REMOVAL OF TERMINAL Α-Galactosyl RESIDUES FROM XENOGENEIC PORCINE ENDOTHELIAL CELLS

Abstract

To determine the role of the terminal alpha-galactosyl residue in the endothelial damage mediated by human xenoreactive natural antibodies (IgM and IgG), we treated porcine endothelial cells in culture with green coffee bean alpha-galactosidase. A practically complete removal of terminal alpha-Gal residues (as evaluated by flow cytometry with Bandeiraea simplicifolia isolectin B4) and concomitant exposure of N-acetyllactosamine were obtained without altering cell viability. A dramatic decrease in IgM and IgG binding (from a pool of human sera) was observed, confirming the key role of the alpha-galactosyl residues. The enzyme treatment did not induce any nonspecific immunoglobulin binding sites, but led to the exposure of new epitopes for a minor fraction of IgM. The main residual IgM and IgG binding could be due to xenoantigens other than the alpha-galactosyl residues. When alpha-galactosidase-treated endothelial cells were used as targets in cytotoxicity experiments, they were less susceptible than untreated cells to complement-mediated cytotoxicity induced by fresh human serum. In contrast, they did not acquire resistance to human IgG-dependent cellular cytotoxicity, despite the decrease in IgG binding. Because it is known that antibody-dependent cytotoxicity mediated by CD16+ NK cells is dependent on IgG1 and IgG3, and not on IgG2 or IgG4, which was confirmed by blocking experiments, we studied the binding of all four subclasses to intact and alpha-galactosidase-treated endothelial cells. Two major subclasses, IgG1 and IgG2, bound to untreated endothelial cells, whereas IgG3 binding was low and IgG4 binding was negligible. A decrease in IgG1, IgG2, and IgG3 binding was observed upon alpha-galactosidase treatment, indicating that antibodies belonging to these three subclasses recognize alpha-galactosyl residues. The decrease in IgG2 binding was more pronounced than the decrease in IgG1 binding. Collectively, these data indicate that IgG1 xenoreactive natural antibodies, including those which are not directed at the alpha-galactosyl residues, could play a major role in the early delayed vascular rejection of pig xenografts.

Combined transgenic expression of α-galactosidase and α1,2-fucosyltransferase leads to optimal reduction in the major xenoepitope Galα(1,3)Gal

“…Thus, treatment of rabbit erythrocytes with ␣-galactosidase decreased the level of Gal␣(1,3)Gal on the cell surface (up to 255-fold using E. coli ␣-galactosidase). Clearly, human-or E. coli-derived ␣-galactosidase can be used to reduce the amount of antigen on erythrocytes, and our results are in agreement with earlier studies using coffee bean ␣-galactosidase (11)(12)(13)(14) or bacteriaderived ␣-galactosidase (10) to remove ␣-galactosyl residues by enzyme treatment. can reduce the amount of Gal␣(1,3)Gal from the cell surface, and the enzyme could be used for the ex vivo treatment of xenograft donor organs; however, this method was cumbersome and would not address the problem of continual resynthesis of the epitope by ␣-galactosidase-treated cells.…”

“…In vitro treatment of pig endothelial cells, lymphocytes, or rabbit erythrocytes with ␣-galactosidase totally eradicated their reaction with human natural antibodies (10)(11)(12)(13)(14). The enzyme also has been perfused into organs before transplantation (10).…”

Molecular Cloning of Endo-β-galactosidase C and Its Application in Removing α-Galactosyl Xenoantigen from Blood Vessels in the Pig Kidney

“…Pig aortic endothelial cells were isolated from a pig as described previously (16). The cells were harvested with trypsin-EDTA (Life Technologies, Inc.) and washed twice with PBS/BSA and resuspended in PBS/BSA.…”

“…As an alternative approach, ␣-galactosidase from the coffee bean was used to remove the ␣-galactosyl antigen from cultured pig endothelial cells. The enzyme-treated cells became resistant to complement-dependent lysis by human sera (15,16). Although very recently the ␣-galactosidase has been applied to remove the ␣-galactosyl antigen from isolated femoral veins from the pig (17), this enzyme has not been applied to treat pig organs, probably because the acidic optimum pH of the enzyme makes it necessary to use enormous amounts of the enzyme.…”

Target Cell Susceptibility to Lysis by Human Natural Killer Cells Is Augmented by α(1,3)-Galactosyltransferase and Reduced by α(1,2)-Fucosyltransferase

“…Porcine endothelium was treated with ␣-galactosidase at concentrations shown to reduce antibody-directed complement lysis of porcine endothelium (32). Briefly, PAEC monolayers were treated with either ␣-galactosidase isolated from the Green coffee been (Sigma) or ␤-galactosidase isolated from Escherichia coli bacteria (Sigma) for 4 h at pH 6.0 or 7.3, respectively.…”