Gong Feng

Properties of a Novel α-galactosidase from B.Fragilis and Its Potential for Human Blood-type B to O Conversion

Enzymatic removal of blood group B antigen is an effective method to develop universal red blood cells (RBCs) by α-galactosidase. Here we investigated the physicochemical properties of a novel α-galactosidase from B. Fragilis and the optimization of enzymatic conversion for RBC of blood group B to O. The results showed that α-galactosidase exhibited maximum activity at a broad optimal pH of 5.6-6.0 and an optimum temperature of 41°C. Furthermore, the enzyme was efficient in complete removal of B antigen. The conditions for B to O blood group conversion were 26°C, pH 6.8 (250 mmol/L glycine and 3 mmol/L NaCl) for 1 h. The structure and function indexes of the converted red blood cells showed no significant difference from those of normal RBCs. Consequently, the novel α-galactosidase described here was more suitable for enzymatic conversion for achieving the goal of producing universal RBCs, which would improve the blood supply while enhancing the safety of clinical transfusions.

Down-regulation of αGal epitopes by co-transfection of α1,3-galactosidase gene and α1,2-fucosyltransferase gene

The polycarbohydrate structure of Galα1-3Galβ1-4GluNAc-R (known as αGal epitopes of xenoantigen), produced by α1-3-galactosyltransferase (α1,3-GT) in the course of animal development, is the major xenoantigen on the cell surface of porcine which causes hyperacute rejection in pig-to-human xenotransplantation. Alpha-1,3-galactosidase (AGL), a hydrolytic enzyme, can remove the terminal α-1,3-galactosyl from the Galα1-3Galβ1-4GluNAc-R structure resulting in cleaning αGal epitopes from the porcine cells. Alpha-1,2-fucosyltransferase (HT) can modify the surface carbohydrate phenotype of porcine cells, bringing about reduction of αGal epitopes expression. In this study, human AGL and HT gene were co-transfected to porcine fetal fibro-blast (PFFb) in equimolar concentration to reduce the xenoantigen. Gene and protein of hAGL and HT were both detected to express at high level by RT-PCR and Western blot, respectively. There was an 84% reduction in αGal xenoantigen and an 82% increase in H antigen as assayed by flow cytometry in the AGL and HT gene co-transfected PFFb. The number and morphology of transgenic PFFb chromosome were normal. Findings indicate that Galα1-3Gal epitopes of PFFb could be down regulated byAGL and HT co-transfection without deleterious effects on the chromosomal profile of the transgenic cell.