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J Biol Chem. 2014 Feb 14;289(7):4001-8. doi: 10.1074/jbc.M113.522284. Epub 2013 Dec 19.

The β-prism lectin domain of Vibrio cholerae hemolysin promotes self-assembly of the β-pore-forming toxin by a carbohydrate-independent mechanism.

Author information

1
From the Divisions of Biochemistry and.

Abstract

Vibrio cholerae cytolysin/hemolysin (VCC) is an amphipathic 65-kDa β-pore-forming toxin with a C-terminal β-prism lectin domain. Because deletion or point mutation of the lectin domain seriously compromises hemolytic activity, it is thought that carbohydrate-dependent interactions play a critical role in membrane targeting of VCC. To delineate the contributions of the cytolysin and lectin domains in pore formation, we used wild-type VCC, 50-kDa VCC (VCC(50)) without the lectin domain, and mutant VCC(D617A) with no carbohydrate-binding activity. VCC and its two variants with no carbohydrate-binding activity moved to the erythrocyte stroma with apparent association constants on the order of 10(7) M(-1). However, loss of the lectin domain severely reduced the efficiency of self-association of the VCC monomer with the β-barrel heptamer in the synthetic lipid bilayer from ∼83 to 27%. Notably, inactivation of the carbohydrate-binding activity by the D617A mutation marginally reduced oligomerization to ∼77%. Oligomerization of VCC(50) was temperature-insensitive; by contrast, VCC self-assembly increased with increasing temperature, suggesting that the process is driven by entropy and opposed by enthalpy. Asialofetuin, the β1-galactosyl-terminated glycoprotein inhibitor of VCC-induced hemolysis, promoted oligomerization of 65-kDa VCC to a species that resembled the membrane-inserted heptamer in stoichiometry and morphology but had reduced global amphipathicity. In conclusion, we propose (i) that the β-prism lectin domain facilitated toxin assembly by producing entropy during relocation in the heptamer and (ii) that glycoconjugates inhibited VCC by promoting its assembly to a water-soluble, less amphipathic oligomer variant with reduced ability to penetrate the bilayer.

KEYWORDS:

Carbohydrate Glycoprotein; Lectin; Lipid-binding Protein; Liposomes; Pore-forming Toxins; Protein Assembly; Vibrio cholerae Hemolysin

PMID:
24356964
PMCID:
PMC3924267
DOI:
10.1074/jbc.M113.522284
[Indexed for MEDLINE]
Free PMC Article

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