Subtilase cytotoxin (SubAB)

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AB5 toxins are an important family of bacterial toxins, so termed because they comprise a catalytic A subunit, non-covalently linked to a pentameric B subunit that binds to specific host cell surface glycans. There are three well-characterised AB5 toxin sub-families: (i) cholera toxin (Ctx) and the closely related E. coli heat labile enterotoxins (LT-I and LT-II); (ii) pertussis toxin (Ptx); and (iii) Shiga toxin (Stx). In each case, these AB5 toxins are key virulence factors of the bacteria that produce them: Vibrio cholerae and enterotoxigenic E. coli [ETEC] (Ctx and LT-I&II, respectively); Bordetella pertussis (Ptx); Shiga toxigenic E. coli [STEC] and Shigella dysenteriae (Stx). A fourth sub-family, subtilise cytotoxin (SubAB), also produced by STEC, has been described relatively recently. The AB5 toxins from each sub-family possess unique properties that arise from differing catalytic activities of the A subunit and/or differing receptor specificities of the B subunit. The A subunits of the Ctx/LT and Ptx families ADP-ribosylate the Gsα and Giα proteins, respectively, disrupting signal transduction pathways. This results in an increase in intracellular cAMP levels and disregulation of ion transport mechanisms. Stx family A subunits have RNA N-glycosidase activity, and inhibit eukaryotic protein synthesis by cleaving a specific adenine base from 28S rRNA, while SubA is a highly specific subtilase-like serine protease that cleaves the essential endoplasmic reticulum chaperone BiP/GRP78. Binding of AB5 toxin B subunits to cognate host glycan receptors triggers internalization by receptor- mediated endocytosis, followed by trafficking to the appropriate intracellular compartment. The glycan receptors for AB5 toxin B subunit pentamers are displayed either on glycolipids (for Ctx/LT and Stx) or on glycoproteins (for Ptx and SubAB). Glycan receptor specificity is critical for the pathogenic process, as it determines host susceptibility, tissue tropism, and the nature and spectrum of the resultant pathology. Knowledge of the molecular/structural basis for B subunit pentamer/glycan interactions is providing a rational framework for design of specific toxin inhibitors with considerable potential as anti-infective therapeutic agents[1].

SubAB is a recently-discovered prototype of a new AB5 toxin sub-family, with a highly novel mode of inducing cytotoxicity[2][3]. It has been selected as a paradigm because the other AB5 toxin sub-families referred to above have been known for many years, and a substantial body of information on toxin-receptor interactions had been built up using conventional biochemical techniques[1][4]. SubB has recently been shown to bind to N-linked glycans displayed on several glycoproteins on the surface of Vero and HeLa cells, including α2β1integrin[5]. However, nothing was known about the identity of the cognate glycan structures prior to accessing the CFG Core H glycan array facilities. Thus, the CFG has enabled seminal studies on SubB-host receptor interactions. Glycan array analysis showed that SubB has a high degree of specificity for glycans terminating with α2-3-linked N-glycolylneuraminic acid (Neu5Gc), with little discrimination for the penultimate moiety[6]. Roughly 20-fold weaker binding was seen with otherwise identical glycans that terminated in α2-3-linked N-acetylneuraminic acid (Neu5Ac), which differs by one hydroxyl group from Neu5Gc. Binding was reduced over 30-fold if the Neu5Gc linkage was changed from α2-3 to α2-6, and 100-fold if the terminal sialic acid was removed. This high specificity for Neu5Gc-terminating glycans is believed to be unique amongst bacterial toxins[6]. Identification of the SubB receptor glycan informed structural analysis of SubB in complex with synthetic oligosaccharides provided by another CFG PI. This showed that Neu5Gc bound to a shallow pocket halfway down the sides of the SubB pentamer, whereas identical experiments using Neu5Ac failed to show any binding[6]. In contrast, CtxB whose receptor is a ganglioside rather than a glycoprotein, has a deep receptor binding pocket located on the base of the pentamer, juxtaposed to the membrane[7]. In the SubB-Neu5Gc complex, Neu5Gc makes key interactions with the side chains of Asp8, Ser12, Glu36 and Tyr78[6]. Neu5Gc differs from Neu5Ac by the addition of a hydroxyl on the methyl group of the N-Acetyl moiety, which makes additional crucial interactions with SubB; namely the extra hydroxyl points towards and interacts with Tyr78OH and also hydrogen bonds with the main chain of Met10. These key interactions could not occur with Neu5Ac, thus explaining the marked preference for Neu5Gc6. The biological relevance of the structural analysis has been confirmed by further interacting residues[6].

Contents

CFG Participating Investigators contributing to the understanding of this paradigm

CFG Participating Investigators (PIs) have made seminal contributions to understanding the biology of this highly potent toxin, in particular the molecular interactions between the binding subunit SubB and cognate host cell glycans described above. These include Xi Chen, Adrienne Paton, James Paton, David Smith, and Ajit Varki. Importantly, on-going collaborations have been established between these PIs as a consequence of involvement in the CFG, and these have already generated one collaborative paper in Nature[6].

Progress toward understanding this GBP paradigm

This section documents what is currently known about SubAB, its carbohydrate ligand(s), and how they interact to mediate cell communication.

Carbohydrate ligands

SubB binds N-linked glycans displayed on glycoproteins on the surface of Vero and HeLa cells, including α2β1integrin[5]. Screening with the CFG glycan array showed that SubB has a high degree of specificity for glycans terminating with α2-3-linked N-glycolylneuraminic acid (Neu5Gc), with little discrimination for the penultimate moiety[6]. Roughly 20-fold weaker binding was seen with otherwise identical glycans that terminated in α2-3-linked N-acetylneuraminic acid (Neu5Ac), which differs by one hydroxyl group from Neu5Gc. Binding was reduced over 30-fold if the Neu5Gc linkage was changed from α2-3 to α2-6, and 100-fold if the terminal sialic acid was removed. This high specificity for Neu5Gc-terminating glycans is believed to be unique amongst bacterial toxins[6].

