Formation of disulfide bonded dimer of mutated heat‐labile enterotoxin in vivo

Hedges, Peter A.; Hardy, Simon

doi:10.1016/0014-5793(96)00082-8

Cited by 3 publications

(3 citation statements)

References 10 publications

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“…However, if the disruption of the ␣1 helix only affected disulfide bond formation and not subsequent protein folding, the mutant protein would have been present in the total cell extract. Earlier studies carried out in the presence of reducing agents also suggest that disulfide bond formation is not necessary for maintaining the integrity of the ␣1 helix (20,41). The failure to observe the presence of the mutant protein in the total cell extract indicates that the ␣1 helix deletion possibly heightens the protease sensitivity of mutant LTB.…”

Section: Discussionmentioning

confidence: 98%

Deletion mutations in N-terminal α1 helix render heat labile enterotoxin B subunit susceptible to degradation

Alone¹,

Malik²,

Krishnan³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli is a heterohexameric protein consisting of an enzymatically active A subunit, LTA, and a carrier pentameric B subunit, LTB. It is clear from the crystal structure of LTB that the N-terminal ␣1 helix lies outside the core structure. However, the function of the N-terminal ␣1 helix of LTB is unknown. The present work was carried out to investigate the effect of site-directed mutagenesis of the ␣1 helix on LTB synthesis. Six amino acids (PQSITE) located at positions 2-7 from the N terminus, including 4 aa from the ␣1 helix, were deleted by site-directed mutagenesis. The deletion resulted in complete inhibition of LTB expression in E. coli when expressed along with its signal sequence. A single amino acid deletion within the ␣1 helix also resulted in loss of expression. However, a single amino acid deletion outside the ␣1 helix did not affect LTB synthesis. Mutant proteins, whose synthesis was not detected in vivo, could be successfully translated in vitro by using the coupled transcriptiontranslation system. Immunoblot analysis, Northern blot analysis, and in vitro transcription-translation data collectively indicate that the lack of synthesis of the mutant proteins is caused by the immediate degradation of the expressed product by cellular proteases rather than by faulty translation of mutant LTB mRNA. Coexpression of the LTA could not rescue the degradation of LTB mutants.gene expression ͉ site-directed mutagenesis ͉ coupled transcription-translation D iarrhea caused by the enterotoxigenic Escherichia coli (ETEC) is a major cause of death in developing countries, especially among children, with an estimated mortality of 1.5 million cases every year (1-3; for review, see ref. 4). Approximately 20% of cases of traveler's diarrhea are caused by ETEC, and thus the organism spreads to the developed countries (4, 5). ETEC produces a number of virulence factors, such as enterotoxins and colonization factors. Of these, the heat-labile (LT) and heat-stable enterotoxins produced by the ETEC are the major virulence factors responsible for its pathogenicity (5, 6). The LT belongs to a family of bacterial proteins designated heat-labile enterotoxins and shares phenotypic and genotypic similarities with other members of the family such as cholera toxin produced by Vibrio cholerae (7, 8; for review, see ref. 9).The mature toxin consists of a single A polypeptide (LTA) and five B polypeptides (LTB) (10-12). LTB and cholera toxin B show a high degree of homology, with 85% conservation of amino acids (13). The genes of the two LT subunits, eltA and eltB, are transcribed as a single polycistronic mRNA (14) and are expressed with signal peptides. The two subunits are synthesized as a precursor protein, and each subunit has its own ribosome-binding site (15-17). After cleavage of the signal peptide, the two subunits of LT are released into the periplasmic space where they spontaneously assemble into a mature holotoxin (18,19). LTA is known to influence LTB oligomeriz...

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Section: Discussionmentioning

confidence: 98%

Deletion mutations in N-terminal α1 helix render heat labile enterotoxin B subunit susceptible to degradation

Alone¹,

Malik²,

Krishnan³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…The A2 fragment ends in a somewhat disordered segment adopting a small helix, where the pore is widening toward the membrane (Figure 7.2A, insert). This segment is more buried in CT compared to LT, where it extends further through the pore (Figure 7.3), potentially explaining the milder symptoms caused by LT compared to CT [27]: A larger number of interactions of the B pentamer with the CTA2 tail compared to LTA2 rationalizes the increased stability and concomitant increase in toxicity observed for holotoxin chimera featuring the 10-amino-acid-residue-stretch from CT (226)(227)(228)(229)(230)(231)(232)(233)(234)(235)(236) [40,41], since holotoxin integrity is important during uptake and transport into intestinal epithelia. At the very tip of the A2 fragment, pointing toward the membrane, is a stretch of highly disordered amino acids with the KDEL-sequence (-Lys-Asp-Glu-Leu-COOH) (LT: RDEL, with Arg replacing Lys), aiding the transport of the toxin to the endoplasmic reticulum (ER) after internalization.…”

Section: A Subunitmentioning

confidence: 95%

“…The toxins spontaneously reoligomerize after acid denaturation followed by neutralization, showing that the holotoxins can assemble into their quaternary structures without the help of chaperones [230][231][232]. Within the periplasm, however, the accumulated free B subunits are more prone to aggregation and require chaperones to shield their hydrophobic surfaces and prevent association with the membrane for efficient assembly of the toxin [233,234]. DsbA was shown to assume such a role in E. coli [235].…”

Section: Toxin Folding and Assemblymentioning

confidence: 99%