Heparin is a highly sulfated carbohydrate produced by mast cells that is widely used as an anticoagulant to prevent deep vein thrombosis, pulmonary embolism and arterial thromboembolism. Heparan sulfate (HS) is structurally related to heparin and decorates the surface of all most all human cells. It interacts with a multitude of proteins including chemokines and cytokines; blood coagulation factors; growth factors and morphogens, proteins involved in complement activation, and cell adhesion and signaling proteins. HS has been implicated in human diseases such as vascular diseases, inflammation, infections and neurodegeneration. It has been postulated that HS encodes information by an ability to recruit proteins in a context-dependent manner. Although the ?HS-sulfate code? hypothesis is conceptually appealing, little is known about the ligand specificities of HS binding proteins and the substrate specificities of HS-biosynthetic enzymes. As a result, it is not known whether isoforms of HS biosynthetic enzymes can create distinctive epitopes that in a cellular context can recruit specific HS- binding proteins. An understanding of the specificities of HS binding also offers opportunities to develop therapeutic modalities. In this program, chemical and chemo-enzymatic methodologies will be developed that will make it possible to prepare a focused library of biologically relevant HS epitopes with and without a 3-O-sulfate moiety. These compounds will be used to develop an HS-microarray to establish ligand requirement of HS-binding proteins. Compounds of interest will be further explored for their ability to interfere in the binding and cellular activation of endothelial cells. We will focus on HS-binding proteins involved in blood coagulation, inflammation and vascular disease. The established structure-activity relationships are expected to provide therapeutic lead compounds. The HS-microarray will also be used as a general platform to discover and characterize HS-binding proteins requiring a 3-O-sulfate. In addition, the synthetic compounds will be used to define substrate requirements of the various 3-O-sulfotransferases. Collectively, our studies will address the question of whether isoforms of 3-O-sulfotransferases can create distinctive epitopes that can recruit specific HS-binding proteins, to mediate various biological processes. 1
Chemical and chemoenzymatic methods will be developed that can provide a library of heparan sulfate molecules with or without a 3-O-sulfate. The focused library will be used (i) to establish ligand requirements of a range of HS-binding proteins; (ii) to engineer cells with specific synthetic HS oligosaccharides to determine biological function; and (iii) to define substrate requirements of 3-O- sulfotransferase isozymes. The results will answer the question whether, in a cellular context, isoforms of HS-biosynthetic enzymes can create unique epitopes that can recruit specific HS-binding protein, and may provide therapeutic lead for inflammatory and vascular diseases. 1
