsslinkers. The M6PgP-conjugated rGAA had 18-fold higher content of M6P glycan than rGAA and displayed greatly increased its uptake by Pompe disease patient fibroblasts [2]. For the first time, a strategy for in vitro mannosyl-phosphorylation of high-mannose type N-glycans has been established employing a recombinant Mnn14 protein (rM14) derived from Saccharomyces cerevisiae. In vitro reaction using rM14 mannosyl-phosphorylated the glycans on rhGAA, which can be converted to have high M6P glycans contents. It will promise the generation of “Biobetter” therapeutic enzymes with improved lysosomal targeting capability.