Abstract
Functional paralogous ABO, GBGT1, A3GALT2, and GGTA1 genes encode blood group A and B transferases (AT and BT), Forssman glycolipid synthase (FS), isoglobotriaosylceramide synthase (iGb3S), and α1,3-galactosyltransferase (GT), respectively. These glycosyltransferases transfer N-acetyl-d-galactosamine (GalNAc) or d-galactose forming an α1,3-glycosidic linkage. However, their acceptor substrates are diverse. Previously, we demonstrated that the amino acids at codons 266 and 268 of human AT/BT are crucial to their distinct sugar specificities, elucidating the molecular genetic basis of the ABO glycosylation polymorphism of clinical importance in transfusion and transplantation medicine. We also prepared in vitro mutagenized ATs/BTs having any of 20 possible amino acids at those codons, and showed that those codons determine the transferase activity and sugar specificity. We have expanded structural analysis to include evolutionarily related α1,3-Gal(NAc) transferases. Eukaryotic expression constructs were prepared of AT, FS, iGb3S, and GT, possessing selected tripeptides of AT-specific AlaGlyGly or LeuGlyGly, BT-specific MetGlyAla, FS-specific GlyGlyAla, or iGb3S and GT-specific HisAlaAla, at the codons corresponding to 266–268 of human AT/BT. DNA transfection was performed using appropriate recipient cells existing and newly created, and the appearance of cell surface oligosaccharide antigens was immunologically examined. The results have shown that several tripeptides other than the originals also bestowed transferase activity. However, the repertoire of functional amino acids varied among those transferases, suggesting that structures around those codons differentially affected the interactions between donor nucleotide-sugar and acceptor substrates. It was concluded that different tripeptide sequences at the substrate-binding pocket have contributed to the generation of α1,3-Gal(NAc) transferases with diversified specificities.
Highlights
Α1,3-Gal(NAc) transferases are comprised of blood group A and B transferases (ATs and BTs), Forssman glycolipid synthases (FSs), isoglobotriaosylceramide synthases, and α1,3-galactosyltransferases (GTs)
Later studies have cast doubt on the hypothesis that iGb3 is the unique self-antigen required for invariant natural killer T (iNKT) cells[18,19,20], it is still possible that autologous glycolipids that are structurally related to iGb3 may contribute to the positive iNKT cell selection
HeLa(FUT2) cells were previously created from HeLa cells to achieve enhanced detection sensitivity of AT and BT activities by retrovirally transducing human FUT2 gene cDNA encoding α1,2-fucosyltransferase that catalyzes the last biosynthetic step of H substance, the acceptor substrate for AT and BT44,45
Summary
Α1,3-Gal(NAc) transferases are comprised of blood group A and B transferases (ATs and BTs), Forssman glycolipid synthases (FSs), isoglobotriaosylceramide synthases (iGb3Ss), and α1,3-galactosyltransferases (GTs). Humans may have functional AT and/or BT, depending on the polymorphic status at the ABO genetic locus, but they carry none of the functional GBGT1, A3GALT2, or GGTA1 genes nor exhibit any of FS, iGb3S, or GT activities[26,27,28,29]. Rare FORS1-positive individuals possess a dominant missense mutation in the GBGT1 gene, resulting in the acquisition of FS activity and the appearance of FORS1 antigen[9,30]. Birds are another example of species-dependent repertoire. In vitro mutagenesis studies from other laboratories[37,38], three-dimensional structural determination of GT and AT/BT with and without substrates[39,40,41,42], as well as structural modeling of α1,3-Gal(NAc) transferases[9,43], have contributed to the better understanding of the structural basis of α1,3-Gal(NAc) transferase functions
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