Microorganisms genetically engineered to have modified N-glycosylation activity
Described herein are methods and genetically engineered cells useful for producing an altered N-glycosylation form of a target molecule. Also described are methods and molecules with altered N-glycosylation useful for treating a variety of disorders such as metabolic disorders.
1. A method of producing an altered N-glycosylation form of a target protein, the method comprising:
providing a Yarrowia lipolytica or an Arxula adeninivorans cell genetically engineered to express a protein capable of promoting mannosyl phosphorylation of N-glycans; and
introducing into the cell a nucleic acid encoding a target protein, wherein the cell produces the target protein in an altered N-glycosylation form, and
wherein the protein capable of promoting mannosyl phosphorylation of N-glycans is selected from the group consisting of:
a wild-type MNN4 polypeptide or a biologically active variant of a wild-type MNN4 polypeptide;
a wild-type PNO1 polypeptide or a biologically active variant of a wild-type PNO1 polypeptide; and
a wild-type MNN6 polypeptide.
2. The method of claim 1 , further comprising isolating the altered N-glycosylation form of the target protein.
3. The method of claim 1 , wherein the target protein is an exogenous protein.
4. The method of claim 1 , wherein the target protein is an endogenous protein.
5. The method of claim 1 , wherein the target protein is a mammalian protein.
6. The method of claim 1 , wherein the target protein is a pathogen protein, a lysosomal protein, a growth factor, a cytokine, a chemokine, or a fusion protein.
7. The method of claim 1 , wherein the target protein is a protein associated with a lysosomal storage disorder (LSD).
8. The method of claim 7 , wherein the lysosomal storage disorder is Gaucher disease, Tay-Sachs disease, Pompe disease, Niemann-Pick disease, or Fabry disease.
9. The method of claim 7 , wherein the target protein is glucocerebrosidase, alpha galactosidase, or galactocerebrosidase.
10. The method of claim 7 , wherein the target protein is selected from the group consisting of alpha-L-iduronidase, beta-D-galactosidase, beta-glucosidase, beta-hexosaminidase, beta-D-mannosidase, alpha-L-fucosidase, arylsulfatase B, arylsulfatase A, alpha-N-acteylgalactosaminidase, aspartylglucosaminidase, iduronate-2-sulfatase, alpha-glucosaminide-N-acetyltransferase, beta-D-glucoronidase, hyaluronidase, alpha-L-mannosidase, alpha-neuraminidase, phosphotransferase, acid lipase, acid ceramidase, sphinogmyelinase, thioesterase, cathepsin K, and lipoprotein lipase.
11. The method of claim 7 , wherein the target protein is human alpha-galactosidase A.
12. The method of claim 1 , further comprising additional processing of the glycoprotein.
13. The method of claim 12 , wherein the additional processing comprises enzymatic or chemical treatment of the altered N-glycosylation form of the target protein.
14. The method of claim 1 , wherein the target protein is a human protein.
15. The method of claim 1 , wherein the cell is further genetically engineered to comprise at least one additional modification of an N-glycosylation activity.
16. The method of claim 15 , wherein the at least one additional modification of an N-glycosylation activity comprises a deficiency in an N-glycosylation activity.
17. The method of claim 16 , wherein the deficiency in an N-glycosylation activity is a deficiency in Outer CHain elongation (OCH 1 ) activity.
18. The method of claim 15 , wherein the at least one additional modification of an N-glycosylation activity comprises expression of a protein having N-glycosylation activity.
19. The method of claim 18 , wherein the expressed protein having N-glycosylation activity is an alpha-mannosidase, or a biologically active variant thereof.
20. The method of claim 18 , wherein the alpha-mannosidase is an alpha 1,2 mannosidase.
21. The method of claim 18 , wherein the alpha-mannosidase is MNS1.
22. The method of claim 18 , wherein the alpha-mannosidase is targeted to the endoplasmic reticulum.
23. An isolated Yarrowia lipolytica or Arxula adeninivorans cell genetically engineered to express a protein capable of promoting mannosyl phosphorylation of N-glycans, wherein the cell is capable of producing a target polypeptide in an altered glycosylation form when a nucleic acid encoding the target polypeptide is introduced into the cell, and
wherein the protein capable of promoting mannosyl phosphorylation of N-glycans is selected from the group consisting of:
a wild-type MNN4 polypeptide or a biologically active variant of a wild-type MNN4 polypeptide;
a wild-type PNO1 polypeptide or a biologically active variant of a wild-type PNO1 polypeptide; and
a wild-type MNN6 polypeptide.
24. The cell of claim 23 , wherein said cell further comprises a nucleic acid encoding a target polypeptide, wherein the cell produces the target polypeptide in an altered N-glycosylation form.
25. The cell of claim 24 , wherein the target polypeptide is a human polypeptide.
26. The cell of claim 24 , wherein the target polypeptide is a polypeptide associated with a lysosomal storage disorder (LSD).
27. The cell of claim 26 , wherein the LSD is selected from the group consisting of Gaucher disease, Tay-Sachs disease, Pompe disease, Niemann-Pick disease, and Fabry disease.
28. The cell of claim 26 , wherein the target polypeptide is selected from the group consisting of glucocerebrosidase, alpha galactosidase, galactocerebrosidase, alpha-L-iduronidase, beta-D-galactosidase, beta-glucosidase, beta-hexosaminidase, beta-D-mannosidase, alpha-L-fucosidase, arylsulfatase B, arylsulfatase A, alpha-N-acteylgalactosaminidase, aspartylglucosaminidase, iduronate- 2 -sulfatase, alpha-glucosaminide-N-acetyltransferase, beta-D-glucoronidase, hyaluronidase, alpha-L-mannosidase, alpha- neuraminidase, phosphotransferase, acid lipase, acid ceramidase, sphinogmyelinase, thioesterase, cathepsin K, lipoprotein lipase, and human alpha- galactosidase A.
