title: Mass spectrometric analysis of recombinant adenylate cyclase toxin from Bordetella pertussis strain 18323/pHSP9.
abstract: The adenylate cyclase toxin-hemolysin (ACT) is a key virulence factor of the whooping cough agent Bordetella pertussis (Bp). The major cytotoxic activity of this 1706-residue protein consists of its capacity to invade a variety of eukaryotic cells directly across their cytoplasmic membrane and to deliver into cells a catalytic adenylate cyclase domain. This causes impairment of immune effector cells and apoptosis of lung macrophages by uncontrolled conversion of ATP to cAMP. The adenylate cyclase toxin-hemolysin acquires biological activity upon post-translational amide-linked palmitoylation of the epsilon-amino group of lysine 983 (K983) by the accessory fatty acyltransferase, CyaC. However, an additional conserved acylation site can be identified in ACT at lysine 860 (K860) and this residue is palmitoylated when recombinant ACT is produced in Escherichia coli (r-Ec-ACT). In this paper we report the double acylation of r-Bp-ACT secreted by a recombinant Bp strain 18323/pHSP9. This strain overproduces ACT from an oligocopy plasmid carrying the entire cya locus of Bordetella pertussis 18323. Palmitoylation of both conserved lysines (K860 and K983) of r-Bp-ACT expressed by this Bp strain was found. In addition, an error in the deduced protein sequence was identified, with Leu being the real residue at position 1001 and not the Val residue given in the published gene sequence. We also discuss these results in comparison with those from recombinant ACT expressed in E. coli strain K12 XL1-Blue. The analytical approach for characterization of the fatty acylation of ACT from strain 18323/pHSP9 consisted of multiple proteolytic digestion procedures (trypsin, Asp-N), microcapillary liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
authors.affiliation: Department of Medicinal Chemistry, Box 357610, University of Washington, Seattle, Washington 98195, USA.
authors.forename: V
authors.initials: V
authors.lastname: Havlícek
authors.forename: L
authors.initials: L
authors.lastname: Higgins
authors.forename: W
authors.initials: W
authors.lastname: Chen
authors.forename: P
authors.initials: P
authors.lastname: Halada
authors.forename: P
authors.initials: P
authors.lastname: Sebo
authors.forename: H
authors.initials: H
authors.lastname: Sakamoto
authors.forename: M
authors.initials: M
authors.lastname: Hackett
mesh_terms: D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000032:analysis*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D001886:Bordetella pertussis / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D010566:Virulence Factors, Bordetella / Q000737:chemistry*
chemical_list: D037361:Adenylate Cyclase Toxin; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D000262:Adenylyl Cyclases