Multivalent Galalpha1,3Gal-substitution makes recombinant mucin-immunoglobulins efficient absorbers of anti-pig antibodies.

Hyperacute organ xenograft rejection can be prevented by removing anti-pig antibodies by extracorporeal absorption prior to transplantation. A novel recombinant absorber of anti-pig antibodies was developed by fusing the cDNA encoding the extracellular part of a mucin-type protein, P-selectin glycoprotein ligand-1, with an antibody Fc fragment cDNA, which upon coexpression with the porcine alpha1,3 galactosyltransferase carried the xenogeneic epitope, Galalpha1,3Gal (Liu J., Qian Y., Holgersson J., Transplantation 1997, 63, 1673-1682). The biochemical characterization of the mucin/Ig and its absorption efficacy compared with that of porcine thyroglobulin and Galalpha1,3Gal-conjugated beads are reported. The carbohydrate portion of the mucin/Ig constituted 43% of its molecular weight and the majority of the Galalpha1,3Gal epitopes were O-linked as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting following N-glycosidase F digestion. Gas chromatography-mass spectrometry of reduced and acetylated saccharides released by alpha-galactosidase treatment revealed that the fusion protein carried approximately 140 mol of terminal, alpha-linked galactose per mole protein. Based on the reduction in pig aortic endothelial cell cytotoxicity, Galalpha1,3Gal-substituted mucin/Igs on agarose beads were, on a carbohydrate molar basis, shown to be approximately 20 times more efficient than agarose-conjugated pig thyroglobulin, and approximately 5000 and 30,000 times more efficient than Galalpha1,3Gal-substituted agarose and macroporous glass beads, respectively. Structural features of the mucin backbone and its carbohydrate core saccharide chains determine the structural context, spatial orientation and spacing of Galalpha1,3Gal epitopes and are likely to explain the superior absorption efficacy of the recombinant mucin-type chimera.