Xenotransplantation: the importance of the Galα1,3Gal epitope in hyperacute vascular rejection

AbstractThe transplantation of organs from other species into humans is considered to be a potential solution to the shortage of human donor organs. Organ transplantation from pig to human, however, results in hyperacute rejection, initiated by the binding of human natural antidonor antibody and complement. The major target antigen of this natural antibody is the terminal disaccharide Galα1,3Gal, which is synthesized by Galβ1,4GlcNAc α1,3-galactosyltransferase. Here we review our current knowledge of this key enzyme. A better understanding of structure, enzyme properties, and expression pattern of α1,3-galactosyltransferase has opened up several novel therapeutic approaches to prevent hyperacute vascular rejection. Cloning, and expression in vitro of the corresponding cDNA, has allowed to develop strategies to induce immune tolerance, and deplete or neutralize the natural xenoreactive antibody. Elucidation of the genomic structure has led to the production of transgenic animals that are lacking α1,3-galactosyltransferase activity. A detailed knowledge of the enzyme properties has formed the basis of approaches to modify donor organ glycosylation by intracellular competition. Study of the expression pattern of α1,3-galactosyltransferase has helped to understand the mechanism of hyperacute rejection in discordant xenotransplantation, and that of complement-mediated, natural immunity against interspecies transmission of retroviruses

Sequence-function relationships in prokaryotic and eukaryotic galactosyltransferases

International audienceGalactosyltransferases are enzymes which transfer galactose from UDP-Gal to various acceptors with either retention of the anomeric configuration to form alpha1,2-, alpha1,3-, alpha1,4-, and alpha1, 6-linkages, or inversion of the anomeric configuration to form beta1, 3-, beta1,4-, and beta1-ceramide linkages. During the last few years, several (c)DNA sequences coding for galactosyltransferases became available. We have retrieved these sequences and conducted sequence similarity studies. On the basis of both the nature of the reaction catalyzed and the protein sequence identity, these enzymes can be classified into twelve groups. Using a sensitive graphics method for protein comparison, conserved structural features were found in some of the galactosyltransferase groups, and other classes of glycosyltransferases, resulting in the definition of five families. The lengths and locations of the conserved regions as well as the invariant residues are described for each family. In addition, the DxD motif that may be important for substrate recognition and/or catalysis is demonstrated to occur in all families but one.Galactosyltransferases are enzymes which transfer galactose from UDP-Gal to various acceptors with either retention of the anomeric configuration to form alpha1,2-, alpha1,3-, alpha1,4-, and alpha1, 6-linkages, or inversion of the anomeric configuration to form beta1, 3-, beta1,4-, and beta1-ceramide linkages. During the last few years, several (c)DNA sequences coding for galactosyltransferases became available. We have retrieved these sequences and conducted sequence similarity studies. On the basis of both the nature of the reaction catalyzed and the protein sequence identity, these enzymes can be classified into twelve groups. Using a sensitive graphics method for protein comparison, conserved structural features were found in some of the galactosyltransferase groups, and other classes of glycosyltransferases, resulting in the definition of five families. The lengths and locations of the conserved regions as well as the invariant residues are described for each family. In addition, the DxD motif that may be important for substrate recognition and/or catalysis is demonstrated to occur in all families but one

Branch specificity of bovine colostrum CMP-sialic acid: N-acetyllactosaminide α 2→6- sialyltransferase. Interaction with biantennary oligosaccharides and glycopeptides of N-glycosylproteins

By use of 500-MHz 1H NMR spectroscopy, the branch specificity of bovine colostrum CMP-NeuAc:Gal β 1→4GlcNAc-R α 2→6- sialyltransferase towards a biantennary glycopeptide and oligosaccharides of the N-acetyllactosamine type, differing in completeness and structure of their core portion, was investigated. In agreement with earlier reports (Van den Eijnden, D. H., Joziasse, D. H., Dorland, L., Van Halbeek H., Vliegenthart, J. F. G., and Schmid, K. (1980) Biochem. Biophys. Res. Commun. 92, 839-845), it appears that the enzyme strongly prefers the galactosyl residue at the Manα 1→ 3Man branch of the biantennary glycopeptide for attachment of the first sialic acid residue. This branch specificity is fully preserved with the structure (formula; see text) Reduction of the reducing N- acetylglucosaminyl residue in this structure, however, leads to a decreased branch specificity, whereas removal of this residue results in a random attachment of sialic acid to the galactoses at both branches. The decrease in branch specificity is accompanied by a reduction in the rate of sialic acid transfer to the galactose at the alpha 1→3 branch. Our results indicate that the presence of the aforementioned N-acetylglucosaminyl residue is a minimal structural requirement for branch specificity of the sialyltransferase. We propose that in the interaction of the sialyltransferase with its substrates, this N-acetylglucosaminyl residue functions as a recognition site mediating the correct positioning of the substrate on the enzyme

Branch specificity of bovine colostrum CMP-sialic acid: N-acetyllactosaminide α 2→6- sialyltransferase. Interaction with biantennary oligosaccharides and glycopeptides of N-glycosylproteins

