The major xenoactive antigens responsible for hyperacute rejection have recently been identified as carbohydrate structures containing terminal alpha Gal1-3Gal sequence (alpha-Gal epitopes). The most common alpha-Gal epitopes are disaccharide alpha Gal1-3Gal1 (1), trisaccharides alphaGal1-3betaGal1-4betaGlcNAc (2) and alphaGal1-3betaGal1-4betaGlc (3), and pentasaccharide alphaGal1-3betaGal1-4betaGlcNAc1-3betaGal1-4betaGlc (4). Alpha-Gal epitopes are abundantly expressed on the cells of most mammals, with the exception of humans, apes and Old World monkeys. Conversely, the natural antibody with specificity to alpha-Gal epitope (anti-Gal) exists only in humans and other Old World primates. The discovery of the interaction of alpha-Gal and anti-Gal has led to experimental attempts to overcome hyperacute rejection by either depleting the recipient's anti-Gal through an alpha-Gal containing affinity column (anti-Gal immunoadsorption approach) or antagonizing anti-Gal by infusing soluble synthetic alpha-Gal oligosaccharides (anti-Gal neutralization approach). Moreover, alpha-Gal-antigen conjugates were shown to enhance antigen presentation by the natural human anti-Gal antibody, and alpha-Gal-immunotoxin glycoconjugates are being used to target B cells which generate anti-Gal antibody. All these biomedical applications require access to a substantial amount of alpha-Gal oligosaccharides as well as synthetically derived alpha-Gal analogs and mimetics with high-affinity to anti-Gal antibodies. Thus, this research program is aimed at development of synthetic technology for large-scale production of alpha-Gal oligosaccharides and at searching for potent alpha-Gal mimetics. 1) Improvement on the synthesis of alpha-Gal epitopes and its multivalent derivatives. The third necessary glycosyltranferase, beta1,3 GlcNAc transferase, will be cloned and overexpressed. Then a one-pot enzymatic system will be developed to synthesize 4 with in situ regeneration of sugar nucleotides UDP-Gal and UDP-GlcNAc through multiple enzyme cycles. To further explore the polyvalent effect of alpha-Gal epitope, structurally defined alpha-Gal oligomers and alpha-Gal dendrimers will be synthesized. The binding of these alpha-Gal derivatives to human anti-Gal will be measured by competition ELISA, flow cytometry, and immunohischemistry assays. 2) Structural studies on alpha-Gal / anti-Gal interaction. Uniformly C-13 labeled alpha-Gal trisaccharide alphaGal1-3betaGal1-4betaGlc, five conformationally constrained alpha-Gal analogs and seven monodeoxy alpha-Gal disaccharide derivatives will be synthesized and used in NMR experiments to determine the active conformation of alpha-Gal bound to monoclonal anti-Gal antibody, and to study the flexibility and controlled rigidity of alpha-Gal epitopes. 3) Alpha-Gal mimetic library and high-throughput screening. A new technology platform (from split synthesis to flow cytometry screening and to MS structural analysis) will be established to generate and screen a large number (greater than ten to the six power) of glycopeptides to identify lead alpha-Gal mimetic sequences. Both natural and unnatural amino acids will be used to construct two combinatorial libraries for screening against different phenotype of anti-Gal for consensus binding structures. In summary, success of this research program will make alpha-Gal oligosaccharides easily accessible, will provide fundamental data on alpha-Gal / anti-Gal interaction and will discover lead potent alpha-Gal mimetic structures that are useful as immunodiagnostic agents, carbohydrate therapeutics, or vaccines.
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