29. The cell of claim 22 , wherein the cell is further genetically engineered to comprise at least one additional modification of an N-glycosylation activity.
30. The cell of claim 24 , wherein the at least one additional modification of an N-glycosylation activity comprises a deficiency in an N-glycosylation activity.
31. The cell of claim 30 , wherein the deficiency in an N-glycosylation activity is a deficiency in Outer CHain elongation (OCH 1 ) activity.
32. The cell of claim 24 , wherein the at least one additional modification of an N-glycosylation activity comprises expression of a protein having N-glycosylation activity.
33. The cell of claim 32 , wherein the expressed protein having N-glycosylation activity is an alpha-mannosidase, or a biologically active variant thereof.
34. The substantially pure culture of claim 33 , wherein said cell further comprises a nucleic acid encoding a target protein, wherein the cell produces the target protein in an altered N-glycosylation form.
a wild-type MNN6 polypeptide.
35. The method of claim 28 , wherein the cell is further genetically engineered to comprise at least one additional modification of an N-glycosylation activity.
36. The method of claim 29 , wherein the at least one additional modification of an N-glycosylation activity comprises a deficiency in an N-glycosylation activity.
37. The method of claim 36 , wherein the deficiency in an N-glycosylation activity is a deficiency in Outer CHain elongation (OCH 1 ) activity.
38. The method of claim 29 , wherein the at least one additional modification of an N-glycosylation activity comprises expression of a protein having N-glycosylation activity.
39. The method of claim 38 , wherein the expressed protein having N-glycosylation activity is an alpha-mannosidase, or a biologically active variant thereof.
40. The method of claim 34 , wherein the target protein is a human protein.
41. The method of claim 34 , wherein the target protein is a protein associated with a lysosomal storage disorder (LSD).
42. The method of claim 41 , wherein the LSD is selected from the group consisting of Gaucher disease, Tay-Sachs disease, Pompe disease, Niemann-Pick disease, and Fabry disease.
43. The method of claim 41 , wherein the target protein is selected from the group consisting of glucocerebrosidase, alpha galactosidase, galactocerebrosidase, alpha-L-iduronidase, beta-D-galactosidase, beta-glucosidase, beta-hexosaminidase, beta-D-mannosidase, alpha-L-fucosidase, arylsulfatase B, arylsulfatase A, alpha-N-acteylgalactosaminidase, aspartylglucosaminidase, iduronate-2-sulfatase, alpha-glucosaminide-N-acetyltransferase, beta-D-glucoronidase, hyaluronidase, alpha-L-mannosidase, alpha-neuraminidase, phosphotransferase, acid lipase, acid ceramidase, sphinogmyelinase, thioesterase, cathepsin K, lipoprotein lipase, and human alpha-galactosidase A.
44. A substantially pure culture of Yarrowia lipolytica or Arxula adeninivorans cells, wherein the culture comprises a cell genetically engineered to express a protein capable of promoting mannosyl phosphorylation of N-glycans, wherein the cell is capable of producing a target polypeptide in an altered glycosylation form when a nucleic acid encoding the target polypeptide is introduced into the cell, and
wherein the protein capable of promoting mannosyl phosphorylation of N-glycans is selected from the group consisting of:
a wild-type MNN4 polypeptide or a biologically active variant of a wild-type MNN4 polypeptide;
a wild-type PNO1polypeptide or a biologically active variant of a wild-type PNO1polypeptide; and a wild-type MNN 6 polypeptide.
45. The substantially pure culture of claim 44 , wherein said cell further comprises a nucleic acid encoding a target polypeptide, wherein the cell procedures the target polypeptide in an altered N-glycosylation form.
46. The culture of claim 45 , wherein the target polypeptide is a human polypeptide.
47. The culture of claim 45 , wherein the target polypeptide is a protein associated with a lysosomal storage disorder (LSD).
48. The culture of claim 44 , wherein the LSD is selected from the group consisting of Gaucher disease, Tay-Sachs disease, Pompe disease, Niemann-Pick disease, and Fabry disease.
49. The culture of claim 47 , wherein the target polypeptide is selected from the group consisting of glucocerebrosidase, alpha galactosidase, galactocerebrosidase, alpha-L-iduronidase, beta-D-galactosidase, beta-glucosidase, beta-hexosaminidase, beta-D-mannosidase, alpha-L-fucosidase, arylsulfatase B, arylsulfatase A, alpha-N-acteylgalactosaminidase, aspartylglucosaminidase, iduronate- 2 -sulfatase, alpha-glucosaminide-N-acetyltransferase, beta-D-glucoronidase, hyaluronidase, alpha-L-mannosidase, alpha- neuraminidase, phosphotransferase, acid lipase, acid ceramidase, sphinogmyelinase, thioesterase, cathepsin K, lipoprotein lipase, and human alpha-galactosidase A.
50. The culture of claim 33 , wherein the cell is further genetically engineered to comprise at least one additional modification of an N-glycosylation activity.
51. The culture of claim 35 , wherein the at least one additional modification of an N-glycosylation activity comprises a deficiency in an N-glycosylation activity.
52. The culture of claim 51 , wherein the deficiency in an N-glycosylation activity is a deficiency in Outer CHain elongation (OCH 1 ) activity.
53. The culture of claim 35 , wherein the at least one additional modification of an N-glycosylation activity comprises expression of a protein having N-glycosylation activity.
54. The substantially pure culture of claim 53 , wherein the expressed protein having N-glycosylation activity is an alpha-mannosidase, or a biologically active variant thereof.