Multivalent Galα1,3Gal‐substitution makes recombinant mucin–immunoglobulins efficient absorbers of anti‐pig antibodies

Abstract

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.

Human Gastric Mucins Differently Regulate Helicobacter pylori Proliferation, Gene Expression and Interactions with Host Cells

“…The BabA ligand Le b but not the SabA ligand sialyl-Le x was able to induce a small, albeit statistically significant increase in H. pylori proliferation. The lack of large effects on H. pylori proliferation from glycoconjugates and recombinant glycoproteins could depend on glycocontext and presentation of the ligands, and that the carbohydrate content is much lower; 20–30% of the total molecular weight for HSA-glycoconjugates and around 40% for recombinant glycoproteins [32], compared to around 80% for mucins [33], resulting in less effect of multivalency and in total less glycans that can interact with H. pylori . However, even when we (over)compensate for differences in total glycan amounts by comparing recombinant protein/HSA-glycoconjugate at 50 µg/mL to human mucins at 10 µg/µL we still found approximately 10-fold larger effects of the human mucins.…”

Characteristics of protein–carbohydrate interactions as a basis for developing novel carbohydrate‐based antirejection therapies

“…The expression of this mucin/Ig chimera together with the porcine α1,3‐galactosyltransferase in COS cells resulted in an adsorber of xenoreactive human antipig antibodies that were, on a molar basis, 5000 and 30 000 times more efficient than Galα1,3Gal‐substituted agarose and macroporous glass beads, respectively 161 , 163 . In order to relate the adsorption capacity with the glycan expression of individual fusion proteins produced in different cell lines, stable CHO‐K1, COS and 293T cells producing this fusion protein were engineered.…”

Mucin Macromolecules in Normal, Adenomatous, and Carcinomatous Colon: Evidence for the Neotransformation

“…[50] Although host immune response to tumour via tumourassociated carbohydrate antigens is weak, these antigens have been used as targets for therapeutic applications, particularly, for suppression of human tumour growth. [178][179][180] Monoclonal antibodies defining tumour-associated antigens with a suitable isotype and affinity have proved to be useful to some extent in suppressing tumour growth in vitro and in vivo, albeit the tumour growth suppression in vivo is highly variable due to multiple, as yet unidentified, factors. An important factor is the intrinsic property of the antigen to which the antibody is directed.…”