Aminocaproic acid

Background & objectives: The proteolytic activity of plasmin promotes migration of pathogenic bacteria through the human extracellular matrix. The human pathogen Streptococcus pneumoniae binds both human plasminogen and plasmin via the surface displayed [alpha]-enolase designated Eno. Electron microscopic studies verified the surface exposition of the glycolytic enzyme [alpha]-enolase and moreover, its ability to reassociate to the cell surface. Carboxyterminal lysine residues of recently described eukaryotic and prokaryotic plasminogen-binding proteins such as SEN of S. pyogenes are involved in interaction with lysine binding sites of kringle domains of plasminogen. In this study, the role of carboxy terminal lysyl residue of eno in plasminogen binding is further analysed.

Methods: Site-directed mutagenesis of eno gene was done using DNA primers with Hind III-restriction enzyme sites for cloning. Purified Eno fusion proteins were separated by SDS-PAGE and human plasminogen binding assay was performed. Radioiodinated ligand binding was done by competitive inhibition assay.

Results: Binding assays performed under reduced conditions indicated also a role of the C-terminal lysyl residues of Eno for plasmin(ogen) binding. Binding of pneumococci to radioiodinated plasminogen was competitively inhibited in the presence of plasminogen, kringle 1-3 (LBS 1) and the lysine-analogon [epsilon]-amino caproic acid indicating the crucial role of lysine-binding sites of plasminogen. However, binding analysis of plasminogen and LBS 1 to wild type Eno and carboxy terminal modified Eno proteins did not reveal any difference in plasminogen-binding activity under native conditions.

Interpretation & conclusion: The present results suggested the presence of a further plasminogen-binding motif in Eno. This hypothesis was confirmed by plasminogen-binding activity of reassociated C-terminal modified enolase to the pneumococcal surface and indicated, therefore, the presence of a further binding motif in Eno for plasminogen binding.

Key words Lysin binding sites * plasminogen * Streptococcus pneumoniae

Streptococcus pneumoniae colonizes the nasopharynx and is also a main cause of upper and lower respiratory tract diseases. A prerequisite for invasiveness is the ability of pneumococci to transmigrate and to disseminate through the epithelial and endothelial layers and to breach the blood-brain barrier. Pathogenic bacteria utilize the proteolytic activity of plasmin to migrate faster through the human extracellular matrix1. The serin protease plasmin is the active form of the 92 kDa zymogen plasminogen and plays a key role in both extrinsic and intrinsic fibrinolysis. Binding of plasminogen and its subsequent activation by the tissue type plasminogen activator tPA has been demonstrated to promote migration of pneumococci through reconstituted basement membranes'. Previous studies showed that S. pneumoniae bind specifically plasmin(ogen) irrespective of serotype and capsule expression via the glycolytic enzyme Eno2. Despite the absence of a signal sequence required for secretion and membrane anchoring motifs, immunoelectron microscopy indicated the presence of Eno on the surface of encapsulated and non - encapsulated pneumococci. Furthermore, soluble Eno protein reassociates to the pneumococcal cell surface thereby enhancing plasminogen acquisition. The carboxy-terminal lysyl residues of Eno at position 433 and 434 were identified as binding site for the kringle motifs of plasmin(ogen). In this report, the role of carboxy-terminal lysyl resdues of Eno in plasminogen binding is further analysed. Plasminogen-binding studies with pneumococci after reassociation of Eno wildtype protein and Eno protein with carboxyterminal amino acid substitutions suggested the presence of a second plasminogen-binding motif.

Material & Methods

Bacterial strains and cell culture: S. pneumoniae were cultured in Todd-Hewitt-broth (Oxoid, Basingstoke, England) supplemented with 0.5 per cent yeast extract (THY) to mid-log phase or grown on blood agar (Merck).

Site directed mutagenesis of the eno gene'. Identification and expression cloning of pneumococcal a-enolase was described previously2. Site-directed mutagenesis of the eno gene was described previously and performed using DNA primers containing Hind III-restriction enzyme sites for cloning and modifications in the reverse primer to introduce the appropriate base substitutions at the 3' end of eno2. This procedure finally resulted in Eno^sup del^, Eno^sup KL^, and Eno^sup LL^ in which both C-terminal lysines were deleted or individually substituted by leucine residues.

Electrophoresis, blot overlay assay and immunoblot analysis: Purified Eno fusion proteins were subjected to SDS-PAGE with 12 per cent gels according to the method described by Laemmli3. The gels were either stained with Coomassie brilliant blue (CBB) or subsequently transferred to a nylon membrane (Immobilon-P, M illipore) using a semi-dry blotting system. Binding of human plasminogen was performed as described previously2.

Competitive inhibition assay: Binding of radioiodinated ligands were performed as described previously2. Briefly, human plasminogen (Sigma, USA) was radioiodinated with ^sup 125^I by a standard chloramin T method4. The binding of 20 ng ^sup 125^I-labelled plasminogen to 4 × 10^sup 8^ wild type pneumococcal cells was determined as pellet bound radioactivity in triple values. Plasminogen binding was expressed as a percentage of total radioactivity added. In competitive inhibition experiments binding to viable pneumococci was measured in the presence of increasing molar excess of unlabelled glu-plasminogen, lysine binding site 1 of plasminogen (kringle 1-3, Sigma) and the lysine analogon [epsilon]-aminocaproic acid (EACA, Merck) in amounts from 0.1 up to 50 µg. Binding to ^sup 125^I-plasminogen was also determined after preincubation of pneumococci to allow reassociation. Eno and Eno^sup KL^ was used in doses of 1, 5, 10, 20 and 50 µg per reaction.

