|[Frontiers in Bioscience 1, e34-41, July 1, 1996]|
PIG alpha1,3GALACTOSYLTRANSFERASE: A MAJOR TARGET FOR GENETIC MANIPULATION IN XENOTRANSPLANTATION
Karen Strahan, Andrew Preece and Kenth Gustafsson
Division of Cell & Molecular Biology, Institute of Child Health, University of London, 30 Guilford St., London WC1N 1EH, UK
Received 06/21/96; Accepted 07/02/96; On-line 07/08/96
The a1,3GT gene was inactivated in the primate ancestors preceding the diversification of the great apes and led to the loss of this epitope (for review see 28). However, two non-expressed pseudo-genes remain on human chromosomes 9 and 12, respectively (30, 31). It is most likely that the copy on chromosome 9 represents the corresponding human gene, vis-à-vis, the expressed mammalian GGTA1 genes since the copy on chromosome 12 (HGT-2) appears to be a processed pseudogene (31). In addition, we have shown that the pig GGTA1 gene is situated in a region of pig chromosome 1 that is syntenic, vis-à-vis, the region harboring the human chromosome 9 copy (23). An interesting possibility, proposed by Galili and colleagues, is that the gene was inactivated in this primate lineage as the result of an intense evolutionary pressure in the form of selection for the presence of antibodies against the Gala1,3Gal structure. This would have occurred at the inevitable expense of the loss of expression of the a1,3GT gene (16). In addition, these authors suggested that the GGTA1 gene was independently inactivated in the respective lineages leading to, on the one hand, apes and man, and on the other hand, Old World monkeys (32). We find the latter interpretation unlikely since both lineages appear to share the same mutation in the form of a stop codon. Although this codon does not necessarily represent the original inactivating mutation, it precedes what has been shown to be the active domain in this enzyme. In addition, Joziasse and colleagues have found that the copy on human chromosome 12 (HGT-2) lacks the different single-base deletions present in the Old World monkey and ape/man lineages of GGTA1 (on chromosome 9 in man). More impor-tantly, the HGT-2 copy does contain the same stop codon as the GGTA1 copy (31, D. Joziasse, personal communication). This indicates that the mutation producing a stop codon preceded the lineage specific mutations. Thus, we favor the interpretation that inactivation of the GGTA1 gene in the common ancestors of both Old World monkeys and apes/man, occurred following the separation of New World monkeys expressing a functional gene product. A shortcoming of this hypothesis is that our own previously constructed phylogenetic tree places the branching point of the human HGT-2 gene before the divergence of all primates in the tree, including New World monkeys expressing the gene (Fig. 2a)(28). However, it must be remembered that when the genetic distances involved are very small, as in this case, mistakes are common in phylogenetic trees. Consequently, based on the observations described above proposing the evolution of the GGTA1 and HGT-2 genes, we have drawn an alternative phylogenetic tree (Fig. 2b). Whatever the exact evolution of the genes involved is, none we can imagine would preclude the explanation that there was a strong selection involved which favored the Gala1,3Gal-negative phenotype. Never-theless, it is also conceivable that the inactivation was merely an 'evolutionary accident'.
Fig. 2 Predicted phylogenetic trees of Gala1,3Gal evolution (not representing the correct genetic distances). a. Phylogenetic tree obtained from partial sequences of a1,3GT's as previously described (28, and references therein), predicting that the HGT-2 gene branched prior to the divergence of New World Monkeys and other primates. b. An abbreviated phylogenetic tree based on a synthesis of the tree in Fig. 2a and actual observations of shared inactivating mutations in the respective genes. In this tree the predicted time point for HGT-2 divergence, involving a very short genetic distance to the nearest branching point, has been disregarded (see 28). Possible wrong order of local gene divergences lead to an alternative suggestion for the evolution of a1,3GT genes. Potential gene inactivation events are depicted by short crossing lines. Species names in parantheses indicate non-expressed genes. OWM: Old World Monkeys, NWM: New World Monkeys (16). HGT-2 represents the human processed a1,3GT pseudogene copy on chromosome 12 (31). Other sequences were from ref. 16, 23, 25, 26, 27, and 30.
If the inactivation of the gene in one primate lineage was brought about by a strong evolutionary pressure, what might the selective agent have been? Many bacteria (33, 34) and some parasites (35-37) commonly carry epitopes to which anti-Gala1,3Gal NAb can bind and assist in opsonization and/or phagocytosis. In addition, it has been suggested that viruses can 'dress up' in this epitope as they are produced from cells expressing the a1,3GT enzyme (38, 39). Takeuchi and colleagues, in collaboration with us, recently showed that e.g. C-type retroviruses produced from murine or canine cells could be readily inactivated by human anti-Gala1-3Gal antibodies (40). Furthermore, expression of the porcine a1,3GT cDNA clone in human cells rendered both these cells as well as the retroviruses produced from them sensitive to human serum. The specificity of these Ab was ascertained following: 1. purification of anti-Gala1,3Gal Ab on a Gal(a1,3)Gal(b1,4)GlcNAc column, and 2. blocking of Ab reactivity using specific disaccharides. We speculated that this mechanism of increased immune reactivity may have given an increased ability of primates carrying high titres of anti-Gala1,3Gal NAb to resist e.g. C-type retroviral infections (40). Incidentally, despite the fact that they appear to be readily transmitted between other mammals, no C-type retroviruses have been found in humans. Since it has been previously shown that other viruses can be adorned with the Gala1,3Gal epitope one may generalize from this finding that other viruses can also be inactivated by human anti-Gala1,3Gal NAb. Thus it becomes apparent that the silencing of the gene in our early ancestors could have been influenced by selective evolutionary forces linked to trans-species viral infections. Alternatively, during primate evolution, some individuals may have enjoyed a selective advantage over others following viral transmissions within the same species. The clinical importance of the Gala1,3Gal epitope in eliciting a human anti-viral response is unknown. The human immune system appears to be able to effectively deal with most intra-species viral infections and, therefore, to eliminate viruses lacking the Gala1,3Gal epitope. Since any viruses produced in Gala1,3Gal-negative pigs are likely to be more resistant to human antibody inactivation, the use of 'clean', i.e. zoonotic-free, pigs as organ donors, should be strongly considered (41, 42).