Suppression of [alpha]1,3-galactosyl epitopes on mammalian cells
Authors:   , Langell John Thomas 
Published: July 2024

Xenotransplantation, the transplantation of tissues between two distinct species, offers a potentially endless supply of donor tissues for the treatment of end-stage organ failure. Unfortunately, discordant xenogeneic organs are hyperacutely rejected by natural antibodies present in all immunocompetent humans. The alpha-1,3-galactosyl ([alpha]1,3Gal) carbohydrate is the main antigen bound by these antibodies. We have attempted to suppress [alpha]1,3Gal expression through two approaches, focusing on separate points in the glycosynthetic pathway. First, we developed a model system designed to suppress [alpha]1,3Gal by cleaving it at or distal to its production, allowing for possible sialic acid capping in the Trans Golgi Network (TGN). To accomplish this, we produced a fusion protein composed of the enzymatic region of coffee bean alpha-1,3-galactosidase linked to an NH2-terminal TGN-targeting peptide derived from rat 2,6-sialyltransferase, and generated NIH-3T3 stable transfectants positive for both expression and activity of the fusion protein. Flow cytometric analysis of these cells using IB4 and L. polyphemus lectins showed a >25% decrease in surface [alpha]1,3Gal levels and a 100% increase in sialic acid. Binding of human natural antibody to these cells is decreased by as much as 55% and complement-mediated cytotoxicity by >20% in the presence of human serum. Our second approach attempted to inhibit the expression of [alpha]1,3Gal on primary porcine aortic endothelial cells (PAEC) using mycophenolic acid (MPA), or 3-azido-3-deoxythymidine (AZT) in vitro. MPA and AZT suppress purine sugar and pyrimidine sugar substrate in glycosynthesis, respectively. PAEC were cultured in the presence of MPA or AZT, and then analyzed to establish the effect of these drugs on cell viability and proliferative potential in culture. These cells were then incubated with IB4 or L. polyphemus lectins and analyzed for [alpha]1,3Gal and sialic acid expression by flow cytometry. Cells were then incubated in human serum and analyzed for natural antibody binding. We found no significant decrease in cell viability at the drug concentrations tested as compared to controls. Although none of the AZT concentrations used inhibited cell expansion, MPA significantly affected expansion at levels above 1[mu]M with complete inhibition at 10[mu]M. MPA and AZT significantly reduced the surface expression of [alpha]1,3Gal and increased surface expression of sialic acid in a dose dependent fashion. Maximum reduction in expression of [alpha]1,3Gal occurred at 10[mu]M MPA(78%) and 10[mu]M AZT(22%) and was accompanied by an increase in sialic acid expression of 62% and 54%, respectively. AZT treated cells also showed a decrease in human antibody binding levels while MPA treated cells exhibited almost twice the binding levels of untreated cells. From this data we conclude that both transgenic and pharmacologic approaches significantly alter the cell surface levels of [alpha]1,3Gal and sialic acid terminal carbohydrates on porcine endothelia, therefore these strategies may prove useful in conditioning porcine tissues for human transplantation.