Soluble GlcNAc phosphotransferase

Abstract

The present invention relates to a soluble GlcNAc phosphotransferase, a method of making the same and a method of phosphorylating with the same.

1 . A method of phosphorylating a protein comprising contacting said protein with a soluble GlcNAc-phosphotransferase; and producing a phosphorylated protein.  
     
     
         2 . The method of  claim 1 , wherein said protein comprises an asparagine-linked oligosaccharide with a high mannose structure.  
     
     
         3 . The method of  claim 1 , wherein said soluble GlcNAc-phosphotransferase comprises the amino acid sequence in SEQ ID NO:2.  
     
     
         4 . The method of  claim 1 , wherein said soluble GlcNAc-phosphotransferase comprises an α subunit, a β subunit and a site-specific proteolytic cleavage site interposed between said α and β subunits, wherein said proteolytic cleavage site is not natural to said GlcNAc-phosphotransferase.  
     
     
         5 . The method of  claim 4 , 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.  
     
     
         6 . The method of  claim 4 , 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.  
     
     
         7 . The method of  claim 4 , wherein said α-subunit comprises amino acids 1-928 of SEQ ID NO:4.  
     
     
         8 . The method of  claim 4 , wherein said β subunit amino acids 1 to 328 of SEQ ID NO:5.  
     
     
         9 . The method of  claim 4 , wherein said soluble GlcNAc-phosphotransferase further comprises a γ subunit.  
     
     
         10 . The method of  claim 9 , 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.  
     
     
         11 . The method of  claim 9 , wherein said γ subunit comprises the amino acid sequence of SEQ ID NO:7.  
     
     
         12 . The method of  claim 1 , 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 Genease I proteolytic cleavage site.  
     
     
         13 . The method of  claim 12 , wherein said site-specific proteolytic cleavage site is a Furin proteolytic cleavage site.  
     
     
         14 . The method of  claim 13 , wherein said Furin proteolytic cleavage site comprises SEQ ID NO:22.  
     
     
         15 . The method of  claim 1 , wherein said protein is a lysosomal hydrolase.  
     
     
         16 . The method of  claim 15 , wherein said lysosomal enzyme is selected from the group consisting of α-glucosidase, α-iduronidase, β-galactosidase A, arylsulfatase, N-acetlygalactosamine-α-sulfatase, β-galactosidase, iduronate 2-sulfatase, ceramidase, galactocerebrosidase, β-glucoronidase, Heparan N-sulfatase, N-Acetyl-α-glucosaminidase, Acetyl CoA-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—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.  
     
     
         17 . The method of  claim 1 , further comprising contacting said phosphoryalated protein with an isolated phospbodiester α-GlcNAcase.  
     
     
         18 . The method of  claim 17 , wherein said phosphodiester α-GlcNAcase comprises the amino acid sequence of SEQ ID NO: 18.  
     
     
         19 . The method of  claim 17 , 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.  
     
     
         20 . The method of  claim 1 , wherein prior to said contacting the method comprises: culturing a host cell which comprises an isolated polynucleotide encoding soluble GlcNAc-phosphotransferase for a time under conditions suitable for expression of the soluble GlcNAc-phosphotransferase; and isolating said soluble GlcNAc-phosphotransferase.  
     
     
         21 . The method of  claim 1 , wherein prior to said contacting the method comprises culturing a host cell which comprises an isolated polynucleotide encoding soluble GlcNAc-phosphotransferase for a time under conditions suitable for expression of the soluble GlcNAc-phosphotransferase, wherein said soluble GlcNAc-phosphotransferase comprises an αsubunit, a β subunit and a site-specific protelytic cleavage site interposed between said α and β subunits, wherein said proteolytic cleavage site is not endogenous to GlcNAc-phosphotransferase;isolating said soluble GlcNAc-phosphotransferase; cleaving said isolated soluble GlcNAc-phosphotransferase with a proteolytic enzyme specific for said proteolytic cleavage site; and mixing said a and βsubunits with a y subunit of GlcNAc-phosphotransferase.  
     
     
         22 . An isolated polypeptide comprising SEQ ID NO:2.  
     
     
         23 . An isolated polynucleotide which encodes the polypeptide of  claim 22 .  
     
     
         24 . An isolated polynucleotide comprising SEQ ID NO: 1.  
     
     
         25 . An isolated polynucleotide, which hybridizes under stringent conditions to the isolated polynucleotide SEQ ID NO:1 or the complement of SEQ ID NO:1.  
     
     
         26 . An 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.  
     
     
         27 . An isolated polynucleotide, which encodes the GlcNAc-phosphotransferase of  claim 26 .  
     
     
         28 . The GlcNAc-phosphotransferase of  claim 26 , wherein said a 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.  
     
     
         29 . The GlcNAc-phosphotransferase of  claim 26 , 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.  
     
