Expression Cloning of a New Member of the ABO Blood Group Glycosyltransferases, iGb3 Synthase, That Directs the Synthesis of Isoglobo-glycosphingolipids

Background: Expression of x 2 glycosphingolipid (PX2) is elevated on erythrocytes from individuals with the rare P/P1/P knegative p phenotype. Results: Globoside-deficient individuals with mutated P synthase (␤1,3GalNAc-T1) lack PX2 and have anti-PX2 in plasma. Transfection of B3GALNT1 induces P and PX2 expression. Conclusion: PX2 synthesized by ␤1,3GalNAc-T1 fulfills blood group criteria. Significance: ␤1,3GalNAc-T1 uses different acceptors to form immunologically distinct glycosphingolipids.

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confidence: 86%

Identification of the Molecular and Genetic Basis of PX2, a Glycosphingolipid Blood Group Antigen Lacking on Globoside-deficient Erythrocytes

Westman

Benktander

Storry

et al. 2015

“…Importantly, while the use of animals deficient in the ␣1,3-galactosyltransferase may appear to be the most attractive option for animal to human xenotransplantation, studies have suggested that some tissues, including the vascular endothelium of deficient animals, may in fact still express minor quantities of Gal␣1-3Gal epitopes (41,42). The most likely explanation for this has been identified as expression of the isogloboside glycolipid (Gal␣1-3Gal␤1-4Glc␤1-Cer) (42), which is controlled by another homologous glycosyltransferase of the CAZy GT6 family, the iGb3 synthase (UDP-Gal:lactosylceramide ␣1,3-galactosyltransferase) (43).…”

Section: Discussionmentioning

confidence: 99%

Identification of a GH110 Subfamily of α1,3-Galactosidases

Liu

Yuan

Bennett

et al. 2008

In search of ␣-galactosidases with improved kinetic properties for removal of the immunodominant ␣1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of ␣-galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454 -464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Gal␣1-3(Fuc␣1-2)Gal, whereas linear oligosaccharides terminated by ␣1,3-linked galactose such as the immunodominant xenotransplantation epitope Gal␣1-3Gal␤1-4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific ␣1,3-galactosidases that act equally well on both branched blood group B and linear ␣1,3Gal structures. We determined by one-dimensional 1 H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known ␣-galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant ␣3Gal xenotransplantation epitope.

“…Most GT-A fold GTs, including those in the GT6 family, require divalent metal ions, such as Mn 2ϩ , for catalytic activity; their metal dependence is linked to a shared DXD sequence motif. Residues of this motif interact with the metal ion and both the ribose and phosphates of the donor substrate to produce an appropriate substrate orientation and conformation for catalysis and to stabilize the UDP leaving group (3,(7)(8)(9)(10).Mammalian members of GT6 are responsible for variations in glycan structures between different species and individuals as the result of selective enzyme inactivation in certain species (␣3GT, Forssman glycolipid synthase, and isogloboside 3 synthase) or the inheritance of multiple alleles at one locus that encode enzymes with different substrate specificity (GTA and GTB) or are inactive (11)(12)(13)(14). The presence of circulating antibodies against glycan structures that are subject to interspecies and individual variability has been linked to resistance to pathogens that also carry the glycans; these antibodies are thought to arise from exposure to potential pathogens, including enveloped viruses and bacteria that carry structurally similar glycans (11).…”

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confidence: 99%

“…Mammalian members of GT6 are responsible for variations in glycan structures between different species and individuals as the result of selective enzyme inactivation in certain species (␣3GT, Forssman glycolipid synthase, and isogloboside 3 synthase) or the inheritance of multiple alleles at one locus that encode enzymes with different substrate specificity (GTA and GTB) or are inactive (11)(12)(13)(14). The presence of circulating antibodies against glycan structures that are subject to interspecies and individual variability has been linked to resistance to pathogens that also carry the glycans; these antibodies are thought to arise from exposure to potential pathogens, including enveloped viruses and bacteria that carry structurally similar glycans (11).…”

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confidence: 99%

Characterization of a Metal-independent CAZy Family 6 Glycosyltransferase from Bacteroides ovatus

Tumbale

Brew

2009

The myriad functions of complex carbohydrates include modulating interactions between bacteria and their eukaryotic hosts. In humans and other vertebrates, variations in the activity of glycosyltransferases of CAZy family 6 generate antigenic variation between individuals and species that facilitates resistance to pathogens. The well characterized vertebrate glycosyltransferases of this family are multidomain membrane proteins with C-terminal catalytic domains. Genes for proteins homologous with their catalytic domains are found in at least nine species of anaerobic commensal bacteria and a cyanophage. Although the bacterial proteins are strikingly similar in sequence to the catalytic domains of their eukaryotic relatives, a metal-binding Asp-X-Asp sequence, present in a wide array of metal ion-dependent glycosyltransferases, is replaced by Asn-X-Asn. We have cloned and expressed one of these proteins from Bacteroides ovatus, a bacterium that is linked to inflammatory bowel disease. Functional characterization shows it to be a metal-independent glycosyltransferase with a 200-fold preference for UDP-GalNAc as substrate relative to UDP-Gal. It efficiently catalyzes the synthesis of oligosaccharides similar to human blood group A and may participate in the synthesis of the bacterial O-antigen. The kinetics for GalNAc transfer to 2-fucosyl lactose are characteristic of a sequential mechanism, as observed previously for this family. Mutational studies indicate that despite the lack of a metal cofactor, there are pronounced similarities in structurefunction relationships between the bacterial and vertebrate family 6 glycosyltransferases. These two groups appear to provide an example of horizontal gene transfer involving vertebrates and prokaryotes.The structures of complex glycans are determined by the specificities of the glycosyltransferases (GTs) 2 that catalyze their biosynthesis. GTs fall into two groups that differ in mechanism, based on whether the anomeric configuration of the donor substrate (␣ for most UDP-sugars) is retained or inverted in the product (1-3). They are classified into 90 different families in the CAZy data base based on sequence similarities (4, 5), but the majority of those that have been structurally characterized fall into one of two fold types, designated GT-A and GT-B (2). The retaining GTs of CAZy family 6 (GT6) have a GT-A fold and catalyze the transfer of either galactose or GalNAc into an ␣-linkage with the 3-OH group of ␤-linked galactose or GalNAc. GT6 includes the histo-blood group A and B GTs (GTA and GTB), the ␣-galactosyltransferase (␣3GT) that catalyzes the synthesis of the xenoantigen or ␣-gal epitope, Forssman glycolipid synthase, isogloboside 3 synthase, and their homologues from other vertebrates (6). GT6 enzymes from vertebrates are type-2 membrane proteins with N-terminal cytosolic domains, a transmembrane helix, a spacer, and a C-terminal catalytic domain (6). Crystallographic studies of recombinant catalytic domains of GTA, GTB, and ␣3GT have provided detailed inform...