Why human anti-Galα1–4Galβ1–4Glc natural antibodies do not recognize the trisaccharide on erythrocyte membrane? Molecular dynamics and immunochemical investigation

Volynsky, Pavel E.; Efremov, Roman G.; Михалев, И И; Dobrochaeva, Kira; Tuzikov, Alexander; Korchagina, Elena; Obukhova, Polina; Rapoport, E. M.; Bovin, Nicolai V.

doi:10.1016/j.molimm.2017.06.247

Cited by 10 publications

(6 citation statements)

References 26 publications

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“…Glycan #11 is an abundant trisaccharide (known as Pk antigen), a key fragment of glycosphingolipid globotriaosylceramide (Gb3Cer) which is a component of erythrocyte and endothelial cell membrane. Antibodies against this trisaccharide were found to bind to synthetic version of the glycan as observed here but not to glycan on cell membrane 38 . The binding difference between synthetic and natural forms of the trisaccharide is probably due to the different spacing and conformation 38 .…”

Section: Resultssupporting

confidence: 49%

“…Antibodies against this trisaccharide were found to bind to synthetic version of the glycan as observed here but not to glycan on cell membrane 38 . The binding difference between synthetic and natural forms of the trisaccharide is probably due to the different spacing and conformation 38 . Our results suggest that ovarian cancer cells may express the trisaccharide in a conformation that are different from those on normal cells and can induce an antibody response in ovarian cancer patients.…”

Section: Resultssupporting

confidence: 49%

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Multiplex glycan bead array for high throughput and high content analyses of glycan binding proteins

Purohit

Guan

et al. 2018

Nat Commun

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Glycan-binding proteins (GBPs) play critical roles in diverse cellular functions such as cell adhesion, signal transduction and immune response. Studies of the interaction between GBPs and glycans have been hampered by the availability of high throughput and high-content technologies. Here we report multiplex glycan bead array (MGBA) that allows simultaneous analyses of 384 samples and up to 500 glycans in a single assay. The specificity, sensitivity and reproducibility of MGBA are evaluated using 39 plant lectins, 13 recombinant anti-glycan antibodies, and mammalian GBPs. We demonstrate the utility of this platform by the analyses of natural anti-glycan IgM and IgG antibodies in 961 human serum samples and the discovery of anti-glycan antibody biomarkers for ovarian cancer. Our data indicate that the MGBA platform is particularly suited for large population-based studies that require the analyses of large numbers of samples and glycans.

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Section: Resultssupporting

confidence: 49%

Section: Resultssupporting

confidence: 49%

Multiplex glycan bead array for high throughput and high content analyses of glycan binding proteins

Purohit

Guan

et al. 2018

Nat Commun

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“…Here, we looked at another, rarely addressed, potential confounder: the RBC membrane cholesterol content. It was shown that excess plasma membrane cholesterol may cloak glycosphingolipids, including the P k antigen, from the binding proteins[ 8 , 29 , 33 ]. Most of these studies focused only on the P k antigen’s role as a Shiga toxin receptor, which is perhaps why the cholesterol aspect has not crossed over to immunohaematologic studies on the P1PK system so far.…”

Section: Discussionmentioning

confidence: 99%

Single nucleotide polymorphisms in A4GALT spur extra products of the human Gb3/CD77 synthase and underlie the P1PK blood group system

et al. 2018

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Contrary to the mainstream blood group systems, P1PK continues to puzzle and generate controversies over its molecular background. The P1PK system comprises three glycosphingolipid antigens: Pk, P1 and NOR, all synthesised by a glycosyltransferase called Gb3/CD77 synthase. The Pk antigen is present in most individuals, whereas P1 frequency is lesser and varies regionally, thus underlying two common phenotypes: P1, if the P1 antigen is present, and P2, when P1 is absent. Null and NOR phenotypes are extremely rare. To date, several single nucleotide polymorphisms (SNPs) have been proposed to predict the P1/P2 status, but it has not been clear how important they are in general and in relation to each other, nor has it been clear how synthesis of NOR affects the P1 phenotype. Here, we quantitatively analysed the phenotypes and A4GALT transcription in relation to the previously proposed SNPs in a sample of 109 individuals, and addressed potential P1 antigen level confounders, most notably the red cell membrane cholesterol content. While all the SNPs were associated with the P1/P2 blood type and rs5751348 was the most reliable, we found large differences in P1 level within groups defined by their genotype and substantial intercohort overlaps, which shows that the P1PK blood group system still eludes full understanding.

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“…S4). Stx binding subunits and antibodies have different affinities for Gb3 and P1 GSLs and vastly differ in size, so these receptors may be less accessible to antibodies (24,47). Taken together, we presume that the p.Q211E mutein indeed produces the P1 GSL less efficiently (hence the decreased anti-P1 binding to protease-treated RBCs), but it exhibits a higher activity toward glycoprotein acceptors in comparison with Gb3/CD77 synthase (hence the increased Stx1B binding).…”

Section: Discussionmentioning

confidence: 82%

Human Gb3/CD77 synthase produces P1 glycotope-capped N-glycans, which mediate Shiga toxin 1 but not Shiga toxin 2 cell entry

Szymczak-Kulus

Weidler

Bereznicka

et al. 2021

Journal of Biological Chemistry

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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, P k ) 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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Why human anti-Galα1–4Galβ1–4Glc natural antibodies do not recognize the trisaccharide on erythrocyte membrane? Molecular dynamics and immunochemical investigation

Cited by 10 publications

References 26 publications

Multiplex glycan bead array for high throughput and high content analyses of glycan binding proteins

Multiplex glycan bead array for high throughput and high content analyses of glycan binding proteins

Single nucleotide polymorphisms in A4GALT spur extra products of the human Gb3/CD77 synthase and underlie the P1PK blood group system

Human Gb3/CD77 synthase produces P1 glycotope-capped N-glycans, which mediate Shiga toxin 1 but not Shiga toxin 2 cell entry

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