     
         30 . The GlcNAc-phosphotransferase of  claim 30 , wherein said cc-subunit comprises amino acids 1-928 of SEQ ID NO:4.  
     
     
         31 . The GlcNAc-phosphotransferase of  claim 26 , wherein said β subunit amino acids 1 to 328 of SEQ ID NO:5.  
     
     
         32 . The GlcNAc-phosphotransferase of  claim 26 , wherein said GlcNAc-phosphotransferase further comprises a γ subunit.  
     
     
         33 . The GlcNAc-phosphotransferase of  claim 32 , 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.  
     
     
         34 . The GlcNAc-phosphotransferase of  claim 32 , wherein said γ subunit comprises the amino acid sequence of SEQ ID NO:7.  
     
     
         35 . The GlcNAc-phosphotransferase of  claim 26 , 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 Genease I proteolytic cleavage site.  
     
     
         36 . The GlcNAc-phosphotransferase of  claim 35 , wherein said site-specific proteolytic cleavage site is a Furin proteolytic cleavage site.  
     
     
         37 . The GlcNAc-phosphotransferase of  claim 36 , wherein said Furin proteolytic cleavage site comprises SEQ ID NO:22.  
     
     
         38 . A vector comprising the isolated polynucleotide of  claim 23 .  
     
     
         39 . A vector comprising the isolated polynucleotide of  claim 24 .  
     
     
         40 . A vector comprising the isolated polynucleotide of  claim 25 .  
     
     
         41 . A vector comprising the isolated polynucleotide of  claim 27 .  
     
     
         42 . A host cell comprising the isolated polynucleotide of  claim 23 .  
     
     
         43 . A host cell comprising the isolated polynucleotide of  claim 24 .  
     
     
         44 . A host cell comprising the isolated polynucleotide of  claim 25 .  
     
     
         45 . A host cell comprising the isolated polynucleotide of  claim 27 .  
     
     
         46 . A method of producing an α and β subunit GlcNAc-phosphotransferase polyprotein comprising culturing the host cell of  claim 42  for a time and under conditions suitable for expression of the α and β subunit GlcNAc-phosphotransferase polyprotein and collecting the α and β subunit GlcNAc-phosphotransferase polyprotein produced.  
     
     
         47 . The method of  claim 46 , wherein prior to said collecting, the αand β GlcNAc-phosphotransferase subunits are cleaved in the host cell by a site specfic protease which is expressed in the cell, wherein said protease is specific for a protease cleavage site positioned between said α and β subunits.  
     
     
         48 . The method of  claim 46 , further comprising after said collecting, the α and β subunits are cleaved with a protease specific for a protease cleavage site positioned between said α and β subunits.  
     
     
         49 . A method of producing an α and β subunit GlcNAc-phosphotransferase polyprotein comprising culturing the host cell of  claim 45  for a time and under conditions suitable for expression of the α and β subunit GlcNAc-phosphotransferase polyprotein and collecting the α and β subunit GlcNAc-phosphotransferase polyprotein produced.  
     
     
         50 . The method of  claim 49 , wherein prior to said collecting, the α and β GlcNAc-phosphotransferase subunits are cleaved in the host cell by a site specfic protease which is expressed in the cell, wherein said protease is specific for a protease cleavage site positioned between said α and β subunits.  
     
     
         51 . The method of  claim 49 , further comprising after said collecting, the α and β subunits are cleaved with a protease specific for a protease cleavage site positioned between said α and β subunits.  
     
     
         52 . A phosphorylated protein obtained by the method of  claim 1 .  
     
     
         53 . A phosphorylated protein obtained by the method of  claim 17 .  
     
     
         54 . A method of treating a patient suffering from a lysosomal storage disease comprising contacting a lysosomal hydrolase with the GlcNAc-phosphotransferase of  claim 26  to produce a lysosomal hydrolase with an N-acetylglucosamine-1-phosphate; removing said N-acetylglucosamine by contacting said lysosomal hydrolase with a phosphodiester α-GlcNAcase to produce a phosphorylated lysosomal hydrolase isolating said phosphorylated lysosomal hydrolase; and administering an amount sufficient to treat said disease the isolated phosphorylated lysosomal hydrolase.  
     
     
         55 . A method of treating a patient suffering from a lysosomal storage disease comprising contacting a lysosomal hydrolase with the GlcNAc-phosphotransferase of  claim 32  to produce a lysosomal hydrolase with an N-acetylglucosamine-1-phosphate; removing said N-acetylglucosamine by contacting said lysosomal hydrolase with a phosphodiester α-GlcNAcase to produce a phosphorylated lysosomal hydrolase isolating said phosphorylated lysosomal hydrolase; and administering an amount sufficient to treat said disease the isolated phosphorylated lysosomal hydrolase.