Rationally designed polysaccharide lyases derived from chondroitinase B and methods of specifically cleaving therewith
The invention relates to rationally designed polysaccharide lyases and uses thereof. In particular, the invention relates to modified chondroitinase B. The modified chondroitinase B enzymes of the invention are useful for a variety of purposes, including cleaving and sequencing polysaccharides such as glycosaminoglycans (GAGs) as well as removing polysaccharides from a solution. The invention also includes methods of inhibiting anticoagulant activity, inhibiting angiogenesis, treating cancer, and inhibiting maternal malarial infection.
1. A method of specifically cleaving chondroitin sulfate, comprising:
contacting chondroitin sulfate with a modified chondroitinase B, and
cleaving the chondroitin sulfate, wherein the modified chondroitinase B has the amino acid sequence of the mature peptide of SEQ ID NO: 2 or conservative substitutions thereof, wherein at least one amino acid residue at a position selected from the group consisting of 116, 184, 213, 219, 245, 250, 271, 272, 296, 298, 318, 333, 363 and 364 of SEQ ID NO: 2 has been substituted or deleted.
2. The method of claim 1 , wherein the chondroitin sulfate is a decasaccharide.
3. The method of claim 1 , wherein the chondroitin sulfate is an octasaccharide, hexasaccharide or a tetrasaccharide.
4. The method of claim 1 , wherein the modified chondroitinase B is a substantially purified recombinant form.
5. The method of claim 1 , wherein the modified chondroitinase B has a modified product profile, wherein the modified product profile of the modified chondroitinase B is at least 10% different than a native product profile of a native chondroitinase B.
6. The method of claim 5 , wherein the modified chondroitinase B has a modified product profile that is at least 20% different than a native product profile of a native chondroitinase B.
7. The method of claim 6 , wherein the modified chondroitinase B has a modified product profile that is at least 50% different than a native product profile of a native chondroitinase B.
8. The method of claim 1 , wherein the modified chondroitinase B has a k cat or K M value for a substrate that is at least 10% different than a native chondroitinase B k cat or K M value.
9. The method of claim 8 , wherein the modified chondroitinase B k cat or K M value is at least 20% different than a native chondroitinase B k cat or K M value.
10. The method of claim 9 , wherein the modified chondroitinase B k cat or K M value is at least 50% different than a native chondroitinase B k cat or K M value.
11. The method of claim 8 , wherein the modified chondroitinase B has the amino acid sequence of the mature peptide of SEQ ID NO: 2 or conservative substitutions thereof, wherein at least one amino acid residue has been substituted and wherein the substituted amino acid is at a position selected from the group consisting of 272, 333, 363 and 364 of SEQ ID NO: 2.
12. The method of claim 1 , wherein the modified chondroitinase B is encoded by a nucleic acid sequence that is at least 97% homologous to the nucleic acid sequence of SEQ ID NO: 1.
13. The method of claim 12 , wherein the modified chondroitinase B is encoded by a nucleic acid sequence that is at least 99% homologous to the nucleic acid sequence of SEQ ID NO: 1.
14. The method of claim 1 , wherein the modified chondroitinase B is immobilized on a solid support membrane.
15. The method of claim 1 , wherein the method is a method of removing a chondroitin sulfate from a chondroitin sulfate containing fluid.
16. The method of claim 1 , wherein the method is a method for sequencing chondroitin sulfate oligosaccharides.
17. The method of claim 1 , wherein the chondroitin sulfate is further contacted with another polysaccharide-degrading enzyme.
18. The method of claim 17 , wherein the polysaccharide-degrading enzyme is chondroitinase ABC, chondroitinase AC or chondroitinase B.
19. A method of specifically cleaving dermatan sulfate, comprising:
contacting dermatan sulfate with a modified chondroitinase B, and
cleaving the dermatan sulfate, wherein the modified chondroitinase B has the amino acid sequence of the mature peptide of SEQ ID NO: 2 or conservative substitutions thereof, wherein at least one amino acid residue at a position selected from the group consisting of 116, 184, 213, 219, 245, 250, 271, 272, 296, 298, 318, 333, 363 and 364 of SEQ ID NO: 2 has been substituted or deleted.
20. The method of claim 19 , wherein the dermatan sulfate is a decasaccharide.
21. The method of claim 19 , wherein the dermatan sulfate is an octasaccharide, hexasaccharide or a tetrasaccharide.
22. The method of claim 19 , wherein the modified chondroitinase B is a substantially purified recombinant form.
23. The method of claim 19 , wherein the modified chondroitinase B has a modified product profile, wherein the modified product profile of the modified chondroitinase B is at least 10% different than a native product profile of a native chondroitinase B.
