2-O sulfatase compositions and methods of analyzing therewith
The invention relates to 2-O sulfatase and uses thereof. In particular, the invention relates to recombinantly produced 2-O sulfatase, functional variants and nucleic acid molecules that encode these molecules. The invention also provides methods of using 2-O sulfatase for a variety of purposes, including degrading and analyzing glycosaminoglycans (GAGs) present in a sample. For instance, 2-O sulfatase may be used for determining the purity, identity, composition and sequence of glycosaminoglycans present in a sample. The invention also relates to methods of inhibiting angiogenesis and cellular proliferation as well as methods for treating cancer, neurodegenerative disease, atherosclerosis and microbial infection using 2-O sulfatase and/or GAG fragments produced by degradation with 2-O sulfatase.
1. A method of analyzing a sample containing a glycosaminoglycan, comprising:
contacting the sample with a recombinantly produced or isolated 2-O sulfatase that has an amino acid sequence set forth as SEQ ID NO: 2 or 4 in an effective amount to analyze the sample, and analyzing the sample.
2. A method of analyzing a sample containing a glycosaminoglycan, comprising:
contacting the sample with a 2-O sulfatase in an effective amount to analyze the sample, and analyzing the sample, wherein the 2-O sulfatase is recombinantly produced by expressing an isolated nucleic acid molecule selected from the group consisting of:
(a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence selected from the group consisting of nucleotide sequences set forth as SEQ ID NOs: 1 and 3, and which code for a 2-O sulfatase that has an activity of the 2-O sulfatase set forth as SEQ ID NO: 2 or 4,
wherein the hybridization conditions are 1) hybridization at 65° C. in hybridization buffer that consists of 3.5×SSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin, 2.5mM NaH2PO4, pH 7, 0.5% sodium dodecyl sulphate (SDS), 2 mM ethylenediaminetetracetic acid (EDTA), wherein SSC is 0.15M sodium chloride/0.015M sodium citrate, pH 7 and 2) washing in 2×SSC at room temperature and then in 0.1×SSC/0.1×SDS at 68° C.
(b) nucleic acid molecules that differ from the nucleic acid molecules of (a) in codon sequence due to degeneracy of the genetic code, and
(c) full complements of (a) or (b).
3. The method of claim 1 , wherein the sample is analyzed with mass spectrometry, gel electrophoresis, capillary electrophoresis or HPLC.
4. The method of claim 1 , wherein the method is a method for determining the purity of the glycosaminoglycan in the sample.
5. The method of claim 1 , wherein the method is a method for determining the presence of a particular glycosaminoglycan in the sample.
6. The method of claim 1 , wherein the method is a method for determining the composition of the glycosaminoglycan in the sample.
7. The method of claim 1 , wherein the method is a method for determining the sequence of saccharide units of the glycosaminoglycan in the sample.
8. The method of claim 1 , further comprising:
contacting the sample with at least one other glycosaminoglycan degrading enzyme.
9. The method of claim 8 , wherein the at least one other glycosaminoglycan degrading enzyme is a heparinase or glycuronidase.
10. The method of claim 8 , wherein the sample is contacted with the at least one other glycosaminoglycan degrading enzyme concomitantly with the 2-O sulfatase.
11. The method of claim 10 , wherein the at least one other glycosaminoglycan degrading enzyme is a glycuronidase.
12. The method of claim 9 , wherein the glycuronidase is Δ 4,5 glycuronidase.
13. The method of claim 11 , wherein the glycuronidase is Δ 4,5 glycuronidase.
14. The method of claim 8 , wherein the sample is contacted with the at least one other glycosaminoglycan degrading enzyme prior to or subsequent to contacting the sample with the 2-O sulfatase.
15. The method of claim 14 , wherein the sample is contacted with the at least one other glycosaminoglycan degrading enzyme prior to contacting the sample with the 2-O sulfatase, and wherein the at least one other glycosaminoglycan degrading enzyme is a heparinase.
16. The method of claim 9 , wherein the at least one other glycosaminoglycan degrading enzyme is heparinase I, heparinase II or heparinase III.
17. The method of claim 15 , wherein the at least one other glycosaminoglycan degrading enzyme is heparinase I, heparinase II or heparinase III.
18. The method of claim 1 , wherein the glycosaminoglycan in the sample contains a 2-O sulfated uronic acid at the non-reducing end.
19. The method of claim 1 , wherein the glycosaminoglycan in the sample contains a highly sulfated glycosaminoglycan.
20. The method of claim 19 , wherein the highly sulfated glycosaminoglycan contains a 6-O sulfated glucosamine.
21. The method of claim 19 , wherein the highly sulfated glycosaminoglycan contains a glucosamine sulfated at the N-position.
22. The method of claim 1 , wherein the glycosaminoglycan in the sample contains a β1→4 linkage.
23. The method of claim 1 , wherein the glycosaminoglycan in the sample is a tetrasaccharide.
24. The method of claim 1 , wherein the glycosaminoglycan in the sample is a decasaccharide.
25. The method of claim 1 , wherein the glycosaminoglycan is a heparin, low molecular weight heparin (LMWH) or heparan sulfate.