Glycosyltransferases catalyze the synthesis of specific oligosaccharide structures on cells and extracellular molecules that have crucial roles in biological recognition processes in normal and pathological states; some also act as membrane-bound carbohydrate receptors and others are important in the regulation of development. However, there is little information available on the structures, interactions, and mechanisms of these important enzymes. Two galactosyltransferases will be investigated that catalyze reactions in which the configuration of the transferred monosaccharide is retained and inverted, respectively. However, shared sequence motifs suggest that, at least parts of these enzymes may be distantly related. Alpha- Galactosyltransferase catalyze the synthesis of a galactosyl- alpha-1,3-galactosyl-beta-OR structure; this enzyme is inactivated in humans and about 1 percent of endogenous antibodies are directed against the product of its action, the alpha-gal epitope, and represent a major barrier to the xenotransplantation of organs. The mechanism of this enzyme and its structural basis will be investigated by kinetic, mutational and collaborative X-ray diffraction analysis of available crystals that diffract to 2.0 Angstrom units resolution. The results will provide information about the basis of specificity in homologous enzymes that catalyze the synthesis of blood group A and B glycans and Forrssman glycolipid. Beta-4- Galactosyltransferase-1, is one of six homologous enzymes in humans that catalyze the transfer of beta-galactose to N- acetylglucosamine or glucose in glycoproteins and glycolipids. The binding of alpha-lactalbumin, a mammary specific protein that is homologous with lysozyme, modulates the specificity of the enzyme to allow it to catalyze lactose biosynthesis (galactose transfer to glucose) during lactation. Structural and mutational approaches will be used to investigate the basis of regulation through this protein-protein interaction. A new homologue of alpha-lactalbumin and lysozyme has been identified as a testis- expressed cDNA. This lacks the lysozyme catalytic site and has some similarities to alpha-lactalbumin, raising the possibility that it could be a novel glycosyltransferase regulatory protein. The activity and structure will be investigated using a recombinant form of this protein.