24. The method of claim 23 , wherein the modified chondroitinase B has a modified product profile that is at least 20% different than a native product profile of a native chondroitinase B.
25. The method of claim 24 , wherein the modified chondroitinase B has a modified product profile that is at least 50% different than a native product profile of a native chondroitinase B.
26. The method of claim 19 , wherein the modified chondroitinase B has a k cat or K M value for a substrate that is at least 10% different than a native chondroitinase B k cat or K M value.
27. The method of claim 26 , wherein the modified chondroitinase B k cat or K M value is at least 20% different than a native chondroitinase B k cat or K M value.
28. The method of claim 27 , wherein the modified chondroitinase B k cat or K M value is at least 50% different than a native chondroitinase B k cat or K M value.
29. The method of claim 26 , wherein the modified chondroitinase B has the amino acid sequence of the mature peptide of SEQ ID NO: 2 or conservative substitutions thereof, wherein at least one amino acid residue has been substituted and wherein the substituted amino acid is at a position selected from the group consisting of 272, 333, 363 and 364 of SEQ ID NO: 2.
30. The method of claim 19 , wherein the modified chondroitinase B is encoded by a nucleic acid sequence that is at least 97% homologous to the nucleic acid sequence of SEQ ID NO: 1.
31. The method of claim 30 , wherein the modified chondroitinase B is encoded by a nucleic acid sequence that is at least 99% homologous to the nucleic acid sequence of SEQ ID NO: 1.
32. The method of claim 19 , wherein the modified chondroitinase B is immobilized on a solid support membrane.
33. The method of claim 19 , wherein the method is a method of removing a dermatan sulfate from a dermatan sulfate containing fluid.
34. The method of claim 19 , wherein the method is a method for sequencing dermatan sulfate oligosaccharides.
35. The method of claim 19 , wherein the dermatan sulfate is further contacted with another polysaccharide-degrading enzyme.
36. The method of claim 35 , wherein the polysaccharide-degrading enzyme is chondroitinase ABC, chondroitinase AC or chondroitinase B.
37. The method of claim 5 , wherein the modified chondroitinase B has the amino acid sequence of the mature peptide of SEQ ID NO: 2 or conservative substitutions thereof, wherein at least one amino acid residue has been substituted and wherein the substituted amino acid is at a position selected from the group consisting of 272, 333 and 364 of SEQ ID NO: 2.
38. The method of claim 23 , wherein the modified chondroitinase B has the amino acid sequence of the mature peptide of SEQ ID NO: 2 or conservative substitutions thereof, wherein at least one amino acid residue has been substituted and wherein the substituted amino acid is at a position selected from the group consisting of 272, 333 and 364 of SEQ ID NO: 2.
39. A method of cleaving a polysaccharide, comprising:
contacting the polysaccharide with a modified chondroitinase B, and
cleaving the polysaccharide, wherein the modified chondroitinase B has the amino acid sequence of the mature peptide of SEQ ID NO: 2 or conservative substitutions thereof, wherein at least one amino acid residue at a position selected from the group consisting of 116, 184, 213, 219, 245, 250, 271, 272, 296, 298, 318, 333, 363 and 364 of SEQ ID NO: 2 has been substituted or deleted.
40. The method of claim 39 , wherein the modified chondroitinase B has the amino acid sequence of the mature peptide of SEQ ID NO: 2 or conservative substitutions thereof, wherein at least one amino acid residue has been substituted and wherein the substituted amino acid is at a position selected from the group consisting of 272, 333, 363 and 364 of SEQ ID NO: 2.
41. The method of claim 39 , wherein the polysaccharide is a tetrasaccharide, hexasaccharide, octasaccharide or decasaccharide.
42. The method of claim 39 , wherein the polysaccharide is a glycosaminoglycan.
43. The method of claim 42 , wherein the glycosaminoglycan is a HLGAG.
44. The method of claim 42 , wherein the glycosaminoglycan is heparin or heparan sulfate.
45. The method of claim 39 , wherein the modified chondroitinase B is a substantially purified recombinant form.
46. The method of claim 39 , wherein the modified chondroitinase B is immobilized on a solid support membrane.
47. The method of claim 39 , wherein the polysaccharide is further contacted with another polysaccharide-degrading enzyme.