Modified heparinase III and methods of sequencing therewith
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 sequencing comprising:
contacting a polysaccharide having 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 to allow cleavage of the polysaccharide by the modified heparinase III, thereby obtaining fragments of the polysaccharide, 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,
analyzing the fragments to identify one or more properties thereof, and
determining the sequence of the polysaccharide based on the one or more properties.
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 3 , wherein the His110 is substituted with alanine.
5. The method of claim 1 , wherein the modified heparinase III has a substitution at His241.
6. The method of claim 5 , wherein the His241 is substituted with alanine.
7. The method of claim 1 , wherein the modified heparinase III has a substitution at His225.
8. The method of claim 7 , wherein the His225 is substituted with alanine.
9. The method of claim 1 , wherein the analyzing step is performed by subjecting the fragments to mass spectrometry (MS), matrix-assisted laser desorption ionization MS (MALDI), capillary electrophoresis (CE), viscosity measurement, and/or total UV absorbance measurement to identify the one or more properties.
10. The method of claim 1 , wherein the modified heparinase III is immobilized on a solid support.
11. The method of claim 10 , wherein the solid support is a sheet, test strip, membrane, bead, test tube, microplate well or the external surface of a rod.