Expression of lysosomal hydrolase in cells expressing pro-N-acetylglucosamine-1-phosphodiester α-N-acetyl glucosimanidase
The present invention provides methods of producing a pro-N-acetylglucosamine-1-phosphodiesterαN-acetyl glucosimanidase (phosphodiester α-GlcNAcase), in mammalian cells deficient in the furin proteolytic enzyme and methods of making lysosomal bydrolases having an N-acetylglucosamine-1-phosphate.
1. A method of producing a lysosomal hydrolase having an oligosaccharide modified with N-acetylglucosamine-1-phosphate comprising
a. introducing a polynucleotide sequence encoding the lysosomal hydrolase in a furin deficient mammalian cell;
b. culturing said furin deficient mammalian cell containing the polynucleotide sequence encoding the lysosomal hydrolase for a time and under conditions suitable for expression of the lysosomal hydrolase; and
c. collecting the lysosomal hydrolase expressed.
2. The method of claim 1 , wherein said lysosomal hydrolase is selected from the group consisting of α-glucosidase, α-L-iduronidase, α-galactosidase A, arylsulfatase, N-acetylgalactosamine-6-sulfatase, β-galactosidase, iduronate 2-sulfatase, ceramidase, galactocerebrosidase, β-glucuronidase, 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, Ganglioside sialidase, Acid β-galactosidase, Hexosaminidase A, Hexosaminidase B, α-fucosidase, α-N-Acetyl galactosaminidase, Glycoprotein Neuraminidase, Aspartylglucosamine amidase, Acid Lipase, Acid Ceramidase, Lysosomal Sphingomyelinase and Sphingomyelinase.
3. The method of claim 1 , further comprising contacting said lysosomal hydrolase having an N-acetylglucosamine-1-phosphate with an active N-acetylglucosamine-1-phosphodiester αN-acetyl glucosimanidase.
4. The method of claim 3 , wherein said N-acetylglucosamine-1-phosphodiester αN-acetyl glucosimanidase comprises an amino acids 56 to 515 of SEQ ID NO:18.
5. The method of claim 3 , wherein said N-acetylglucosamine-1-phosphodiester αN-acetyl glucosimanidase is encoded by a nucleotide sequence comprising SEQ ID NO:17 or a nucleotide sequence that hybridizes under stringent conditions to the complement of SEQ ID NO:17.
6. The method of claim 3 , further comprising purifying said lysosomal hydrolase after said contacting.
7. The method of claim 1 , further comprising culturing said mammalian cell culture in the presence of a α1,2-mannosidase inhibitor.
8. The method of claim 7 , wherein said α1,2-mannosidase inhibitor comprises both deoxymannojirimycin and kifunensine.
9. A lysosomal hydrolase produced by the method of claim 1 .
10. A method of producing a lysosomal hydrolase having an oligosaccharide N-acteylglucosamine-1-phosphate comprising
a. a step for expressing a lysosomal hydrolase in a furin deficient mammalian cell; and
b. a step for collecting the lysosomal hydrolase expressed.
11. The method of claim 10 , wherein said lysosomal hydrolase is selected from the group consisting of α-glucosidase, α-L-iduronidase, α-galactosidase A, arylsulfatase, N-acetylgalactosamine-6-sulfatase, β-galactosidase, iduronate 2-sulfatase, ceramidase, galactocerebrosidase, β-glucuronidase, 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, Ganglioside sialidase, Acid β-galactosidase, Hexosaminidase A, Hexosaminidase B, α-fucosidase, α-N-Acetyl galactosaminidase, Glycoprotein Neuraminidase, Aspartylglucosamine amidase, Acid Lipase, Acid Ceramidase, Lysosomal Sphingomyelinase and Sphingomyelinase.
12. The method of claim 8 , further comprising a step for removing the N-acetylglucosamine from said lysosomal hydrolase.
13. The method of claim 12 , further comprising a step for purifying said lysosomal hydrolase.
14. A lysosomal hydrolase produced by the method of claim 10 .