Heparinase III and uses thereof
The invention relates to heparinase III and mutants thereof. Modified forms of heparinase III having reduced enzymatic activity which are useful for a variety of purposes, including sequencing of heparin-like glycosaminoglycans (HLGAGs), removing active heparan sulfate from a solution, inhibition of angiogenesis, etc. have been discovered according to the invention. The invention in other aspects relates to methods of treating cancer and inhibiting tumor cell growth and/or metastasis using heparinase III, or products produced by enzymatic cleavage by heparinase III of HLGAGs.
1. A substantially pure heparinase III, comprising:
a polypeptide having the amino acid sequence of the mature peptide of SEQ ID NO: 2, wherein at least one histidine residue selected from the group consisting of His 36, His105, His110, His139, His152, His225, His234, His241, His424, His469, and His539 has been substituted with a residue selected from the group consisting of alanine, serine, tyrosine, threonine, and lysine.
2. A substantially pure heparinase III comprising:
a modified heparinase III having a modified product profile, wherein the modified product profile of the modified heparinase III is at least 10% different than a native product profile of a native heparinase III,
wherein the substantially pure heparinase III has the amino acid sequence of the mature peptide of SEQ ID NO: 2 where at least one histidine residue selected from the group consisting of His 36, His105, His110, His139, His152, His225, His234, His241, His424, His469, and His539 has been substituted with a residue selected from the group consisting of alanine, serine, tyrosine, threonine, and lysine.
3. A substantially pure heparinase III comprising:
a modified heparinase III that can cleave a heparan sulfate substrate having modified heparinase III k cat value, wherein the modified heparinase III k cat value is at leas 10% different than a native heparinase III k cat value,
wherein the substantially pure heparinase III has the amino acid sequence of the mature peptide of SEQ ID NO: 2 where at least one histidine residue selected from the group consisting of His 36, His105, His110, His139, His152, His225, His234, His241, His424, His469, and His539 has been substituted with a residue selected from the group consisting o alanine, serine, tyrosine, threonine, and lysine.
4. A pharmaceutical preparation comprising a sterile formulation of the substantially pure heparinase III of claim 1 and a pharmaceutically acceptable carrier.
5. An immobilized substantially pure modified heparinase III comprising:
a modified heparinase III as in claim 1 , and
a solid support, wherein the modified heparinase III is immobilized on the solid support.
6. The substantially pure heparinase III of claim 1 , wherein the polypeptide has at least one substitution within a histidine residue selected from the group consisting of His 110, His225, and His241.
7. The substantially pure heparinase III of claim 1 , wherein the polypeptide has a substitution at His110.
8. The substantially pure heparinase III of claim 1 , wherein the polypeptide has a substitution at His241.
9. The substantially pure heparinase III of claim 1 , wherein the polypeptide has a substitution at His225.
10. The substantially pure heparinase III of claim 9 , wherein the His225 is substituted with alanine.