This project is intended to determine the feasibility of converting human blood type A and type B erythrocytes to universal donor blood type O erythrocytes by treatment with blood group-specific glycosidases purified from the cell-free culture supernatants of 2 commensal, non-pathogenic strains of human enteric bacteria. The ultimate goal is to provide a safe and cost-effective means of converting erythrocytes from blood type A and B donors, which tend to accumulate in excess of demand, to blood type O erythrocytes, whose demand frequently exceeds supply. The rationale is based upon the fact that human blood type A and type B antigen specificities are conferred by single sugar glycosides: alpha-N-acetyl-D-galactosamine and alpha-D-galactose, respectively, at the outer, non-reducing end of oligosaccharide chains of membrane glycoproteins and glycolipids which confer blood type O(H) antigen specificity to the cell membranes of type O red cells. Cleavage of the linkage between either of these sugars and the underlying O(H) antigenic structure changes the antigen specificity from blood type A or B to O. Cleavage requires 2 glycosidases, one specific for the type A linkage (an alpha-N-acetylgalactosaminidase), and one for the type B linkage (an alpha-D-galactosaminidase). The investigator has isolated 2 strains of fecal bacteria, R. torques strain IX-70 and R. gnavus strain VI-268, each of which produces one of these glycosidases as an extracellular enzyme. The blood group A-degrading enzyme has been purified very nearly to homogeneity while the B-degrading enzyme appears close to being purified.
The specific aims of this project are: 1) to complete the purification of the B-degrading alpha-(1-3)-D-galactosidase and to fully characterize the substrate specificities of both glycosidases; 2) to characterize the actions of each purified glycosidase on blood type A or B erythrocytes with respect to alterations of membrane glycoconjugates, membrane blood group antigens, membrane integrity, and the ability of enzyme treatment to alter binding of complement-activating antibodies to the cell membrane; 3) if warranted by the results in Aim 2, to measure the survival of enzyme-treated cells labelled with 51-Cr in the circulation of a small number of adult volunteers; and 4) to clone the genome of each glycosidase into recombinant DNA for large scale production of each purified enzyme.
| Hoskins, L C; Boulding, E T; Larson, G (1997) Purification and characterization of blood group A-degrading isoforms of alpha-N-acetylgalactosaminidase from Ruminococcus torques strain IX-70. J Biol Chem 272:7932-9 |
| Hoskins, L C; Larson, G; Naff, G B (1995) Blood group A immunodeterminants on human red cells differ in biologic activity and sensitivity to alpha-N-acetylgalactosaminidase. Transfusion 35:813-21 |
| Falk, P; Hoskins, L C; Lindstedt, R et al. (1991) Deantigenation of human erythrocytes by bacterial glycosidases--evidence for the noninvolvement of medium-sized glycosphingolipids in the Dolichos biflorus lectin hemagglutination. Arch Biochem Biophys 290:312-9 |
