Removal of α-Gal Epitopes in Aortic Valve and Pericardium of Pig Using Green Coffee Bean α-Galactosidase

Background: It is currently thought that tissue valve degeneration is related to an animal's immune response, which is mainly due to cell surface α-Gal epitopes. Cell surface α-Gal epitopes are known to be degraded by the enzyme called green coffee bean α-galactosidase. It is also well known that α-Gal epitopes are immunologically stained by Griffonia Simplicifolia isolectin type B4. We know that many commercially available tissue valves are made of aortic valves and pericardial tissue of pig. So, we investigated whether α-Gal epitopes of the aortic valve and pericardial tissue of a pig can be removed by green coffee bean α-galactosidase, and we did so by comparing immunologic staining of the tissues before and after the enzyme treatment. Material and method: After treating fresh porcine aortic valve and pericardial tissue with green coffee bean α-galactosidase at concentrations of 0.5 unit/mL, 1.0 unit/mL, 2.0 unit/mL, respectively, under the condition of pH 6.5, temperature 4 o C and 24 hours of incubation, each sample was stained with Griffonia Simplicifolia isolectin type B4 immunofluorescent labeling. We then examined whether the α-Gal epitopes were reduced or abolished in each consecutive concentration of green coffee bean α-galactosidase by comparing the degree of the Griffonia Simplicifolia isolectin B4 staining in each sample. Result: In the pig aortic valve tissue, a 1.0 unit/mL concentration of green coffee bean α-galactosidase at pH 6.5, 4 o C and reaction for 24 hours was enough for complete removal of α-Gal epitopes from the cell surface on the immunostaining with Griffonia Simplicifolia isolectin B4. On the other hand, more α-Gal epitopes were present in the pig pericardial tissue on Griffonia Simplicifolia isolectin B4 staining before the enzyme treatment, and 1.0 unit/mL of galactosidase was not sufficient for complete removal of α-Gal from the tissue. 2.0 units/mL of green coffee bean α-galactosidase was needed to completely remove the α-Gal epitopes from the pericardial tissue on immunostaining. Conclusion: The α-Gal epitopes of the pig's aortic valve and pericardial tissue were successfully stained with Griffonia Simplicifolia isolectin B4. We could remove nearly all the α-Gal epitopes using green coffee bean α-galactosidase at the concentration of 1.0 unit/mL in the aortic valve of pig, and 2.0 unit/mL was need to nearly completely remove all the α-Gal epitopes in the pericardial tissue of pig under the condition of pH 6.5, 4 o C and 24 hours of reaction time. In the near future, removal of α-Gal epitopes in the pig's aortic valve and pericardial tissue will become a powerful tool for the improvement of the tissue valve durability. It needs