Heparinase III and methods of specifically cleaving therewith
View Patent ↗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 method of cleaving comprising:
contacting a linear polysaccharide with a disaccharide repeat unit of a uronic acid [α-L-iduronic acid (I) or β-D-glucuronic acid (G)] linked 1, 4 to α-D-hexosamine (H) with a protein comprising a modified heparinase III in vitro, wherein the modified heparinase III has 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 His36, 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. The method of claim 1 , wherein the modified heparinase III has at least one substitution at a histidine residue selected from the group consisting of His110, His225 and His241.
3. The method of claim 1 , wherein the modified heparinase III has a substitution at His110.
4. The method of claim 1 , wherein the modified heparinase III has a substitution at His241.
5. The method of claim 1 , wherein the modified heparinase III has a substitution at His225.
6. The method of claim 5 , wherein the His225 is substituted with alanine.
7. The method of claim 1 , wherein the modified heparinase III is in a composition that comprises a pharmaceutically acceptable carrier.
8. The method of claim 1 , wherein the modified heparinase III is immobilized on a solid support.
9. The method of claim 8 , wherein the solid support is a sheet, test strip, membrane, bead, test tube, microplate well or the external surface of a rod.