Results

Competitive inhibition of plasminogen binding to Streptococcus pneumoniae: In competitive inhibition experiments the effects of plasmin(ogen), LBS 1 and the lysine analogon EACA on binding of ^sup 125^I-plasminogen to S. pneumoniae R6x was analysed. The results of the inhibition studies revealed that unlabelled plasminogen and also the EBS 1 of the plasminogen molecule containing lysine-binding sites are sufficient to inhibit in a dose depending manner binding of plasminogen to pneumococci (Fig.1). The value of the 50 per cent inhibitory dose for glu-plasminogen (0.17 µM) was in the same order of magnitude as those determined for EBS 1 (0.54 µM). The substrate analogon EACA also showed competitive inhibition of plasminogen binding with an increased value of 5.82 µM for 50 per cent inhibitory dose. The results suggested that a lysine-mediated interaction contributes to Eno-plasminogen interaction.

Role of C-terminal lysyl residues for plasminogenbinding activity under reducing conditions: To elucidate the role of C-terminal lysyl residues of Eno for plasminogen-binding activity, the lysyl residues at position 433 and 434 (Eno^sup LL^) or the terminal lysine (Eno^sup KL^) were substituted by leucine residues. The Eno proteins were seperated under reducing conditions on a SDS-PAGE and plasminogen-binding activity of wild type Eno and mutated Eno proteins was determined by blot overlay analysis.

The results indicated that the plasminogen-binding activity of both mutated Eno proteins was significantly reduced compared to wild type Eno (Fig.2).

Plasminogen-binding after reassociation of Eno on thepneumococcal cell surface: Previous binding studies with radioiodinated plasminogen showed, that reassociation of soluble Eno protein to cell surface of S. pneumonias enhanced pneumococcal plasminogen-binding activity. In binding assays with radioiodinated plasminogen the effect of reassociated Eno wild type protein compared to C-terminal substituted EnoKL protein on plasminogen binding to pneumococci was determined. The results of the binding assay demonstrated plasminogen-binding activity of both, reassociated wild type Eno and Eno^sup KL^ (Fig.3). These results supported the idea of a further important plasminogen-binding site of Eno which contributes to the Eno-plasminogen interaction.

Discussion

Plasmin(ogen) binding interaction is mediated by recognition of C-terminal lysine residues of eukaryotic enolases by the lysine binding sites of plasminogen5. The binding of S. pneumoniae to radioiodinated plasminogen is inhibited by unlabelled plasminogen and the LBS 1. These results suggested that the kringle 1-3 of the plasminogen molecule are involved in plasminogen-binding activity.

A dose dependent inhibition of plasminogen binding to pneumococci by the lysyl analogen EACA indicated that the lysine binding sites of the kringle domains seem to be important for binding of Eno. Interactions of plasmin(ogen) with aC-terminal lysyl residue have been described previously for another streptococcal plasmin(ogen)-binding protein5 and also for putative eukaryotic plasmin(ogen) receptors7,8. The amino acid sequence of the pneumococcal [alpha]-enolase is terminated by two lysine residues. Leucine substitutions for either the terminal lysine or both the penultimate and terminal lysyl residues significantly reduced plasminogen binding in blot overlay assays. These observations suggested the contribution of exposed carboxy-terminal lysines in plasminogen binding to Eno. However, results of binding experiments using the characteristic property of Eno to reassociate to the pneumococcal cell surface, showed a dose dependent binding of plasminogen to both reassociated wildtype Eno and C-terminal modified EnoKL protein. These results suggested the contribution of a further binding site in Eno in plasminogen binding in addition to the C-terminal lysyl residues. Pneumococcal [alpha]-enolase shows a 93 per cent homology to the streptococcal surface associated enolase (SEN) of S. pyogenes9. The presence of a further binding site in these enolases was suggested by Scatchard analysis which revealed a non-linear plot and resulted, therefore, in two dissociation constants for plasminogen binding to SEN6. This study underlined the importance of the second interaction site for plasminogen-binding under more physiological conditions. Since plasminogen binding and subsequent activation by host factors facilitate migration and dissemination of microorganisms through biological matrices and membranes10,11, the elucidation of the proposed binding site in Eno is pivotal to understand the host-pathogen interaction.

References

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Simone Bergmann, Manfred Rohde, Gursharan S. Chhatwal & Sven Hammerschmidt

Department of Microbial Pathogenicity, GBF-German Research Centre for Biotechnology 38124 Braunschweig, Germany

Received August 7, 2003

Reprint requests: Dr. Sven Hammerschmidt, Research Center for Infectious Diseases, University of Wurzburg, Rontgenring 11, 97070 Wurzburg, Germany

e-mail: S.Hammerschmidt@mail.uni-wuerzburg.de

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