Complex carbohydrate moieties play an important role in many ligand/receptor binding events. For example, P-selectin ligand (see W095/30001) found on the surface of neutrophils contains sialyl Lewis x (sLe.sub.x) moieties which are essential to binding of the ligand to its receptor, P-selectin, on the surface of vascular endothelium. The interaction of P-selectinligand and P-selectin is involved in the development of inflammatory responses, including both those which produce desired immune responses and those which cause disease states. As a result, many complex carbohydrate moieties, including sLe.sub.x, are currently under investigation for use as therapeutics to interfere with detrimental ligand/receptor binding events.
Complex carbohydrates are usually synthesized with the aid of enzymes which can convert more easily obtainable starting materials and intermediates into necessary moieties which are more difficult to produce by non-enzymatic chemical synthesis. For example, sLe.sub.x contains an essential fucose moiety which can be added enzymatically starting with GDP-fucose, which is readily obtained from GDP-mannose using the appropriate enzymes. As a result, the identification and isolation of a wide variety of enzymes for such purpose has been a continuing goal for those interested in carbohydrate production. In particular, the identification of enzymes capable of coverting GDP-mannose to GDP-fucose has been sought to aid in the development of production methods for sLe.sub.x and other fucose- containing carbohydrates.
One route of synthesis which is used in the productionof fucose-containing carbohydrates is through the conversion of GDP-mannose to GDP-fucose, which can then be used to incorporate the fucose moiety into the synthetic product. This conversion involves multiple steps, each of which can be catalyzed by an appropriate enzyme. The first step in this process is the conversion of GDP-mannose to GDP-4-keto-6-deoxy-mannose. In vivo this conversion is made by the action of an GDP-mannose 4,6-dehydratase.
A GDP-mannose dehydratase enzyme has been cloned and isolated from E. coli (GenBank accession number U38473). However, the enzymes from different species may have different in vivo and in vitro characteristics and activities, both beneficial and detrimental, which may affect their relative usefulness for production of complex carbohydrates. Nevertheless, no human dehydratase enzyme has yet been identified.
Certain disease states have also been associated with defects in the action of such dehydratases. For example, the human disease leukocyte adhesion deficiency II (LADII) may be due to a defect in the dehydratase enzyme. The correlation of other dehydratase enzymes with disease states will allow the examination of therapies for such conditions.
Since these dehydratase enzymes are also responsible for the in vivo production of fucose-containing carbohydrate moieties, blocking their activity through the use of antagonists or other means can provide another route for interruption of unwanted inflammatory responses and other conditions dependent upon the production of fucose-containing carbohydrate moieties. Isolated dehydratases can be used as a target to screen for inhibitors of enzyme activity which can be used for this purpose.
Therefore, it would be desirable to identify, clone and isolate additional dehydratase enzymes to expand the panoply of enzymes available to the carbohydrate synthetist. It would also be desirable to identify additional dehydratase which are associated with certain disease states to allow the examination and development of treatments for such conditions. It would also be deisrable to identify and isolate additional human dehydratase enzymes which can serve as targets for screening inhibitors.
USPTO Backgrounds
USPTO Backgrounds