Molecular Basis of Evolutionary Loss of the α1,3-Galactosyltransferase Gene in Higher Primates

Abstract

Galactose-␣1,3-galactose (␣Gal) epitopes, the synthesis of which requires the enzyme product of ␣1,3-galactosyltransferase (␣1,3GT), are sugar chains on the cell surface of most mammalian species. Notable exceptions are higher primates including Old World monkeys, apes, and humans. The ␣Gal-negative species as well as mice with deletion of the ␣1,3GT gene produce abundant anti␣Gal antibodies. The evolutionary loss of ␣Gal epitopes has been attributed to point mutations in the coding region of the gene. Because no transcripts could be found in the higher primate species with Northern blot analysis, a potential alternative explanation has been loss of upstream regulation of the gene. Here, we have demonstrated that the rhesus promoter is functional. More importantly, a variety of full-length transcripts were detected with sensitive PCR-based methods in the tissues of rhesus monkeys, orangutans, and humans. Five crucial mutations were delineated in the coding region of the human and rhesus and three in the orangutan, any one of which could be responsible for inactivation of the ␣1,3GT gene. Two of the mutations were shared by all three higher primates. These findings, which elucidate the molecular basis for the evolutionary loss of ␣Gal expression, may have implications in medical research.Most mammals express the cell surface carbohydrate epitope galactose-␣1,3-galactose (␣Gal), 1 with notable exceptions that include Old World monkeys, apes, and humans (1). With the loss of the ␣Gal epitopes, the synthesis of which is dependent on enzyme product of the ␣1,3-galactosyltransferase (␣1,3GT) gene, higher primates produce anti-␣Gal antibodies (2) that are responsible for the hyperacute rejection of organs transplanted from ␣Gal-positive donors (3).In 1989, Joziasse et al. (4) reported the sequence of fulllength cDNA clone of the bovine ␣1,3GT gene and demonstrated the presence of this gene in the DNA of human cell lines. Using an 804-bp fragment derived from bovine fulllength cDNA as a probe, they also detected mRNA transcripts in bovine and marmoset (New World monkey) but not in human or African green monkey cell lines (4). The absence of ␣1,3GT mRNA has since been widely viewed as a feature of all Old World monkeys, apes, and humans (5, 6). The molecular basis for the inactivation of the ␣1,3GT gene in the ␣Gal-negative species has been attributed to a mutation(s) localized to a partial sequence of exon 9 (4, 5, 7).Stimulated by the current interest in producing transgenic pigs for clinical use as tissue and organ xenograft donors, the cDNA for the ␣1,3GT gene of the ␣Gal-positive pig was isolated (8), and the coding regions were characterized (9 -12). Further, the full genomic organization of the porcine ␣1,3GT, gene including its upstream regulatory region, was completed (12). A CpG island (i.e. a C connected by a 3Ј-5Ј phosphodiester bond to a G) characteristic of housekeeping genes was observed around exon 1 of the pig ␣1,3GT gene (12). It is of interest that a CpG island was not present in the mouse gene (13-15...

CD1d-restricted iNKT cells, the ‘Swiss-Army knife’ of the immune system

“…When the lysosomal glycosphingolipid (GSL) isoglobotrihexosylceramide (iGb3) was described as an endogenous, and perhaps the selecting, ligand for iNKT cells [66], it faced a good deal of skepticism, mainly because humans are believed to lack functional α1,3 Galactosyltransferase [67,68], an enzyme thought to be critical to the synthesis of iGb3. Two papers published last year raised some doubts regarding the role of iGb3 as a self-antigen.…”

Gene- and cell-based therapeutics for type I diabetes mellitus

“…To what extent apoptosis or senescence play a role in this is uncertain. 200,201 The importance of this breakthrough is underscored by the fact that the major target of xenoreactive antibodies, which promote an acute rejection of porcine tissues, is the epitope that is synthesized by this enzyme. While this is the major porcine xenoantigen, it is almost certain that other minor porcine epitopes will contribute, perhaps not to acute rejection, but to delayed or chronic xenograft rejection and these are challenges that must be surmounted in the future.…”

Anti-Gal and Anti-Non Gal Antibody Barriers in Xenotransplantation

“…The 1,3GT gene (also referred to as Ggta1) is expressed in mammalian cells but is inactive in humans, apes and Old World monkeys (Galili et al 1988b;Thall et al 1991). This inactivation is primarily the result of various deletions in the Ggta1 gene causing frame shift mutations in the open reading frame and the generation of pre-mature stop codons (Larsen et al, 1990;Joziasse et al, 1992;Galili & Swanson, 1992;Koike et al, 2002). Studies evaluating the expression of this pseudo-gene in humans by PCR have demonstrated its low transcription (Koike et al, 2002), however, since the protein molecule is truncated, it is devoid of catalytic activity.…”

“…This inactivation is primarily the result of various deletions in the Ggta1 gene causing frame shift mutations in the open reading frame and the generation of pre-mature stop codons (Larsen et al, 1990;Joziasse et al, 1992;Galili & Swanson, 1992;Koike et al, 2002). Studies evaluating the expression of this pseudo-gene in humans by PCR have demonstrated its low transcription (Koike et al, 2002), however, since the protein molecule is truncated, it is devoid of catalytic activity. Truncation studies in the New World monkey 1,3GT have indicated that deletion of as few as three amino acids at the C-terminus is sufficient to result in complete lose of catalytic activity (Henion et al, 1994).…”