A new strategy for defining critical functional groups on heparan sulfate.
Heparan sulfate (HS) is a sulfated polysaccharide present on cell surfaces and in the extracellular matrix. Accumulating evidence shows that HS plays key roles in many biological systems by interacting with various proteins in a structural-specific manner. Due to technical difficulties, however, the understanding of critical functional groups on HS for protein interaction is vague. We report a rapid, convenient, sensitive, and inexpensive strategy using in vitro modification with pure enzymes and gel mobility shift assay to study the subject. We demonstrated the requirements of 3-O, 6-O sulfates and the minimal length of oligosaccharide for antithrombin III (AT-III) binding. We regenerated the binding sites for AT-III on completely desulfated N-resulfated heparin and revealed the critical modification enzymes. This new strategy could be used to identify critical functional groups on HS and to generate HS library and biologically active HS, providing information applicable to the design of HS drugs, such as anticoagulant reagents and viral infection blockers. The binding assay with fibroblast growth factors and receptors confirmed the general usefulness of this approach.
Wu
Zhengliang L
ZL
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Zhang
Lijuan
L
Beeler
David L
DL
Kuberan
B
B
Rosenberg
Robert D
RD
D000818:Animals; D000990:Antithrombin III / Q000378:metabolism; D001665:Binding Sites; D019556:COS Cells; D002240:Carbohydrate Sequence; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D005346:Fibroblast Growth Factors / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry* / Q000502:physiology*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D015238:Sulfotransferases / Q000378:metabolism
D000990:Antithrombin III; D005346:Fibroblast Growth Factors; D006497:Heparan Sulfate; D009844:Oligosaccharides; D017468:Receptors, Fibroblast Growth Factor; D015238:Sulfotransferases