Methods for producing highly phosphorylated lysosomal hydrolases

1. 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 10 6 pmol/h/mg to produce a modified lysosomal hydrolase.

2. The method of claims 1 , further comprising purifying said modified lysosomal hydrolase after said contacting.

3. The method of claim 1 , wherein said N-acetylglucosamine-phosphotransferase catalyzes the transfer of N-acetylglucosamine-1-phosphate from UDP-N-Acetylglucosamine to a mannose on the hydrolase.

4. The method of claim 1 , wherein said lysosomal hydrolase is a recombinant hydrolase.

5. The method of claim 1 , wherein said lysosomal hydrolase is selected from the group consisting of α-glucosidase, α-iduronidase, α-galactosidase A, arylsulfatase, N-acetylgalactosamine-6-sulfalase, β-galactosidase, iduronate 2-sulfatase, ceramidase, galactocerebrosidase, B-glucoronidase, Heparan N-sulfatase, N-Acetyl-α-glucosaminidase, Acetyl CoA-α-glucosaminide N-acetyl transferase, N-acetyl-glucosamine-6 sulfatase, Galactose 6-sulfatase, Arylsulfatase A, Arylsulfatase B, Arylsulfatase C, Arylsulfatase A Cerebroside, Ganglioside, Acid β-galactosidase G M1 Galglioside, Acid β-galactosidase, Hexosaminidase A, Hexosaminidase B, α-fucosidase, α-N-Acetyl galactosaminidase, Glycoprotein Neuraminidase, Aspartyiglucosamine amidase, Acid Lipase, Acid Ceramidase, Lysosomal Sphingomyelinase, Sphingomyelinase, and Glucocerebrosidase β-Glucosidase.

6. The method of claim 1 , further comprising 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.

7. The method of claim 1 , wherein said N-acetylglucosamine-1-phosphotransferase has a specific activity of at least 5×10 6 pmol/h/mg.

8. The method of claim 1 , wherein said N-acetylglucosamine-1-phosphotransferase has a specific activity of at least 12×10 6 pmol/h/mg.

9. The method of claim 6 , wherein said isolated N-acetylglucosamine-1-phosphodiester α-N-Acetylglucosaminidase has a specific activity of at least about 1000 units/mg.

10. The method of claim 6 , wherein said isolated N-acetylglucosamine-1-phosphodiester α-N-Acetylglucosaminidase has a specific activity of at least about 472,000 units/mg.

11. The method of claim 1 , wherein the N-acetylglucosamine-1-phosphotransferase comprises an α subunit, a β subunit, and a γ subunit; and wherein the α and β subunits are encoded by a DNA molecule comprising SEQ ID NO:20; and the γ subunit is encoded by a DNA molecule comprising nucleotides 96 to 941 of SEQ ID NO:5.

12. The method of claim 1 , wherein the N-acetylglucosamine-1-phosphotransferase comprises an α subunit, a β subunit, and a γ subunit; and wherein the α and β subunits are encoded by a DNA molecule which hybridizes under stringent conditions to the complement of SEQ ID NO:20; and the γ subunit is encoded by a DNA molecule which hybridizes under stringent conditions to the complement of nucleotides 96 to 941 of SEQ ID NO:5; wherein the combination of the a subunit, a β subunit, and a γ subunit yields a protein with the activity to catalyze the transfer of N-acetylglucosamine-1-phosphate from UDP-N-Acetylglucosamine to a mannose on the hydrolase.

13. The method of claim 1 , wherein the lysosomal hydrolase is α-glucosidase.

14. The method of claim 1 , wherein the lysosomal hydrolase is α-iduronidase.

15. The method of claim 1 , wherein the lysosomal hydrolase is α-galactosidase A.

16. A modified lysosomal hydrolase produced by the method of claim 1 .

17. 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.

18. The method of claim 17 , wherein said method further comprises purifying the phosphorylated lysosomal hydrolase.

19. The method of claim 17 , wherein said lysosomal hydrolase is selected from the group consisting of α-glucosidase, α-iduronidase, α-galactosidase A, arylsulfatase, N-acetylgalactosamine-6-sulfatase, β-galactosidase, iduronate 2-sulfatase, ceramidase, galactocerebrosidase, B-glucoronidase, Heparan N-sulfatase, N-Acetyl-α-glucosaminidase, Acetyl CoA-α-glucosaminide N-acetyl transferase, N-acetyl-glucosamine-6 sulfatase, Galactose 6-sulfatase, Arylsulfatase A, Arylsulfatase B, Arylsulfatase C, Arylsulfatase A Cerebroside, Ganglioside, Acid β-galactosidase G M1 Galglioside, Acid β-galactosidase, Hexosaminidase A, Hexosaminidase B, α-fucosidase, α-N-Acetyl galactosaminidase, Glycoprotein Neuraminidase, Aspartylglucosamine amidase, Acid Lipase, Acid Ceramidase, Lysosomal Sphingomyelinase, Sphingomyelinase, and Glucocerebrosidase β-Glucosidase.

20. The method of claim 17 , wherein said N-acetylglucosamine-1-phosphodiester α-N-Acetylglucosaminidase catalyzes the removal of N-acetylglucosamine from N-acetylglucosamine phosphomannose diester to generate a terminal mannose 6-phosphate on said lysosomal hydrolase.

