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Titolo:
Mass spectrometric analysis of recombinant adenylate cyclase toxin from Bordetella pertussis strain 18323/pHSP9
Autore:
Havlicek, V; Higgins, LA; Chen, WB; Halada, P; Sebo, P; Sakamoto, H; Hackett, M;
Indirizzi:
Univ Washington, Dept Med Chem, Seattle, WA 98195 USA Univ Washington Seattle WA USA 98195 Dept Med Chem, Seattle, WA 98195 USA Acad Sci Czech Republ, Inst Microbiol, CZ-14220 Prague 4, Czech Republic Acad Sci Czech Republ Prague Czech Republic 4 0 Prague 4, Czech Republic Inst Pasteur, Lab Biol & Genet Paludisme, Paris, France Inst Pasteur Paris France ur, Lab Biol & Genet Paludisme, Paris, France
Titolo Testata:
JOURNAL OF MASS SPECTROMETRY
fascicolo: 4, volume: 36, anno: 2001,
pagine: 384 - 391
SICI:
1076-5174(200104)36:4<384:MSAORA>2.0.ZU;2-J
Fonte:
ISI
Lingua:
ENG
Soggetto:
ESCHERICHIA-COLI; FATTY-ACYLATION; HEMOLYSIN HLYA; IN-VIVO; ACTIVATION; ELECTROSPRAY; PROTEIN; CYAC;
Keywords:
recombinant adenylate cyclase toxin; Bordetella pertussis; microcapillary liquid chromatography/tandem mass spectrometry; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Physical, Chemical & Earth Sciences
Citazioni:
20
Recensione:
Indirizzi per estratti:
Indirizzo: Hackett, M Univ Washington, Dept Med Chem, Box 357610, Seattle, WA 98195 USA Univ Washington Box 357610 Seattle WA USA 98195 e, WA 98195 USA
Citazione:
V. Havlicek et al., "Mass spectrometric analysis of recombinant adenylate cyclase toxin from Bordetella pertussis strain 18323/pHSP9", J MASS SPEC, 36(4), 2001, pp. 384-391

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 wasidentified, 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-night mass spectrometry. Copyright (C) 2001 John Wiley & Sons, Ltd.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 21/09/20 alle ore 12:53:20