Molecular cloning of endo-beta -galactosidase C and its application in removing alpha -galactosyl xenoantigen from blood vessels in the pig kidney.

Abstract Galα1–3Gal is the major xenoantigenic epitope responsible for hyperacute rejection upon pig to human xenotransplantation. Endo-β-galactosidase C from Clostridium perfringens destroys the antigenic epitope by cleaving the β-galactosidic linkage in the Galα1–3Galβ1–4GlcNAc structure. Based on partial peptide sequences of the enzyme, we molecularly cloned the enzyme gene, which encodes a protein with a predicted molecular mass of about 93 kDa. The deduced protein sequence of the enzyme has limited homology in the C-terminal half with endo-β-galactosidase from Flavobacterium keratolyticus and β-1,3-glucanases. The enzyme expressed in Escherichia coli removed the α-galactosyl epitope nearly completely from pig erythrocytes and from pig aortic endothelial cells. The enzyme-treated endothelial cells in culture were greatly reduced in cell surface antigens, which were recognized by IgM, IgG, or IgA in human sera, and became much less susceptible to complement-mediated cytotoxicity caused by human sera. When the pig kidney was perfused with the enzyme, the vascular endothelial cells became virtually devoid of the α-galactosyl epitope, with concomitant decrease in binding to IgM in human plasma. These results demonstrated that the recombinant endo-β-galactosidase C is a valuable aid in xenotransplantation.