Modifying the sugar icing on the transplantation cake

Abstract

As a transplant surgeon, my interest in glycobiology began through my research into ABO-incompatible allotransplantation, and grew when my goal became overcoming the shortage of organs from deceased human donors by the transplantation of pig organs into patients with terminal organ failure (xenotransplantation/cross-species transplantation). The major target for human "natural" (preformed) anti-pig antibodies is galactose-α(1,3)-galactose (the "Gal" epitope), which is expressed on many pig cells, including the vascular endothelium. The binding of human IgM and IgG antibodies to Gal antigens initiates the process of hyperacute rejection, resulting in destruction of the pig graft within minutes or hours. This major barrier has been overcome by the production of pigs in which the gene for the enzyme α(1,3)-galactosyltransferase (GT) has been deleted by genetic engineering, resulting in GT knockout (GTKO) pigs. The two other known carbohydrate antigenic targets on pig cells for human anti-pig antibodies are (i) the product of the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene, i.e., N-glycolylneuraminic acid, and (ii) the product of the β1,4 N-acetylgalactosaminyltransferase gene, i.e., the Sd(a) antigen. Expression of these two has also been deleted in pigs. These genetic manipulations, together with others directed to overcoming primate complement and coagulation activation (the latter of which also relates to glycobiology) have contributed to the prolongation of pig graft survival in nonhuman primate recipients to many months rather than a few minutes. Clinical trials of the transplantation of pig cells are already underway and transplantation of pig organs may be expected within the relatively near future.

Keywords: ABO-incompatibility; N-glycolylneuraminic acid; galactose-α(1,3)-galactose; glycobiology; pig; xenotransplantation; β(1,4)N-acetylgalactosaminyltransferase.

Fig. 1.

Carbohydrate structure on human and pig RBCs. Pig RBCs express Gal epitopes on oligosaccharides that are similar in structure to the human blood type B oligosaccharide (which has a fucose side arm).

Fig. 2.

Evolutionary time scale of mammals during the past 125 million years. All mammals originally synthesized the Gal sugar, which was made by the enzyme GT. Evidence suggests that ∼10–25 million years ago the Old World higher primates were afflicted by a lethal infection caused by a microorganism that also expressed the Gal determinant. Only those primates that could produce anti-Gal antibodies survived. This necessitated suppression of the synthesis of the Gal sugar by mutation of the gene for the enzyme GT. (Modified from Galili 1998.)

Fig. 3.

(Left to right) Expression of Gal and NeuGc on aortas from wild-type, GTKO/CD46 and GTKO/CD46/CMAHKO pigs, and also on a human aorta. Expression of Gal was determined by staining with the isolectin B4 from Bandeiraea simplicifolia, and expression of NeuGc by staining with a chicken-derived anti-NeuGc immunohistochemistry set. Therefore, it is not possible to make a direct quantitative comparison of the level of expression between the two oligosaccharides. However, Gal (green) is expressed mainly on the vascular endothelium (indicated by red arrowheads), whereas NeuGc (red) is much more widely expressed in all layers, including the vascular endothelium. (Cell nuclei—blue; Gal—green; NeuGc—red. Magnification ×200.) (Figure kindly provided by W. Lee, MD).

Fig. 4.

Human IgM (A) and IgG (B) antibody binding to pig and human aortic endothelial cells by flow cytometry (n = 6). Human IgM and IgG binding to GTKO/CD46 pAECs was significantly decreased compared with wild-type (WT, i.e., genetically unmodified) pAECs (*P < 0.05), and was further decreased to GTKO/CD46/CMAHKO pAECs (*P < 0.05). There was significantly greater IgM binding to GTKO/CD46/CMAHKO pAECs than to human AECs, but there was no statistical significance in the extent of IgG binding between them. (Figure kindly provided by H. Hara, MD, PhD)