METHODS FOR DETERMINATION OF HS EPITOPES THAT BIND ANTITHROMBIN III
Heparin and heparan sulfate (HS) are subclasses of a heterogeneous family of anionic glycans called glycosaminoglycans. Heparin and HS are made of the repeating disaccharide units HexAβ/α4GlcNα4. One of the most important features of heparin/HS is the ability to interact with a wide array of proteins. More than a hundred heparin binding proteins have been discovered and the number is increasing (1). These proteins are involved in a wide range of physiological processes including blood coagulation, cell proliferation, differentiation and migration, cell-cell adhesion, inflammation and pathogen invasion (2). Heparin and HS exert a regulatory role on these processes by modulating the activity of the proteins with which they interact. A growing body of evidence suggests that binding of proteins to heparins is not random but requires defined sequences within the heparin chain. Based on the knowledge of heparin structure whereby sulfo and carboxyl groups are arranged at defined intervals and in a specific orientation, the general paradigm is that a protein requires a minimal structure that displays optimal functional group arrangement for specific interactions. The specificity of these interactions can be restrictive, meaning that one or a few structures are able to bind the protein. Conversely, some proteins can exhibit more relaxed criteria such that different patterns of substitutions are tolerated (3).
Antithrombin III (ATIII) belongs to the serpin (serine protease inhibitors) superfamily of proteases (4). Heparin binding to ATIII induces a conformational change in this protein which results in the inhibition of downstream proteins (such as FXa) involved in the blood coagulation cascade. A pentasaccharide that has been sequenced is the minimal sequence required to bind ATIII, enhancing its inhibitory activity towards Factor Xa (5).
References:
1.Conrad, H. E. (1998) Heparin Binding Proteins, Academic Press,New York
2. Capila, I., and Linhardt, R. J. (2002) Heparin-protein interactions. Angew. Chem., Int. Ed. 41, 391–412.
3. Esko, J. D., and Selleck, S. B. (2002) Order out of chaos: Assembly of ligand binding sites in heparan sulfate. Annu. ReV. Biochem.71, 435–471.
4. Goldsmith, E. J., and Mottonen, J. (1994) Serpins: The uncut version. Structure 2, 241–244.
5. Lindahl, U., Thunberg, .L., Backstrom, G., Riesenfeld, J., Nordling, K., and Bjork, I. (1984) Extension and structural variability of the antithrombin-binding sequence in heparin. J. Biol. Chem. 259, 12368–12376.
