| Glycosaminoglycans (GAGS), long polysaccharide chains composed of repeating disaccharide units containing a hexosamine, and various binding proteins play key roles throughout the vertebrate body such as mediating and/or regulating adhesion, signaling, coagulation and inflammation. The extracellular matrix and cell surfaces contain various GAGS such as hyaluronan (HA) and chondroitin. Multiple GAG/protein interactions occur, but their exact binding specificities remain unknown.; The HA synthase, PmHAS, and the chondroitin synthase, PmCS, from the Gram-negative bacterium Pasteurella multocida, polymerize the glycosaminoglycan (GAG) sugar chains HA or chondroitin, respectively. We have shown that oligosaccharides and polysaccharides of certain non-cognate GAGS (including sulfated and iduronic acid-containing forms) are elongated by PmHAS (e.g., chondroitin elongated by PmHAS) or PmCS.; In vivo, the bacterial enzymes only process unsulfated polymers, thus it is not expected that the PmCS and PmHAS catalysts would exhibit such relative relaxed sugar specificity by acting on a variety of animal-derived sulfated or epimerized GAGs. However, this feature allows the chemoenzymatic synthesis of a variety of chimeric GAG polymers, including mimics of proteoglycan complexes.; We have been determining binding specificity of oligosaccharides utilizing solid-phase GAG oligosaccharide libraries made by in situ solid-phase or liquid-phase chemoenzymatic synthesis. Short sugar chains composed of hyaluronan, chondroitin or combinations of both GAGs (5-20 monosaccharides in length) were synthesized in either a stepwise fashion in a 96-well plate format using the dual-action transferase Pasteurella catalysts, PmHAS and PmCS, or in liquid-phase utilizing mutant Pasteurella catalysts with the capability of only single-sugar transferase activity. These enzymes enabled us to create a unique library of hybrid oligosaccharides varying in both composition and length (e.g. HA4-C 2, C4-HA2, C7, HA13). These oligosacchandes were probed with various proteins including cartilage-derived aggrecan fragments, and inflammation molecule, tumor necrosis factor stimulated gene-6 (TSG-6), and a cell surface receptor for HA, CD44.; Our results indicate that GAG binding specificity of the various proteins depends on the length and the composition of sugar chains. Therefore, the development of more potent and/or selective ligands for the binding proteins should be possible. Unnatural oligosaccharides and oligosaccharides of specific length should be beneficial for future medical used such as tissue engineering (e.g., biomaterials for construction of scaffolds for possible bone regeneration and repair) and novel therapeutics (e.g., anticancer agents and angiogenesis modulators and drug targeting agents), respectively. |