21. The method of claim 17 , wherein the N-acetylglucosamine-1-phosphodiester α-N-Acetylglucosaminidase is encoded by a DNA molecule comprising nucleotides 151 to 1548 of SEQ ID NO:7.

22. The method of claim 17 , wherein the N-acetylglucosamine-1-phosphodiester α-N-Acetylglucosaminidase is encoded by a DNA molecule which hybridizes under stringent conditions to the complement of nucleotides 151 to 1548 of SEQ ID NO:7.

23. The method of claim 17 , wherein the lysosomal hydrolase is α-glucosidase.

24. The method of claim 17 , wherein the lysosomal hydrolase is α-iduronidase.

25. The method of claim 17 , wherein the lysosomal hydrolase is α-galactosidase A.

26. A composition comprising the modified lysosomal hydrolase of claim 16 and a carrier.

27. 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 10 6 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.

28. The method of claim 27 , further comprising purifying said phosphorylated lysosomal hydrolase after said contacting with the isolated N-acetylglucosamine-1-phosphodiester α-N-Acetylglucosaminidase.

29. The method of claim 27 , further comprising purifying said modified lysosomal hydrolase prior to said contacting with the isolated N-acetylglucosamine-1-phosphodiester α-N-Acetylglucosaminidase.

30. The method of claim 27 , wherein said lysosomal hydrolase is selected from the group consisting of α-glucosidase, α-iduronidase, α-galactosidase A, arylsulfatase, N-acetylgalactosamine-6-sUlfatase, β-galactosidase, iduronate 2-sulfatase, ceramidase, galactocerebrosidase, B-glucoronidase, Heparan N-sulfatase, N-Acetyl-α-glucosaminidase, Acetyl CoA-α-glucosaminide N-acetyl transferase, N-acetyl-glucosamine-6 sulfatase, Galactose 6-sulfatase, Arylsulfatase A, Arylsulfatase B, Arylsulfatase C, Arylsulfatase A Cerebroside, Ganglioside, Acid β-galactosidase G M1 Galglioside, Acid β-galactosidase, Hexosaminidase A, Hexosaminidase B, α-fucosidase, α-N-Acetyl galactosaminidase, Glycoprotein Neuraminidase, Aspartylglucosamine amidase, Acid Lipase, Acid Ceramidase, Lysosomal Spbingomyelinase, Sphingomyelinase, and Glucocerebrosidase β-Glucosidase.

31. The method of claim 27 , wherein said N-acetylglucosamine-1-phosphotransferase has a specific activity of at least 5×10 6 pmol/h/mg.

32. The method of claim 27 , wherein said N-acetylglucosamine-1-phosphotransferase has a specific activity of at least 12×10 6 pmol/h/mg.

33. The method of claim 27 , wherein the phosphorylated lysosomal hydrolase comprises at least 6% bis-phosphorylated oligosaccharides.

34. The method of claim 27 , wherein the phosphorylated lysosomal hydrolase comprises at least 100% bis-phosphorylated oligosaccharides.

35. The method of claim 27 , wherein the phosphorylated lysosomal hydrolase comprises at least 5 mannose 6-phosphates.

36. The method of claim 27 , wherein the N-acetylglucosamine-1-phosphotransferase comprises an α subunit, a β subunit, and a γ subunit; and wherein the α and β subunits are encoded by a DNA molecule comprising SEQ ID NO:20; and the γ subunit is encoded by a DNA molecule comprising nucleotides 96 to 941 of SEQ ID NO:5.

37. The method of claim 27 , wherein the N-acetylglucosamine-1-phosphodiester α-N-Acetylglucosaminidase is encoded by a DNA molecule comprising nucleotides 151 to 1548 of SEQ ID NO:7.

38. The method of claim 27 , wherein the N-acetylglucosamine-1-phosphotransferase comprises an a subunit, a β subunit, and a γ subunit; and wherein the α and β subunits are encoded by a DNA molecule which hybridizes under stringent conditions to the complement of SEQ ID NO:20; and the γ subunit is encoded by a DNA molecule which hybridizes under stringent conditions to the complement of nucleotides 96 to 941 of SEQ ID NO:5; wherein the combination of the α subunit, a β subunit, and a γ subunit yields a protein with the activity to catalyze the transfer of N-acetylglucosamine-1-phosphate from UDP-N-Acetylglucosamine to a mannose on the hydrolase.

39. The method of claim 27 , wherein the N-acetylglucosamine-1-phosphodiester α-N-Acetylglucosaminidase is encoded by a DNA molecule which hybridizes under stringent conditions to the complement of nucleotides 151 to 1548 of SEQ ID NO:7.

40. The method of claim 27 , wherein the lysosomal hydrolase is α-glucosidase.

41. The method of claim 27 , wherein the lysosomal hydrolase is α-iduronidase.

42. The method of claim 27 , wherein the lysosomal hydrolase is α-galactosidase A.

43. A phosphorylated lysosomal hydrolase produced by the method of claim 27 .

44. A composition comprising the phosphorylated lysosomal hydrolase produced by the method of claim 27 .

45. The method of claim 27 , wherein said isolated N-acetylglucosamine-1-phosphodiester α-N-Acetylglucosaminidase has a specific activity of at least about 1000 units/mg.

46. The method of claim 27 , wherein said isolated N-acetylglucosamine-1-phosphodiester α-N-Acetylglucosaminidase has a specific activity of at least about 472,000 units/mg.