Methods for determining oligosaccharide binding
The invention relates to methods for detecting and characterizing enzymatic modifications of oligosaccharides, such as heparan sulfate, and their interaction with binding partners, such as proteins, using an oligosaccharide-binding partner binding assay, such as a gel mobility shift assay. The instant invention relates to a rapid, convenient, sensitive and inexpensive method for identifying or studying oligosaccharide-binding partner interactions, identifying and characterizing structural features on oligosaccharides, identifying and characterizing binding partners, identifying agents capable of interfering with, enhancing, or facilitating the binding of an oligosaccharide to its binding partner, diagnosing conditions associated with altered oligosaccharide-binding partner binding, and generating oligosaccharide libraries and kits therefor.
1. A method for determining the requirement for a functional group on a modified oligosaccharide for binding to a binding partner, the method comprising the steps of:
a) contacting a modified oligosaccharide comprising at least one functional group, which provides a binding characteristic with a binding partner to form a modified oligosaccharide-binding partner complex;
b) contacting an oligosaccharide which is an unmodified oligosaccharide of step (a) with a binding partner, wherein said unmodified oligosaccharide does not form an oligosaccharide binding partner complex; and
c) detecting the presence of said modified oligosaccharide-binding partner complex, upon subjecting the oligosaccharides in (a) and (b) to an assay, wherein said assay consists of an electrophoretic mobility shift assay consisting of a native gel, wherein said detection is a direct detection comprising the visualization and optionally quantification of a region in the gel comprising the modified oligosaccharide-binding partner complex; and
whereby a detection of said modified oligosaccharide-binding partner complex of (a) and a lack of detection of an unmodified oligosaccharide-binding partner complex of (b) indicates a requirement for said functional group, for binding of the modified oligosaccharide to a binding partner.
2. The method of claim 1 , wherein said modified oligosaccharide or oligosaccharide comprises between 4 and 300 saccharides.
3. The method of claim 1 , wherein said modified oligosaccharide or oligosaccharide comprises at least a pentasaccharide.
4. The method of claim 1 , wherein said modified oligosaccharide is manufactured in vitro.
5. The method of claim 1 , wherein said modified oligosaccharide or oligosaccharide is derived from an in vivo tissue sample.
6. The method of claim 1 , wherein said modified oligosaccharide is modified in vitro.
7. The method of claim 1 , wherein said modified oligosaccharide is modified in vitro with 3-O-sulfotransferase-1 and/or 6-O-sulfotransferase-1.
8. The method of claim 1 , wherein said modified oligosaccharide is modified in vitro with at least one sulfotransferase selected from the group consisting of 2-O-sulfotransferase, 3-O-sulfotransferase-2, 3-O-sulfotransferase-3A, 3-O-sulfotransferase-4, 6-O-sulfotransferase-2A, 6-O-sulfotransferase-2B, and 6-O-sulfotransferase-3.
9. The method of claim 1 , wherein said modified oligosaccharide is modified in vitro with an N-deacetylase/N-sulfotransferase (NDST).
10. The method of claim 9 , wherein said NDST is selected from the group consisting of NDSTI, NDST2, NDST3, and NDST4.
11. The method of claim 1 , wherein said binding partner is a protein.
12. The method of claim 1 , wherein said binding partner is selected from the group consisting of antithrombin III (AT-III), a fibroblast growth factor (FGF), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor (VEGF), placental growth factor (PIGF), heparinbinding EGF-like growth factor, hepatocyte growth factor (HGF), transforming growth factorbeta (TGF-beta), platelet-derived growth factor (PDGF), pleiotrophin, platelet factor-4 (PF-4), interleukin-8 (IL-8), macrophage inflammatory protein-1 (MIP-1), interferon-g-inducible protein-10 (IP-10), interferon-gamma (IFN-gamma), and HIV-Tat transactivating factor.
13. The method of claim 1 , wherein said binding partner is a peptide.
14. The method of claim 1 , wherein said binding partner is an antibody.
15. The method of claim 1 , wherein said binding partner comprises a detectable label.
16. The method of claim 1 , wherein said modified oligosaccharide or oligosaccharide comprises a detectable label.
17. The method of claim 15 or 16 , wherein said detectable label is a radioactive label.
18. The method of claim 15 or 16 , wherein said detectable label is a non-radioactive label selected from the group consisting of biotin, fluorescein, and green fluorescent protein.
19. The method of claim 1 , wherein said gel is selected from the group consisting of a vertical gel, a horizontal gel, and a capillary gel.
20. The method of claim 1 , wherein said gel is subjected to autoradiography.
21. The method of claim 1 , wherein said method further comprises characterizing the binding between said modified oligosaccharide and said binding partner.
22. The method of claim 1 , wherein said method further comprises identifying an agent capable of altering the binding of an oligosaccharide with a binding partner.
23. The method of claim 1 , wherein said method further comprises determining the binding affinity for said modified oligosaccharide with said binding partner.
24. The method of claim 1 , wherein said method further comprises determining the shortest oligosaccharide that can bind to a binding partner.
25. The method of claim 1 , wherein said method further comprises identifying or characterizing said binding partner.
26. The method of claim 1 , wherein said method further comprises screening a library of modified and/or unmodified oligosaccharides for binding to said binding partner.
27. The method of claim 1 , wherein said method further comprises identifying and/or characterizing an enzyme that modifies said unmodified oligosaccharide prior to contacting step (a).
28. The method of claim 1 , wherein said method further comprises synthesis of said oligosaccharides, wherein said synthesis is carried out prior to contacting step (a).