Soluble GlcNAc phosphotransferase
View Patent ↗The present invention relates to a soluble GlcNAc phosphotransferase, a method of making the same and a method of phosphorylating with the same.
1. An isolated polypeptide comprising SEQ ID NO:2.
2. An N-acetylglycosamine-1-phosphotransferase (GlcNAc-phosphotransferase) comprising an α subunit, a β subunit and a site-specific proteolytic cleavage site interposed between said α and β subunits, wherein said site-specific proteolytic cleavage site is not endogenous to GlcNAc-phosphotransferase,
wherein said α subunit is encoded by nucleotides 165 to 2948 of SEQ ID NO:3, or a sequence that hybridizes under stringent conditions to the complement of nucleotides 165 to 2948 of SEQ ID NO:3, wherein said stringent conditions comprise hybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C. and washing in 0.1×SSC at 65° C. and which encodes a protein when combined with a β subunit protein encoded by nucleotides 2949 to 2932 of SEQ ID NO:3 has GlcNAc-phosphotransferase activity; and
wherein said β-subunit is encoded by nucleotides 2949 to 3932 of SEQ ID NO:3, or a sequence that hybridizes under stringent conditions to the complement of nucleotides 2949 to 3932 of SEQ ID NO:3, wherein said stringent conditions comprise hybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C. and washing in 0.1×SSC at 65° C. and which encodes a protein when combined with an α subunit protein encoded by nucleotides 165 to 2948 of SEQ ID NO:3 has GlcNAc-phosphotransferase activity.
3. The GlcNAc-phosphotransferase of claim 2 , wherein said α-subunit comprises amino acids 1-928 of SEQ ID NO:4.
4. The GlcNAc-phosphotransferase of claim 2 , wherein said β subunit comprises amino acids 1 to 328 of SEQ ID NO:5.
5. The GlcNAc-phosphotransferase of claim 2 , wherein said GlcNAc-phosphotransferase further comprises a γ subunit.
6. The GlcNAc-phosphotransferase of claim 5 , wherein said γ subunit comprises the amino acid sequence of SEQ ID NO:7.
7. The GlcNAc-phosphotransferase of claim 2 , wherein said site-specific proteolytic cleavage site is selected from the group consisting of a Furin proteolytic cleavage site, a Factor Xa proteolytic cleavage site, a Enterokinase proteolytic cleavage site, and a proteolytic cleavage site comprising SEQ ID NO:25.
8. The GlcNAc-phosphotransferase of claim 7 , wherein said site-specific proteolytic cleavage site is a Furin proteolytic cleavage site.
9. The GlcNAc-phosphotransferase of claim 8 , wherein said Furin proteolyfic cleavage site comprises SEQ ID NO:24.
10. A method of phosphorylating a lysosomal hydrolase protein comprising an asparagine-linked oligosaccharide with a high mannose structure, the method comprising contacting said lysosomal hydrolase protein with the isolated polypeptide of claim 1 for a time and under conditions suitable to produce a phosphorylated lysosomal hydrolase protein.
11. The method of claim 10 , wherein said lysosomal hydrolase protein is selected from the group consisting of α-glucosidase, α-L-iduronidase, α-galactosidase A, arylsulfatase, N-acetlygalactosamine-α-sulfatase, β-galactosidase, iduronate 2-sulfatase, ceramidase, galactocerebrosidase, β-glucoronidase, Heparan N-sulfatase, N-Acetyl-α-glucosaminidase, 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, Aspartylgucosamine amidase, Acid Lipase, Acid Ceramidase, Sphingomyelinase, and Glucocerebrosidase.
12. The method of claim 10 , further comprising contacting said phosphoryalated protein with an isolated N-acetylglucosamine-1-phosphodiester-N-Acetyglucosaminidase (phosphodiester α-GlcNAcase).
13. The method of claim 12 , wherein said phosphodiester α-GlcNAcase comprises the amino acid sequence of SEQ ID NO:18.
14. The method of claim 12 , wherein said phosphodiester α-GlcNAcase 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, wherein said stringent conditions comprise hybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C. and washing in 0.1×SSC at 65° C., and which encodes a protein with phosphodiester α-GlcNAcase activity.
15. The method of claim 10 , wherein prior to said contacting the method comprises: culturing a host cell which comprises a polynucleotide encoding the polypeptide for a time under conditions suitable for expression of the polypeptide; and isolating said polypeptide.
16. A method of phosphorylating a lysosomal hydrolase protein comprising an asparagine-linked oligosaccharide with a high mannose structure, the method comprising contacting said protein with the GlcNAc phosphotransferase of claim 2 for a time and under conditions suitable to produce a phosphorylated protein.
17. The method of claim 16 , wherein said α-subunit comprises amino acids 1-928 of SEQ ID NO:4.
18. The method of claim 16 , wherein said β subunit comprises amino acids 1 to 328 of SEQ ID NO:5.
19. The method of claim 16 , wherein said GlcNAc-phosphotransferase further comprises a γ subunit.
20. The method of claim 19 , wherein said γ subunit is encoded by SEQ ID NO:6, or a nucleotide sequence that hybridizes under stringent conditions to the complement of SEQ ID NO:6, wherein said stringent conditions comprise hybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C. and washing in 0.1×SSC at 65° C.
21. The method of claim 19 , wherein said γ subunit comprises the amino acid sequence of SEQ ID NO:7.
22. The method of claim 16 , wherein said site-specific proteolytic cleavage site is selected from the group consisting of a Furin proteolytic cleavage site, a Factor Xa proteolytic cleavage site, a Enterokinase proteolytic cleavage site, and a proteolytic cleavage site comprising SEQ ID NO:25.
23. The method of claim 22 , wherein said site-specific proteolytic cleavage site is a Furin proteolytic cleavage site.
24. The method of claim 23 , wherein said Furin proteolytic cleavage site comprises SEQ ID NO:24.
25. The method of claim 16 , wherein said lysosomal hydrolase protein is selected from the group consisting of α-glucosidase, α-L-iduronidase, α-galactosidase A, arylsulfatase, N-acetlygalactosamine-α-sulfatase, β-galactosidase, iduronate 2-sulfatase, ceramidase, galactocerebrosidase, β-glucoronidase, Heparan N-sulfatase, N-Acetyl-α-glucosaminidase, 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, Sphingomyelinase, and Glucocerebrosidase.
26. The method of claim 16 , further comprising contacting said phosphoryalated protein with an isolated phosphodiester α-GlcNAcase.
27. The method of claim 26 , wherein said phosphodiester α-GlcNAcase comprises the amino acid sequence of SEQ ID NO:18.
28. The method of claim 26 , wherein said phosphodiester α-GlcNAcase 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, wherein said stringent conditions comprise hybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C. and washing in 0.1×SSC at 65° C.
29. The method of claim 16 , wherein prior to said contacting the method comprises: culturing a host cell which comprises a polynucleotide encoding the GlcNAc-phosphotransferase for a time under conditions suitable for expression of the GlcNAc-phosphotransferase; and isolating said GlcNAc-phosphotransferase.