Carbohydrate-mediated Regulation of Interaction of Vibrio cholerae Hemolysin with Erythrocyte and Phospholipid Vesicle

Abstract

Vibrio cholerae hemolysin is an extracellular pore-forming monomeric protein with a native molecular weight of about 60,000. In this study, we showed that the hemolysin interacted with immobilized phospholipids and cholesterol and formed oligomers in vesicles constituted from phospholipids alone with a stoichiometry identical to those produced in rabbit erythrocyte membrane. However, the hemolysin bound to glycoproteins with terminal beta1-galactosyl residues and an association constant of 9.4 x 10(7) M(-1) was estimated for the hemolysin-asialofetuin complex by solid phase binding assay. Oligomerization of the hemolysin in lipid bilayer converted the sugar-binding monomer to a lectin with strong carbohydrate-dependent hemagglutinating activity accompanied by inactivation of hemolytic activity and loss in ability to interact with phospholipids. There was no evidence for erythrocyte surface carbohydrates playing an essential role in interaction of the hemolysin with the cell. However, specific glycoproteins inhibited hemolysis of rabbit erythrocytes as well as interaction of the hemolysin with phospholipid. The results suggest (i) V. cholerae hemolysin is a monomer with distinct domains associated with specific binding to carbohydrates and interaction with lipids, (ii) the pore-forming property depends solely on the protein-lipid interaction with no evidence for involvement of sugars, and (iii) specific sugars can down-regulate the ability of the hemolysin to form pores in lipid bilayers.

Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins

“…The VCC protoxin (17) consists of a core cytolytic domain containing the single prestem (pore-forming) loop, an amino-terminal 15-kDa prodomain, and two carboxyl-terminal domains with lectin-like folds that may aid in binding to carbohydrate receptors (18). Functional studies indicate that proteolytic activation must first occur within a consecutive string of protease sites that connect the prodomain to the toxin core (19).…”

Oligomerization of Vibrio Cholerae Hemolysin Induces CXCR3 Upregulation and Activation of B-1a Cell

“…The HlyA monomer and oligomer were purified from a non-O1 V. cholerae strain V 2 as described before (2,8).…”

“…In contrast, the HlyA oligomer (2), though recognized by the same TLR, results in nuclear translocation of NF-κB and induction of IgA. In this paper, we evaluated B-1a cell proliferation, expression of B cell activation marker CD25 (3), and upregulation of the costimulatory molecules, CD80-CD86, as hallmark of cell activation and prelude to HlyA oligomer-induced IgA response.…”

Structural Requirements of Cholesterol for Binding to Vibrio cholerae Hemolysin

“…Therefore, it also seems reasonable to propose that cholesterol is a binding site for VCH. However, it has been shown that VCH also binds to saccharides (21), glycophorin B (27), and sphingolipids (32), respectively, present on target membranes; this suggests that target membranes have multiple binding components for the VCH molecule.…”