Mutational Analysis of Ganglioside GM₁-Binding Ability, Pentamer Formation, and Epitopes of Cholera Toxin B (CTB) Subunits and CTB/Heat-Labile Enterotoxin B Subunit Chimeras

Abstract

Variants of cholera toxin B subunit (CTB) were made by bisulfite-and oligonucleotide-directed mutagenesis of the ctxB gene. Variants were screened by a radial passive immune hemolysis assay (RPIHA) for loss of binding to sheep erythrocytes (SRBC). Variant CTBs were characterized for the formation of immunoreactive pentamers, the ability to bind ganglioside GM 1 in vitro, and reactivity with a panel of monoclonal anti-CTB antibodies. Substitutions at eight positions (i.e., positions 22, 29, 36, 45, 64, 86, 93, and 100) greatly reduced the yield of immunoreactive CTB. RPIHA-negative substitution variants that formed immunoreactive pentamers were obtained for residues 12, 33, 36, 51, 52 ؉ 54, 91, and 95. Tyrosine-12 was identified as a novel residue important for GM 1 binding since, among all of the novel variants isolated with altered RPIHA phenotypes, only CTB with aspartate substituted for tyrosine at position 12 failed to bind significantly to ganglioside GM 1 in vitro. In contrast, CTB variants with single substitutions for several other residues (Glu-51, Lys-91, and Ala-95) that participate in GM 1 binding, based on the crystal structure of CTB and the oligosaccharide of GM 1 , were not appreciably altered in their ability to bind GM 1 in vitro, even though they showed altered RPIHA phenotypes and did not bind to SRBC. Hybrid B genes made by fusing ctxB and the related Escherichia coli heat-labile enterotoxin eltB genes at codon 56 produced CTB variants that had 7 or 12 heat-labile enterotoxin B residue substitutions in the amino or carboxyl halves of the monomer, respectively, each of which which also bound GM 1 as well as wild-type CTB. This collection of variant CTBs in which 47 of the 103 residues were substituted was used to map the epitopes of nine anti-CTB monoclonal antibodies (MAbs). Each MAb had a unique pattern of reactivity with the panel of CTB variants. Although no two of the epitopes recognized by different MAbs were identical, most of the single amino acid substitutions that altered the immunoreactivity of CTB affected more that one epitope. The tertiary structures of the epitopes of these anti-CTB MAbs are highly conformational and may involve structural elements both within and between CTB monomers. Substitution of valine for alanine at positions 10 and 46 had dramatic effects on the immunoreactivity of CTB, affecting epitopes recognized by eight or six MAbs, respectively.Cholera toxin (CT), an enterotoxin produced by Vibrio cholerae, is the prototype for the immunologically and structurally related family of heat-labile enterotoxins (LTs) produced by Vibrio cholerae and Escherichia coli (11). These toxins are composed of A and B subunits in a 1:5 molar ratio. Upon secretion to the periplasm in E. coli or V. cholerae, cholera toxin B (CTB) monomers spontaneously assemble into pentameric CTB and, in the presence of CTA, assemble into stable holotoxin. Free CTA and pentameric CTB, however, will not assemble into holotoxin. Delivery of the enzymatically active A subunit to the cytosol of sensit...

Cholera Holotoxin Assembly Requires a Hydrophobic Domain at the A-B5Interface: Mutational Analysis and Development of an In Vitro Assembly System

“…pARCT5 is an arabinose-inducible clone derived from pAR3 (21) expressing an operon containing the ctxA and ctxB genes with signal sequences derived from the LTIIb B gene and using the T7g10 Shine-Dalgarno sequence (15,16). pARCT5 is a derivative of pARCT4 containing a unique, translationally silent PstI site within ctxA1 (13,14).…”

Low-density Lipoprotein Receptor Deficiency Causes Impaired Osteoclastogenesis and Increased Bone Mass in Mice because of Defect in Osteoclastic Cell-Cell Fusion

“…6). In wild-type mononuclear preosteoclasts and multinucleated osteoclasts, Atp6v0d2 staining was more intense on the plasma membrane than in the perinuclear region, and a large amount of Atp6v0d2 was colocalized with the plasma membrane marker GM-1 ganglioside, which is a receptor of cholera toxin subunit B (56). In contrast, in LDLR Ϫ/Ϫ preosteoclasts, Atp6v0d2 remained abundant in the cytoplasm and in the perinuclear region, and little colocalization with GM-1 ganglioside was observed (Fig.…”

Glycosphingolipid Functions

“…CTx binding was the first described GSL receptor function (Heyningen 1974). The B subunit-GM1 oligosaccharide has been cocrystallized to resolve the binding site (Merritt et al 1994), but within this site, only tyrosine 12 was found crucial for GM1 binding (Jobling and Holmes 2002). B subunit-GM1 binding mediates the internalization of the holotoxin and its subsequent retrograde transport through endosomes, trans-Golgi network, and Golgi to the endoplasmic reticulum, in which the A subunit separates and is transported through the Sec61 translocon into the cytosol (Schmitz et al 2000).…”