title: Mutations in the A subunit affect yield, stability, and protease sensitivity of nontoxic derivatives of heat-labile enterotoxin.
abstract: Heat-labile toxin (LT) is a protein related to cholera toxin, produced by enterotoxigenic Escherichia coli strains, that is organized as an AB5 complex. A number of nontoxic derivatives of LT, useful for new or improved vaccines against diarrheal diseases or as mucosal adjuvants, have been constructed by site-directed mutagenesis. Here we have studied the biochemical properties of the nontoxic mutants LT-K7 (Arg-7-->Lys), LT-D53 (Val-53-->Asp), LT-K63 (Ser-63-->Lys), LT-K97 (Val-97-->Lys), LT-K104 (Tyr-104-->Lys), LT-K114 (Ser-114-->Lys), and LT-K7/K97 (Arg-7-->Lys and Val-97-->Lys). We have found that mutations in the A subunit may have profound effects on the ability to form the AB5 structure and on the stability and trypsin sensitivity of the purified proteins. Unstable mutants, during long-term storage at 4 degrees C, showed a decrease in the amount of the assembled protein in solution and a parallel appearance of soluble monomeric B subunit. This finding suggests that the stability of the B pentamer is influenced by the A subunit which is associated with it. Among the seven nontoxic mutants tested, LT-K63 was found to be efficient in AB5 production, extremely stable during storage, resistant to proteolytic attack, and very immunogenic. In conclusion, LT-K63 is a good candidate for the development of antidiarrheal vaccines and mucosal adjuvants.
authors.affiliation: IRIS, the Chiron Biocine Immunobiological Research Institute Siena, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Magagnoli
authors.forename: R
authors.initials: R
authors.lastname: Manetti
authors.forename: M R
authors.initials: MR
authors.lastname: Fontana
authors.forename: V
authors.initials: V
authors.lastname: Giannelli
authors.forename: M M
authors.initials: MM
authors.lastname: Giuliani
authors.forename: R
authors.initials: R
authors.lastname: Rappuoli
authors.forename: M
authors.initials: M
authors.lastname: Pizza
mesh_terms: D010450:Endopeptidases / Q000378:metabolism; D004768:Enterotoxins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D009154:Mutation; D013379:Substrate Specificity
chemical_list: D004768:Enterotoxins; D010450:Endopeptidases