title: alpha-galactosyl (Galalpha1-3Galbeta1-4GlcNAc-R) epitopes on human cells: synthesis of the epitope on human red cells by recombinant primate alpha1,3galactosyltransferase expressed in E.coli.
abstract: Developing methods for in vitro synthesis of the carbohydrate structure Galalpha1-3Galbeta1-4GlcNAc-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 alpha-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 alpha1,3galactosyltransferase (rec. alpha1,3GT) expressed in bacteria. Escherichia coli was transformed with cDNA of the luminal portion of New World monkey rec. alpha1,3GT 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. alpha1,3GT produced in COS cells. Red cells were pre-treated with sialidase for exposure of N-acetyllactosamine acceptors, then subjected to rec. alpha1,3GT 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.
authors.affiliation: Department of Microbiology and Immunology, The Medical College of Pennsylvania, Philadelphia, USA.
authors.forename: U
authors.initials: U
authors.lastname: Galili
authors.forename: F
authors.initials: F
authors.lastname: Anaraki
mesh_terms: D000818:Animals; D001483:Base Sequence; D002144:Callithrix; D002240:Carbohydrate Sequence; D004912:Erythrocytes / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D005690:Galactose / Q000276:immunology; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D037102:Lectins / Q000378:metabolism; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000276:immunology; D037121:Plant Lectins; D011994:Recombinant Proteins / Q000378:metabolism; D014312:Trisaccharides / Q000096:biosynthesis* / Q000276:immunology; D014612:Vaccines
chemical_list: C045475:Griffonia simplicifolia lectins; D037102:Lectins; D037121:Plant Lectins; D011994:Recombinant Proteins; D014312:Trisaccharides; D014612:Vaccines; C076569:alpha-galactosyl epitope; D005700:Galactosyltransferases; C037959:beta-D-galactosyl-N-acetylglucosaminylglycopeptide alpha-1,3-galactosyltransferase; D005690:Galactose