alpha-Galactosyl (Ga1 alpha 1-3Gal beta 1-4GlcNAc-R) epitopes on human cells: Synthesis of the epitope on human red cells by recombinant primate alpha 1,3galactosyltransferase expressed in E-coli

Developing methods for in vitro synthesis of the carbohydrate structure Gal alpha 1-3Gal beta 1-4GlcNAc-R (termed the alpha-galactosyl epitope) on human tumour cells may be of potential clinical significance in cancer immunotherapy, Tumour vaccines with this epitope would be opsonized in vivo by the natural anti-Gal antibody, which is present in large amounts in humans, and which interacts specifically with a-galactosyl epitopes. Binding of anti-Gal to alpha-galactosyl epitopes on tumour cell membranes is likely to increase uptake of the cell membranes by antigen-presenting cells, such as macrophages, via the adhesion of the Fc portion of anti-Gal to Fc receptors on these cells, This, in turn, may increase processing and presentation of tumour-associated antigens by antigen-presenting cells, and induce an effective immune response against tumour cells with these antigens, The present study describes a method for the synthesis of alpha-galactosyl epitopes on human cells (red cells used as a model) by recombinant alpha-1,3galactosyltransferase (rec. alpha 1,3GT) expressed in bacteria, Escherichia coli was transformed with cDNA of the luminal portion of New World monkey rec, alpha 1,3GT linked to six histidines (His)(6) at the N-terminus, The enzyme produced by the bacteria was isolated from bacterial lysates on a nickel-Sepharose column and eluted with imidazole, This recombinant enzyme displayed acceptor specificity similar to that of rec. alpha 1,3GT produced in COS cells, Red cells were pre-treated with sialidase for exposure of N-acetyllactosamine accepters, then subjected to rec, alpha 1,3GT activity, This enzyme synthesized at least 4 x 10(4) alpha-galactosyl epitopes/red cell. These epitopes were found to be accessible for binding of anti-Gal, as well as Bandeiraea simplicifolia IB4 lectin, It is argued that the method presented can be used for the synthesis of alpha-galactosyl epitopes on membranes of autologous tumour vaccines in humans.