Lysosomal storage disorders (LSDs) are a group of hereditary metabolic disorders characterized by the accumulation of undigested macromolecules in lysosomes. This build-up stems from an impaired activity of lysosomal catabolic enzymes and leads to cell dysfunction and progressive clinical manifestations. Deficiencies in these enzymes can be corrected by Enzyme Replacement Therapy (ERT), provided that the administered enzyme can be targeted to the lysosomes of the diseased cells. A major lysosomal targeting pathway entails the modification of cargo proteins with mannose-6-phosphate (Pi-6-Man) in the Golgi. LSDs are currently treated with lysosomal enzymes produced in mammalian cells. These biopharmaceuticals contain relatively low levels of mannose-6-phosphate, although this glycotope is required for lysosomal delivery. High dosing is therefore required, with high cost for a suboptimal therapeutic effect.
We present a novel glycosidase from C. cellulans that 'uncaps' yeast-type mannose-Pi-6-mannose-modified N-glycans to the mannose-6-phosphate-substituted mammalian-type. In a case study, the Pompe's disease treatment enzyme, lysosomal α-glucosidase was produced in a glyco-engineered Yarrowia lipolytica, and processed with the new uncapping enzyme to generate a highly mannose-6-phosphate modified form of α-glucosidase that was efficiently taken up in Pompe disease patient fibroblasts. We further present the X-ray structure of the uncapping enzyme and its complex with a receptor analogue, providing insight in its unique substrate specificity.
