The Seeberger Lab

3.

Synthesis and Biochemical Studies of Glycosaminoglycans

Heparin-like glycosaminoglycans (HLGAGs) are the most acidic naturally occurring biopolymers. These complex polysaccharides, found in the extracellular matrix, play a key role in regulating the biological activity of several proteins in the coagulation cascade along with many other processes of biomedical importance including growth factor interactions, virus entry, and angiogenesis.

The relationship between structure and function of HLGAGs is still very poorly understood due to the complexity and heterogeneity of these polymers. It has become increasingly evident that defined lengths and sequences of HLGAGs are responsible for binding to a particular protein and modulating its biological activity. Defined HLGAG oligosaccharides would constitute valuable molecular tools to gain a detailed understanding of the structure-activity relationship of heparin. Currently available synthetic methods for the preparation of HLGAGs are extremely lengthy and lack generality.

We established a framework for the synthesis of any desired HLGAG sequence by developing a modular, general synthetic strategy for the preparation of heparin-like glycosaminoglycans. In the context of this synthesis we have devised completely new routes for the synthesis of uronic acid4 and differentially protected glucosamine building blocks. A novel method for controlling the stereoselectivity of glycosylation reactions by conformational locking of the glycosyl acceptor was introduced to selectively install a-glucosamine linkages. This transformation was the key in preparing a set of disaccharide modules that were utilized in the synthesis of heparin oligosaccharides. Using the modular assembly approach defined HLGAG tetra- and hexasaccharides related to the anti-thrombin III binding sequence were prepared. Currently, the modular protocol is applied to the solid-phase synthesis of a defined dodecasaccharide sequence recognized by specific growth factors using disaccharide building blocks.

Orgueira, H.A.; Bartolozzi, A.; Schell, P.H.; Seeberger, P.H.; Stereocontrol of Glycosylation Reactions by Conformational Locking of the Glycosyl Acceptor in Disaccharide Formation; Angew. Chem. Int. Ed. 2002, 41, 2128-2131.

3-O-Sulfates are the rarest substituent of heparan sulfate and are ideally suited to selectively regulate biologic activities. Individual isoforms of heparan sulfate D-glucosaminyl 3-O-sulfotransferase (3-OST) exhibit sequence specific action, which creates heparan structures with distinct biologic functions. For example, 3-OST-1 preferentially generates antithrombin-binding sites; whereas, 3-OST-3 isoforms create binding sites for the gD envelope protein of herpes simplex virus 1 (HSV-1), which enables viral entry. 3-OST enzymes are comprised of a presumptive sulfotransferase domain and a divergent amino-terminal region. To localize determinants of sequence specificity, we conducted domain swaps between cDNAs. We established that the sequence specific properties of 3-OSTs are defined by a self-contained sulfotransferase domain, and are not directly influenced by the divergent amino-terminal region.

Currently we are in the process of cloning 3-OST-5, an isoform identified from the human genome sequence. Tissue and substrate specificity will be determined upon overexpression of this protein. Determination of the substrate specificities of different isoforms of 3-O-sulfotransferases is ongoing by using defined HLGAG oligosaccharide sequences.

Yabe, T.; Shukla, D.; Spears, P.; Rosenberg, R.D.; Seeberger, P.H.; Shworak, N.W. A Portable Sulfotransferase Domain Determines Sequence Specificity of Heparan Sulfate 3-O-Sulfotransferases., Biochem. J. 2001, 359, 235-241.