Removal of alpha-Gal Epitopes in Aortic Valve and Pericardium ofPig Using Green Coffee Bean alpha-Galactosidase

Park, Seongsik; Kim, Woong Han; Kim, Kyung Hwan; Lee, Chang Ha; Choi, Sun Young; Lee, Cheul; Oh, Sam Sae; Kim, Kwan Chang; Kim, Yong Jin

Abstract

BACKGROUND: It is currently thought that tissue valve degeneration is related to an animal's immune response, which is mainly due to cell surface alpha-Gal epitopes. Cell surface alpha-Gal epitopes are known to be degraded by the enzyme called green coffee bean alpha-galactosidase. It is also well known that alpha-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 alpha-Gal epitopes of the aortic valve and pericardial tissue of a pig can be removed by green coffee bean alpha-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 alpha-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 4degrees C and 24 hours of incubation, each sample was stained with Griffonia Simplicifolia isolectin type B4 immunofluorescent labeling. We then examined whether the alpha-Gal epitopes were reduced or abolished in each consecutive concentration of green coffee bean alpha-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 alpha-galactosidase at pH 6.5, 4degrees C and reaction for 24 hours was enough for complete removal of alpha-Gal epitopes from the cell surface on the immunostaining with Griffonia Simplicifolia isolectin B4. On the other hand, more alpha-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 alpha-Gal from the tissue. 2.0 units/mL of green coffee bean alpha-galactosidase was needed to completely remove the alpha-Gal epitopes from the pericardial tissue on immunostaining.
CONCLUSION
The alpha-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 alpha-Gal epitopes using green coffee bean alpha-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 alpha-Gal epitopes in the pericardial tissue of pig under the condition of pH 6.5, 4degrees C and 24 hours of reaction time. In the near future, removal of alpha-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 to be determined if alpha-galactosidase treated pig tissue is immune to human anti-Gal antibody or anti-Gal monoclonal antibodies.