Suppression of the ??-1,3-Galactosyl Epitope by Golgi Expression of a Coffee Bean ??-1,3-Galactosidase Transgene

Abstract

725 The α-1,3-galactosyl epitope (α-1,3Gal) is thought to be the main target antigen for human natural antibodies to porcine xenografts. Several methods have been employed to modify α-1,3Gal expression and have been partially successful in reducing the levels of α-1,3Gal as measured by IB4 lectin binding. However, antibody/complement-mediated cytotoxicity remains unchanged or increased in these models. Discrepancies between α-1,3Gal expression and functional antibody activity may be attributed to the currently unpredictable, tissue specific changes in Golgi glycosylation. To help elucidate this problem, we have developed a model designed to suppress α-1,3Gal in the late stage of Golgi glycosylation. Our aim was to cleave the α-1,3Gal epitope at or distal to its production in the Golgi, allowing for increased sialic acid capping in the Trans Golgi Network (TGN). Sialic acid has been shown to confer resistance to complement-mediated lysis of pathogens and mammalian cells. A fusion protein composed of the enzymatic region of the coffee bean α-1,3-galactosidase gene linked to an amino terminal TGN targeting peptide derived from the rat 2,6-sialyltransferase was designed and produced. Next, stable NIH-3T3 transfectants positive for both expression and activity of the fusion protein were generated. Flow analysis of these cells using IB4 and L. polyphemus lectins demonstrated a >30% decrease in surfaceα-1,3Gal levels and a 100% increase in sialic acid expression. Although binding of xenoreactive natural antibodies to these cells remained unaltered, a >10% decrease in complement-mediated cytotoxicity as measure by propidium iodide exclusion was noted. From these data, we conclude that TGN expression of the galactosidase fusion protein was able to partially suppressα-1,3Gal, causing a shift in surface carbohydrate expression. More importantly, expression of the coffee bean α-1,3-Galactosidase transgene resulted in an increased resistance to complement mediated lysis not seen in previous transgenic models.