title: Functional and structural studies on different forms of the adenylate cyclase toxin of Bordetella pertussis.
abstract: A comparison was made of the cytotoxic activity and secondary structural features of four recombinant forms of adenylate cyclase toxin (CyaA). These forms were fully functional CyaA, CyaA lacking adenylate cyclase enzymatic activity (CyaA*), and non-acylated forms of these toxins, proCyaA and proCyaA*. At a toxin concentration>1 microg/ml, CyaA* was as cytotoxic towards J774.2 cells as CyaA and mediated cell killing at a faster rate than CyaA. At concentrations<0.5 microg/ml, CyaA* was less cytotoxic than CyaA and, at <0.1 microg/ml of CyaA*, no activity was detected. CyaA, but not CyaA*, was able to induce caspase 3/7 activity, a measure of apoptosis. ProCyaA and proCyaA* had no detectable cytotoxic or apoptotic activity. CyaA caused 50% inhibition of the zymosan-stimulated oxidative burst at 0.003 microg/ml, whereas a approximately 500-fold greater toxin concentration of CyaA* or proCyaA was needed for 50% inhibition. ProCyaA* was inactive. CyaA is a calcium-binding protein and far UV circular dichroism (CD), near UV CD and fluorescence spectra analyses showed that all the forms of CyaA had similar overall structures at different calcium concentrations up to 5.0 mM. At 7.5 mM CaCl2, the far UV spectrum of CyaA altered significantly, indicating a change in secondary structure associated with high beta-sheet content or a beta-aggregated state, whereas the spectrum of CyaA* showed only a slight alteration at this calcium concentration. Near UV CD and fluorescence studies were consistent with a rearrangement of secondary structural elements in the presence of CaCl2 for all CyaA forms. There was a marked dependence on protein concentration of the far UV spectra of these CyaA forms, implying an interaction between individual molecules at higher protein concentrations.
authors.affiliation: Division of Infection and Immunity, University of Glasgow, Institute of Biomedical and Life Sciences, Glasgow Biomedical Research Centre, 120 University Place, Glasgow G12 8TA, UK.
authors.forename: Gordon Y C
authors.initials: GY
authors.lastname: Cheung
authors.forename: Sharon M
authors.initials: SM
authors.lastname: Kelly
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Jess
authors.forename: Sandra
authors.initials: S
authors.lastname: Prior
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Price
authors.forename: Roger
authors.initials: R
authors.lastname: Parton
authors.forename: John G
authors.initials: JG
authors.lastname: Coote
mesh_terms: D000215:Acylation; D037361:Adenylate Cyclase Toxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D001886:Bordetella pertussis / Q000201:enzymology* / Q000235:genetics; D002460:Cell Line; D002942:Circular Dichroism; D005453:Fluorescence; D008264:Macrophages; D051379:Mice; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases