Two Paralogous Gb3/CD77 Synthases in Birds Show Different Preferences for Their Glycoprotein and Glycosphingolipid Substrates.
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ABSTRACT:
SUBMITTER: Bereznicka A
PROVIDER: S-EPMC8466213 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Glycolipids are present on the surfaces of all living cells and thereby represent targets for many protein receptors, such as lectins. Understanding the interactions between lectins and glycolipids is essential for investigating the functions of lectins and the dynamics of glycolipids in living membranes. This review focuses on lectins binding to the glycosphingolipid globotriaosylceramide (Gb3), an attractive host cell receptor, particularly for pathogens and pathogenic products. Shiga toxin (Stx), from Shigella dysenteriae or Escherichia coli, which is one of the most virulent bacterial toxins, binds and clusters Gb3, leading to local negative membrane curvature and the formation of tubular plasma membrane invaginations as the initial step for clathrin-independent endocytosis. After internalization, it is embracing the retrograde transport pathway. In comparison, the homotetrameric lectin LecA from Pseudomonas aeruginosa can also bind to Gb3, triggering the so-called lipid zipper mechanism, which results in membrane engulfment of the bacterium as an important step for its cellular uptake. Notably, both lectins bind to Gb3 but induce distinct plasma membrane domains and exploit mainly different transport pathways. Not only, several other Gb3-binding lectins have been described from bacterial origins, such as the adhesins SadP (from Streptococcus suis) and PapG (from E. coli), but also from animal, fungal, or plant origins. The variety of amino acid sequences and folds demonstrates the structural versatilities of Gb3-binding lectins and asks the question of the evolution of specificity and carbohydrate recognition in different kingdoms of life.
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Project description:Rare polyagglutinable NOR erythrocytes contain three unique globoside (Gb4Cer) derivatives, NOR1, NOR(int), and NOR2, in which Gal(?1-4), GalNAc(?1-3)Gal(?1-4), and Gal(?1-4)GalNAc(?1-3)Gal(?1-4), respectively, are linked to the terminal GalNAc residue of Gb4Cer. NOR1 and NOR2, which both terminate with a Gal(?1-4)GalNAc- sequence, react with anti-NOR antibodies commonly present in human sera. While searching for an enzyme responsible for the biosynthesis of Gal(?1-4)GalNAc, we identified a mutation in the A4GALT gene encoding Gb3/CD77 synthase (?1,4-galactosyltransferase). Fourteen NOR-positive donors were heterozygous for the C>G mutation at position 631 of the open reading frame of the A4GALT gene, whereas 495 NOR-negative donors were homozygous for C at this position. The enzyme encoded by the mutated gene contains glutamic acid instead of glutamine at position 211 (substitution Q211E). To determine whether this mutation could change the enzyme specificity, we transfected a teratocarcinoma cell line (2102Ep) with vectors encoding the consensus Gb3/CD77 synthase and Gb3/CD77 synthase with Glu at position 211. The cellular glycolipids produced by these cells were analyzed by flow cytometry, high-performance thin-layer chromatography, enzymatic degradation, and MALDI-TOF mass spectrometry. Cells transfected with either vector expressed the P1 blood group antigen, which was absent from untransfected cells. Cells transfected with the vector encoding the Gb3/CD77 synthase with Glu at position 211 expressed both P1 and NOR antigens. Collectively, these results suggest that the C631G mutation alters the acceptor specificity of Gb3/CD77 synthase, rendering it able to catalyze synthesis of the Gal(?1-4)Gal and Gal(?1-4)GalNAc moieties.
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Project description:Human Gb3/CD77 synthase (α1,4-galactosyltransferase) is the only known glycosyltransferase that changes acceptor specificity because of a point mutation. The enzyme, encoded by A4GALT locus, is responsible for biosynthesis of Gal(α1-4)Gal moiety in Gb3 (CD77, Pk antigen) and P1 glycosphingolipids. We showed before that a single nucleotide substitution c.631C > G in the open reading frame of A4GALT, resulting in replacement of glutamine with glutamic acid at position 211 (substitution p. Q211E), broadens the enzyme acceptor specificity, so it can not only attach galactose to another galactose but also to N-acetylgalactosamine. The latter reaction leads to synthesis of NOR antigens, which are glycosphingolipids with terminal Gal(α1-4)GalNAc sequence, never before described in mammals. Because of the apparent importance of position 211 for enzyme activity, we stably transfected the 2102Ep cells with vectors encoding Gb3/CD77 synthase with glutamine substituted by aspartic acid or asparagine, and evaluated the cells by quantitative flow cytometry, high-performance thin-layer chromatography and real-time PCR. We found that cells transfected with vectors encoding Gb3/CD77 synthase with substitutions p. Q211D or p. Q211N did not express Pk, P1 and NOR antigens, suggesting complete loss of enzymatic activity. Thus, amino acid residue at position 211 of Gb3/CD77 synthase is critical for specificity and activity of the enzyme involved in formation of Pk, P1 and NOR antigens. Altogether, this approach affords a new insight into the mechanism of action of the human Gb3/CD77 synthase.
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Project description:Globotriaosylceramide (Gb3), a glycosphingolipid found in the plasma membrane of animal cells, is the endocytic receptor of the bacterial Shiga toxin. Using x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), lipid monolayers containing Gb3 were investigated at the air-water interface. XR probed Gb3 carbohydrate conformation normal to the interface, whereas GIXD precisely characterized Gb3's influence on acyl chain in-plane packing and area per molecule (APM). Two phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), were used to study Gb3 packing in different lipid environments. Furthermore, the impact on monolayer structure of a naturally extracted Gb3 mixture was compared to synthetic Gb3 species with uniquely defined acyl chain structures. XR results showed that lipid environment and Gb3 acyl chain structure impact carbohydrate conformation with greater solvent accessibility observed for smaller phospholipid headgroups and long Gb3 acyl chains. In general, GIXD showed that Gb3 condensed phospholipid packing resulting in smaller APM than predicted by ideal mixing. Gb3's capacity to condense APM was larger for DSPC monolayers and exhibited different dependencies on acyl chain structure depending on the lipid environment. The interplay between Gb3-induced changes in lipid packing and the lipid environment's impact on carbohydrate conformation has broad implications for glycosphingolipid macromolecule recognition and ligand binding.
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Project description:The human Gb3/CD77 synthase, encoded by the A4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Galα1→4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, Pk) and the P1 antigen. Gb3 is the major receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli. A single amino acid substitution (p.Q211E) ramps up the enzyme's promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise to NOR1 and NOR2 GSLs. Human Gb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Galα1→4Galβ1→4GlcNAc-R) on a complex type N-glycan and a synthetic N-glycoprotein (saposin D). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminated N-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays show that Stx1 can use P1 N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonical GSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology.
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