| 1. |
- Ringdahl, Ulrika, et al.
(författare)
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Molecular co-operation between protein PAM and streptokinase for plasmin acquisition by Streptococcus pyogenes.
- 1998
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 273:11, s. 6424-6430
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial surface-associated plasmin formation is believed to contribute to invasion, although the underlying molecular mechanisms are poorly understood. To define the components necessary for plasmin generation on group A streptococci we used strain AP53 which exposes an M-like protein ("PAM") that contains a plasminogen-binding sequence with two 13-amino acid residues long tandem repeats (a1 and a2). Utilizing an Escherichia coli-streptococcal shuttle vector, we replaced a 29-residue long sequence segment of Arp4, an M-like protein that does not bind plasminogen, with a single (a1) or the combined a1a2 repeats of PAM. When expressed in E. coli, the purified chimeric Arp/PAM proteins both bound plasminogen, as well as plasmin, and when used to transform group A streptococcal strains lacking the plasminogen-binding ability, transformants with the Arp/PAM constructs efficiently bound plasminogen. Moreover, when grown in the presence of plasminogen, both Arp/PAM- and PAM-expressing streptococci acquired surface-bound plasmin. In contrast, plasminogen activation failed to occur on PAM- and Arp/PAM-expressing streptococci carrying an inactivated streptokinase gene: this block was overcome by exogenous streptokinase. Together, these results provide evidence for an unusual co-operation between a surface-bound protein, PAM, and a secreted protein, streptokinase, resulting in bacterial acquisition of a host protease that is likely to spur parasite invasion of host tissues.
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| 2. |
- Wistedt, AC, et al.
(författare)
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Identification of a plasminogen-binding motif in PAM, a bacterial surface protein.
- 1995
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Ingår i: Molecular Microbiology. - 1365-2958. ; 18:3, s. 569-578
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Tidskriftsartikel (refereegranskat)abstract
- Surface-associated plasmin(ogen) may contribute to the invasive properties of various cells. Analysis of plasmin(ogen)-binding surface proteins is therefore of interest. The N-terminal variable regions of M-like (ML) proteins from five different group A streptococcal serotypes (33, 41, 52, 53 and 56) exhibiting the plasminogen-binding phenotype were cloned and expressed in Escherichia coli. The recombinant proteins all bound plasminogen with high affinity. The binding involved the kringle domains of plasminogen and was blocked by a lysine analogue, 6-aminohexanoic acid, indicating that lysine residues in the M-like proteins participate in the interaction. Sequence analysis revealed that the proteins contain common 13-16-amino-acid tandem repeats, each with a single central lysine residue. Experiments with fusion proteins and a 30-amino-acid synthetic peptide demonstrated that these repeats harbour the major plasminogen-binding site in the ML53 protein, as well as a binding site for the tissue-type plasminogen activator. Replacement of the lysine in the first repeat with alanine reduced the plasminogen-binding capacity of the ML53 protein by 80%. The results precisely localize the binding domain in a plasminogen surface receptor, thereby providing a unique ligand for the analysis of interactions between kringles and proteins with internal kringle-binding determinants.
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