Role of staphylokinase in the acquisition of plasmin(ogen)-dependent enzymatic activity by staphylococci.

In this study, the role of the staphylococcal plasminogen activator, staphylokinase (SAK), was analyzed for its ability to mediate acquisition of cell-associated plasmin-like activity by staphylococci in the presence of a source of human plasminogen. A panel of staphylococcal strains isolated from humans was tested for the presence of the SAK gene, secretion of the plasminogen activator, and the ability to acquire enzymatic activity when incubated with purified human plasminogen or serum. When SAK was compared with the eukaryotic plasminogen activators, urokinase and tissue plasminogen activator, only SAK could mediate acquisition of cell-associated enzymatic activity by staphylococci without first generating significant fluid-phase plasmin. These studies provide evidence for a novel mechanism by which SAK-producing S. aureus can acquire an unregulatable host plasmin-like activity that might contribute to their invasive potential.

Binding of human serum amyloid P-component to phosphocholine.

Human serum amyloid P-component (SAP) and C-reactive protein (CRP) are structurally similar pentraxins composed of five identical subunits in a disc-like configuration and display Ca(2+)-dependent binding reactivity to a variety of unrelated ligands. CRP is generally classified and defined as a phosphocholine (PC)-binding protein, whereas SAP is identified as a polysaccharide-binding protein. We examined the PC-binding activity of human SAP and compared it to human CRP since many of the biological activities of CRP are triggered upon PC-binding. SAP was able to bind to immobilize PC in a saturable, Ca(2+)-dependent manner but with lower avidity than CRP in direct competitive binding assays. The affinity of the binding of SAP to soluble [14C]PC was slightly lower than the affinity of CRP; however, the valence of SAP was only one PC-binding site/pentraxin or 2/protein vs 5 such sites per CRP molecule. Both SAP and CRP displayed a similar binding preference for PC vs phosphoethanolamine (PE). Two monoclonal antibodies (mAb) generated against the PC-binding site of SAP also reacted with the PC-binding site of CRP and inhibited PC-binding by both pentraxins. A mAb specific for the PC-binding site on CRP also inhibited SAP binding to PC. SAP was also recognized by two anti-idiotypic mAb that shared reactivity with the TEPC-15 PC-binding myeloma protein and the PC-binding site of CRP. Both pentraxins could be isolated from human serum by affinity chromatography on either PC- or PE-substituted agarose beads. The findings indicate that SAP is also a PC-binding protein.

Specificity of the binding interaction between human serum amyloid P-component and immobilized human C-reactive protein.

C-reactive protein (CRP) and serum amyloid P-component (SAP) are two members of a group of plasma proteins termed pentraxins, which are composed of five identical noncovalently linked subunits that display Ca(2+)-dependent binding to a wide variety of substrates. Purified human SAP binds to CRP, only when the latter is immobilized, in a Ca(2+)-dependent manner under physiological conditions. Externally labeled SAP rapidly binds to two distinct forms of immobilized CRP (direct and phosphorylcholine captured) with a relatively high affinity (KD = 5 nM) at a molar ratio of specifically bound SAP/CRP = 0.3. Studies of binding inhibition using monoclonal antibodies to CRP or synthetic peptides of CRP revealed that residues 134-148 and the COOH-terminal region (residues 191-206) were recognized by SAP. A fragment of CRP consisting of the COOH-terminal 60 residues within each subunit was also selectively bound by SAP. The ability of immobilized CRP to bind SAP was distinguished from CRP's lectin-like binding reactivity since deglycosylated SAP retained its binding reactivity for CRP and sugars that inhibit CRP's lectin-like binding activity failed to inhibit binding. A peptide from trypsin digested SAP composed of residues 144-199 retained CRP binding activity, implicating the COOH-terminal region of SAP as the CRP recognition site.

Human serum amyloid P-component (SAP) selectively binds to immobilized or bound forms of C-reactive protein (CRP).

The two homologous human pentraxins, C-reactive protein (CRP) and serum amyloid P-component (SAP), specifically bind to each other only when the CRP is in an immobilized form bound to one of its ligands or to an antibody. CRP did not bind to immobilized SAP. The binding of SAP to immobilized forms of CRP was Ca(2+)-dependent and of sufficient affinity to occur in the presence of serum or purified serum proteins. SAP bound preferentially to a synthetic peptide corresponding to the Ca(2+)-binding region of CRP. Monoclonal antibodies to a synthetic peptide corresponding to the Ca(2+)-binding region selectively inhibited the binding interaction. Proteolytic cleavage of CRP between residues 146 and 147 within the Ca2+ binding region abolished the SAP-binding site; however, the intact subunits of the pentameric CRP were capable of binding SAP. The significance of the binding interaction is that it may serve as the basis for localization of SAP to sites of tissue damage or repair, sites where CRP is selectively deposited.

A cell attachment peptide from human C-reactive protein.

The serum acute phase reactant, C-reactive protein (CRP), is selectively deposited at sites of tissue damage and degraded by neutrophils into biologically active peptides. A synthetic peptide corresponding to residues 27-38 present in each of the five identical subunits of CRP mediated cell attachment activity in vitro. Although the CRP-derived peptide contains a Tuftsin (TKPR)-like sequence at its amino-terminus, the Tuftsin tetrapeptide itself, as well as several synthetic peptides of CRP, failed to inhibit the cell-attachment activity to the CRP-derived peptide. Peptides containing the sequences responsible for the cell attachment activity of the extracellular matrix proteins, fibronectin (Fn) and laminin, failed to inhibit the CRP-derived peptide cell attachment activity. However, the addition of the RGDS and RGDSPASSLP cell-binding peptides of Fn to cells enhanced attachment to the active peptide from CRP. In the converse experiment, the cell-binding peptide of CRP did not influence cell attachment to Fn or laminin. A peptide corresponding to the same stretch of amino acid residues within the homologous Pentraxin, serum amyloid P-component (SAP), displayed nearly identical cell-attachment activity. Several monoclonal antibodies (mAb) specific for the CRP-derived cell-binding peptide neutralized its cell-attachment activity. These mAbs reacted with intact CRP and neutralized the cell-binding activity of CRP itself. The findings suggest that a peptide with cell-binding activity could be generated from the breakdown of CRP and then contribute directly to cellular events leading to tissue repair.