By use of 500-MHz 1H NMR spectroscopy, the branch specificity of bovine colostrum CMP-NeuAc:Gal β 1→4GlcNAc-R α 2→6- sialyltransferase towards a biantennary glycopeptide and oligosaccharides of the N-acetyllactosamine type, differing in completeness and structure of their core portion, was investigated. In agreement with earlier reports (Van den Eijnden, D. H., Joziasse, D. H., Dorland, L., Van Halbeek H., Vliegenthart, J. F. G., and Schmid, K. (1980) Biochem. Biophys. Res. Commun. 92, 839-845), it appears that the enzyme strongly prefers the galactosyl residue at the Manα 1→ 3Man branch of the biantennary glycopeptide for attachment of the first sialic acid residue. This branch specificity is fully preserved with the structure (formula; see text) Reduction of the reducing N- acetylglucosaminyl residue in this structure, however, leads to a decreased branch specificity, whereas removal of this residue results in a random attachment of sialic acid to the galactoses at both branches. The decrease in branch specificity is accompanied by a reduction in the rate of sialic acid transfer to the galactose at the alpha 1→3 branch. Our results indicate that the presence of the aforementioned N-acetylglucosaminyl residue is a minimal structural requirement for branch specificity of the sialyltransferase. We propose that in the interaction of the sialyltransferase with its substrates, this N-acetylglucosaminyl residue functions as a recognition site mediating the correct positioning of the substrate on the enzyme

Branch specificity of bovine colostrum CMP-sialic acid: Galβ 1→4GlcNAc-Rα2→6-sialyl-transferase. Sialylation of bi-, tri-, and tetraantennary oligosaccharides and glycopeptides of the N-acetyllactosamine type

Using 500-MHz 1H NMR spectroscopy we have investigated the branch specificity that bovine colostrum CMP-NeuAc:Galβ1→4GlcNAc-Rα2→6-sialyltransferase shows in its sialylation of bi-, tri-, and tetraantennary glycopeptides and oligosaccharides of the N- acetyllactosamine type. The enzyme appears to highly prefer the galactose residue at the Galβ1→4GlcNAcβ1→2Manα1→3 branch for attachment of the 1st mol of sialic acid in all the acceptors tested. The 2nd mol of sialic acid becomes linked mainly to the Galβ1→4GlcNAcβ1→2Manα1→6 branch in bi- and triantennary substrates, but this reaction invariably proceeds at a much lower rate. Under the conditions employed, the Galβ1→4GlcNAcβ1→6Manα1→6 branch is extremely resistant to α2→6-sialylation. A higher degree of branching of the acceptors leads to a decrease in the rate of sialylation. In particular, the presence of the Galβ1→4GlcNAcβ1→6Manα1→6 branch strongly inhibits the rate of transfer of both the 1st and the 2nd mol of sialic acid. In addition, it directs the incorporation of the 2nd mol into tetraantennary structures toward the Galβ1→4GlcNAcβ1→4Manα1→3 branch. In contrast, the presence of the Galβ1→4GlcNAcβ1→4Manα1→3 branch has only minor effects on the rates of sialylation and, consequently, on the branch preference of sialic acid attachment. Results obtained with partial structures of tetraantennary acceptors indicate that the Manβ1→4GlcNAc part of the core is essential for the expression of branch specificity of the sialyltransferase. The sialylation patterns observed in vivo in glycoproteins of different origin are consistent with the in vitro preference of α2→6-sialyltransferase for the Galβ1→4GlcNAcβ1→2Manα1→3 branch. Our findings suggest that the terminal structures of branched glycans of the N- acetyllactosamine type are the result of the complementary branch specificity of the various glycosyltransferases that are specific for the acceptor sequence Galβ1→4GlcNAc-R

Branch specificity of bovine colostrum CMP-sialic acid: Galβ 1→4GlcNAc-Rα2→6-sialyl-transferase. Sialylation of bi-, tri-, and tetraantennary oligosaccharides and glycopeptides of the N-acetyllactosamine type

Using 500-MHz 1H NMR spectroscopy we have investigated the branch specificity that bovine colostrum CMP-NeuAc:Galβ1→4GlcNAc-Rα2→6-sialyltransferase shows in its sialylation of bi-, tri-, and tetraantennary glycopeptides and oligosaccharides of the N- acetyllactosamine type. The enzyme appears to highly prefer the galactose residue at the Galβ1→4GlcNAcβ1→2Manα1→3 branch for attachment of the 1st mol of sialic acid in all the acceptors tested. The 2nd mol of sialic acid becomes linked mainly to the Galβ1→4GlcNAcβ1→2Manα1→6 branch in bi- and triantennary substrates, but this reaction invariably proceeds at a much lower rate. Under the conditions employed, the Galβ1→4GlcNAcβ1→6Manα1→6 branch is extremely resistant to α2→6-sialylation. A higher degree of branching of the acceptors leads to a decrease in the rate of sialylation. In particular, the presence of the Galβ1→4GlcNAcβ1→6Manα1→6 branch strongly inhibits the rate of transfer of both the 1st and the 2nd mol of sialic acid. In addition, it directs the incorporation of the 2nd mol into tetraantennary structures toward the Galβ1→4GlcNAcβ1→4Manα1→3 branch. In contrast, the presence of the Galβ1→4GlcNAcβ1→4Manα1→3 branch has only minor effects on the rates of sialylation and, consequently, on the branch preference of sialic acid attachment. Results obtained with partial structures of tetraantennary acceptors indicate that the Manβ1→4GlcNAc part of the core is essential for the expression of branch specificity of the sialyltransferase. The sialylation patterns observed in vivo in glycoproteins of different origin are consistent with the in vitro preference of α2→6-sialyltransferase for the Galβ1→4GlcNAcβ1→2Manα1→3 branch. Our findings suggest that the terminal structures of branched glycans of the N- acetyllactosamine type are the result of the complementary branch specificity of the various glycosyltransferases that are specific for the acceptor sequence Galβ1→4GlcNAc-R