title: Deletion mutations in N-terminal alpha1 helix render heat labile enterotoxin B subunit susceptible to degradation.
abstract: 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 alpha1 helix lies outside the core structure. However, the function of the N-terminal alpha1 helix of LTB is unknown. The present work was carried out to investigate the effect of site-directed mutagenesis of the alpha1 helix on LTB synthesis. Six amino acids (PQSITE) located at positions 2-7 from the N terminus, including 4 aa from the alpha1 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 alpha1 helix also resulted in loss of expression. However, a single amino acid deletion outside the alpha1 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 transcription-translation 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.
authors.affiliation: Gene Regulation Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Pankaj V
authors.initials: PV
authors.lastname: Alone
authors.forename: Gunjan
authors.initials: G
authors.lastname: Malik
authors.forename: Anuja
authors.initials: A
authors.lastname: Krishnan
authors.forename: Lalit C
authors.initials: LC
authors.lastname: Garg
mesh_terms: D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D054307:Enterotoxigenic Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D004768:Enterotoxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014158:Transcription, Genetic
chemical_list: D001427:Bacterial Toxins; D017931:DNA Primers; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; C027538:heat-labile enterotoxin, E coli; D008274:Magnesium