Methods for producing highly phosphorylated lysosomal hydrolases
AssigneeGenzyme Glycobiology Research Institute, Inc.
InventorWilliam M. Canfield
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Abstract
The present invention provides highly phosphorylated lysosomal hydrolases, methods of modifying lysosomal hydrolases with the lysosomal targeting pathway enzymes GlcNAc-phosphotransferase and/or phosphodiester α-GlcNAcase.
Claims46
A method of modifying a lysosomal hydrolase comprising contacting said lysosomal hydrolase with an isolated N-acetylglucosamine-1-phosphotransferase, which has a specific activity of at least 106 pmol/h/mg to produce a modified lysosomal hydrolase.
A method of preparing a phosphorylated lysosomal hydrolase comprising contacting said lysosomal hydrolase with an isolated N-acetylglucosamine-1-phosphodiester α-N-Acetylglucosaminidase, which has a specific activity of at least about 472,000 units/mg and which catalyzes the removal of N-acetylglucosamine from said lysosomal hydrolase and generates a terminal mannose 6-phosphate on said hydrolase, and wherein said lysosomal hydrolase comprises a N-acetylglucosamine phosphomannose diester.
A method of preparing a phosphorylated lysosomal hydrolase comprising:
- contacting said lysosomal hydrolase with an isolated N-acetylglucosamine-phosphotransferase, which has a specific activity of at least 106 pmol/h/mg to produce a modified lysosomal hydrolase;
- and
- contacting said modified lysosomal hydrolase with an isolated N-acetylglucosamine-1-phosphodiester α-N-Acetylglucosaminidase, which catalyzes the removal of N-acetylglucosamine from said modified lysosomal hydrolase and generates a terminal mannose 6-phosphate on said hydrolase.
Dependencies are inferred from the claim text.