Cellular expression of GBP and ligands

Subtilase cytotoxin, SubAB, is produced by Shiga toxigenic E. coli. Other members of the AB5 toxin family are expressed as follows: Cholera toxin (Ctx) is produced by Vibrio cholerae, the heat-labile enterotoxins LT-I and LT-II are produced by enterotoxigenic E. coli (ETEC), pertussis toxin (Ptx) is produced by Bordetella pertussis, and shiga toxin (Stx) is produced by Shiga toxigenic E. coli (STEC) and Shigella dysenteriae.
AB5 toxin family members bind to glycan receptors in the host. The glycan receptors for AB5 toxin B subunit pentamers are displayed either on glycolipids (for Ctx/LT and Stx) or on glycoproteins (for Ptx and SubAB).

Biosynthesis of ligands

Synthesis of CMP-NeuGc from CMP-NeuAc is mediated by cytidine monophospho-N-acetylneuraminic acid hydroxylase, encoded by the Cmah gene, which is functional in mice but not in humans[8]. Transfer of the NeuGc to glycan acceptors is mediated by multiple 2,3-sialyltransferases (Database), which work with either N-acetyl- or N-glycolylneuraminic acid donors[9].

Structure

Structural analysis of SubB in complex with synthetic oligosaccharides showed that Neu5Gc binds to a shallow pocket halfway down the sides of the SubB pentamer, whereas identical experiments using Neu5Ac failed to show any binding[6]. In contrast, CtxB, whose receptor is a ganglioside rather than a glycoprotein, has a deep receptor binding pocket located on the base of the pentamer, juxtaposed to the membrane[7]. In the SubB-Neu5Gc complex, Neu5Gc makes key interactions with the side chains of Asp8, Ser12, Glu36 and Tyr78[6]. Neu5Gc differs from Neu5Ac by the addition of a hydroxyl on the methyl group of the N-Acetyl moiety, which makes additional crucial interactions with SubB; namely, the extra hydroxyl points towards and interacts with Tyr78OH and also hydrogen bonds with the main chain of Met10. These key interactions could not occur with Neu5Ac, thus explaining the marked preference for Neu5Gc6.

Biological roles of GBP-ligand interaction

Glycan receptor specificity is critical for the pathogenic process, as it determines host susceptibility, tissue tropism, and the nature and spectrum of the resultant pathology.

CFG resources used in investigations

The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the CFG database search results for SubAB and subtilase.

Glycan profiling


Glycogene microarray

SubAB is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.

Knockout mouse lines

Not applicable.

Glycan array

The CFG glycan array was fundamental in the identification and characterization of SubB receptor specificity. This information then permitted structural analysis of protein-glycan complexes. Glycan array analysis was also critical for investigating the influence of mutation of SubB residues predicted to be critical for Neu5Gc-specific binding on the repertoire of glycan structures engaged by the toxin. To see all glycan array results for subtilase cytotoxin, click here.

Related GBPs

References

  1. ↑ 1.0 1.1 Fan, E. et al. (2000). AB5 toxins: structures and inhibitor design. Curr. Opin. Struct. Biol. 10: 680-686.
  2. ↑ Paton, A.W., Srimanote, P., Talbot, U.M., Wang, H., and Paton, J.C. (2004). A new family of potent AB5 cytotoxins produced by Shiga toxigenic Escherichia coli. J. Exp. Med. 200: 35-46.
  3. ↑ Paton, A.W., Beddoe, T., Thorpe, C.M., Whisstock, J.C., Wilce, M.C.J., Rossjohn, J., Talbot, U.M. and Paton J.C. (2006). AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP. Nature 443: 548-552.
  4. ↑ Merrit, E. A. and Hol, W. G. J. (1995). AB5 toxins. Curr. Opin. Struct. Biol. 5: 165-171.
  5. ↑ 5.0 5.1 Yahiro, K., Morinaga, N., Satoh, M., Matsuura, G., Tomonaga, T., Nomura, F., Moss, J., Noda, M. (2006). Identification and characterization of receptors for vacuolating activity of subtilase cytotoxin. Mol. Microbiol. 62: 480–490.
  6. ↑ 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 Byres, E., Paton, A.W., Paton, J.C., Löfling, J.C., Smith, D.F., Wilce, M.C.J., Talbot, U.M., Chong, D.C., Yu, H., Huang, S., Chen, X., Varki, N.M., Varki, A., Rossjohn, J., and Beddoe, T. (2008). Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin. Nature. 456: 648-652.
  7. ↑ 7.0 7.1 Merritt, E.A.; Sarfarty, S.; Jobling, M.G.; Chang, T.; Holmes, R.K.; Hirst, T.R.; Hol, W.G. Structural studies of receptor binding by cholera toxin mutants. Protein Sci. 1997, 6, 1516–1528.
  8. ↑ Irie, A, Koyama, S, Kozutsumi, Y, Kawasaki, T and Suzuki, A (1998) The Molecular Basis for the Absence of N-Glycolylneuraminic Acid in Humans. J Biol Chem 273, 15866-15871
  9. ↑ Higa, HH and Paulson, JC (1986) Sialylation of glycoprotein oligosaccharides with N-Acetyl-, N-Glycolyl-, and N-O-Diacetylneuraminic Acids. J Biol Chem 260, 8836-8849

Acknowledgements

The CFG is grateful to the following PIs for their contributions to this wiki page: Joseph Barbieri, James Paton

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