| Autor: |
Hartford, Orla M., Wann, Elisabeth R., Höök, Magnus, Foster, Timothy J. |
| Zdroj: |
Journal of Biological Chemistry; January 2001, Vol. 276 Issue: 4 p2466-2473, 8p |
| Abstrakt: |
Clumping factor A (ClfA) is a cell surface-associated protein of Staphylococcus aureusthat promotes binding of this pathogen to both soluble and immobilized fibrinogen (Fg). Previous studies have localized the Fg-binding activity of ClfA to residues 221–559 within the A region of this protein. In addition, the C-terminal part of the A region (residues 484–550) has been implicated as being important for Fg binding. In this study, we further investigate the involvement of this part of ClfA in the interaction of this protein with Fg. Polyclonal antibodies generated against a recombinant protein encompassing residues 500–559 of the A region inhibited the interaction of both S. aureusand recombinant ClfA with immobilized Fg in a dose-dependent manner. Using site-directed mutagenesis, two adjacent residues, Glu526and Val527, were identified as being important for the activity of ClfA. S. aureusexpressing ClfA containing either the E526A or V527S substitution exhibited a reduced ability to bind to soluble Fg and to adhere to immobilized Fg. Furthermore,bacteria expressing ClfA containing both substitutions were almost completely defective in Fg binding. The E526A and V527S substitutions were also introduced into recombinant ClfA (rClfA-(221–559)) expressed in Escherichia coli. The single mutant rClfA-(221–559) proteins showed a significant reduction in affinity for both immobilized Fg and a synthetic fluorescein-labeled C-terminal γ-chain peptide compared with the wild-type protein, whereas the double mutant rClfA-(221–559) protein was almost completely defective in binding to either species. Substitution of Glu526and/or Val527did not appear to alter the secondary structure of rClfA-(221–559) as determined by far-UV circular dichroism spectroscopy. These data suggest that the C terminus of the A region may contain at least part of the Fg-binding site of ClfA and that Glu526and Val527may be involved in ligand recognition. |
| Databáze: |
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