Methods for producing highly phosphorylated lysosomal hydrolases

A phosphorylated purified lysosomal hydrolase comprising a mannose 6-phosphate, which comprises at least 6% bis-phosphorylated oligosaccharides.

A method of preparing a phosphorylated lysosomal hydrolase comprising contacting said lysosomal hydrolase with an isolated N-acetylglucosamine-1-phosphodiester α-N-Acetylglucosamindase, wherein said lysosomal hydrolase comprises a terminal mannose 6-phosphate, and said N-acetylglucosamine-1-phosphodiester α-N-Acetylglucosamindase comprises the amino acid sequence in SEQ ID NO:6.

A method of preparing a phosphorylated lysosomal hydrolase comprising:

• contacting said lysosomal hydrolase with an isolated N-acetylglucosamine-phosphotransferase to produce a modified lysosomal hydrolase, wherein said N-acetylglucosamine-phosphotransferase comprises SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3;
• and
• contacting said modified lysosomal hydrolase with an isolated N-acetylglucosamine-1-phosphodiester α-N-Acetylglucosamindase, wherein said N-acetylglucosamine-1-phosphodiester α-N-Acetylglucosamindase comprises the amino acid in SEQ ID NO:6.

A method of modifying a lysosomal hydrolase comprising contacting said lysosomal hydrolase with an isolated N-acetylglucosamine-1-phosphotransferase to produce a modified lysosomal hydrolase, wherein said N-acetylglucosamine-phosphotransferase comprises an amino acid sequence that is at least 70% identical to SEQ ID NO:1, an amino acid sequence that is at least 70% identical to SEQ ID NO:2, and an amino acid sequence that is at least 70% identical to SEQ ID NO:3, and said N-acetylglucosamine-phosphotransferase catalyzes the transfer of N-acetylglucosamine-1-phosphate from UDP-N-Acetylglucosamine to a mannose on the lysosomal hydrolase.