C-reactive protein, inflammation, and innate immunity.

The circulating acute phase reactant C-reactive protein (CRP) has traditionally been characterized as an effector of nonclonal host resistance since it activates the classical complement cascade and mediates phagocytosis, but it is also capable of regulating inflammation. The three-dimensional structure of human CRP has revealed the molecular basis for complement activation and binding of phosphate monoesters. CRP gene expression by liver hepatocytes in response to cytokines (IL-1beta and IL-6) released in tissues requires several transcription factors which interact. Elevated levels of CRP are a prognostic marker for coronary artery disease; however, the role of CRP in atheriosclerosis remains unknown. CRP also mediates direct host protection to some microbial pathogens via its opsonic activity through certain Fcgamma-receptors. The CRP response may be one of the links between nonspecific innate immunity and specific clonal immunity.

C-reactive protein in the arterial intima: role of C-reactive protein receptor-dependent monocyte recruitment in atherogenesis.

Infiltration of monocytes into the arterial wall is an early cellular event in atherogenesis. Recent evidence shows that C-reactive protein (CRP) is deposited in the arterial intima at sites of atherogenesis. In this study, we demonstrate that CRP deposition precedes the appearance of monocytes in early atherosclerotic lesions. CRP is chemotactic for freshly isolated human blood monocytes. A specific CRP receptor is demonstrated on monocytes in vitro as well as in vivo, and blockage of the receptor by use of a monoclonal anti-receptor antibody completely abolishes CRP-induced chemotaxis. CRP may play a major role in the recruitment of monocytes during atherogenesis.

Role of STAT3 and C/EBP in cytokine-dependent expression of the mouse serum amyloid P-component (SAP) and C-reactive protein (CRP) genes.

Inflammation is accompanied by a rapid increase in blood levels of acute phase proteins synthesized by hepatocytes in response to cytokines. Although C-reactive protein (CRP) levels increase dramatically in most mammals, the major acute phase protein in the mouse is the homologous pentraxin, serum amyloid P-component (SAP), whereas CRP is a minor acute phase reactant. The molecular basis for the pronounced difference in SAP and CRP gene expression in the mouse is unknown. Transfection of ++/Li mouse hepatoma cells with CAT-reporter constructs containing the 5'-flanking region of the mouse CRP gene indicated that transcription was stimulated by either IL-6, or IL-6 plus IL-1, when > or =360 bp of the 5'-proximal DNA was present. Examination of the 5'-flanking region of the mouse SAP gene revealed that the region between -433 and -397 from the transcription start site responded to IL-1 and IL-6 by binding both STAT3 and C/EBPbeta. This responsive region consisted of two adjacent C/EBPbeta consensus sites that overlap with two STAT3 consensus sites and was found to bind C/EBPbeta at an upstream site of -427 to -409 and STAT3 at a downstream site of -415 to -397. By contrast, the 360 bp promoter of the CRP gene was bound only by STAT3 at consensus sites at -93, -142, -173, and -287 from the start site; however, a single consensus site for C/EBP at -75 was not recognized. STAT3 appears to be necessary for both mouse SAP and CRP gene transcription since overexpression of an inactive, deletion mutant of STAT3 inhibited transcription of both genes. The results indicate that both STAT3 and C/EBPbeta participate in mouse SAP gene expression, whereas only STAT3 is involved in mouse CRP gene expression. The findings for mouse SAP gene expression are consistent with the reported interaction between these two transcription factors for human CRP gene transcription.

Regulation of phagocytic leukocyte activities by C-reactive protein.

The classic acute phase reactant C-reactive protein (CRP) is classified as an effector of innate host resistance because it activates the classical complement cascade and is opsonic. The latter action occurs via specific CRP receptors (CRP-R) that have recently been identified as both FcgammaRI and FcgammaRII on human phagocytic leukocytes. New findings also suggest an anti-inflammatory role for CRP because it modulates endotoxin shock and inhibits chemotaxis and the respiratory burst of neutrophils. CRP inhibited phorbol myristate acetate-induced superoxide (O2-) production more efficiently than the fMLP-triggered response. An examination of the inhibition of the protein kinase C (PKC)-dependent assembly of the NADPH oxidase complex revealed that both phosphorylation and translocation of PKC-beta2 to the membrane were inhibited by a threshold acute phase dose of approximately 50 microg/mL CRP. Translocation to the membrane and serine-phosphorylation of the major cytosolic p47-phox component of the NADPH oxidase complex was inhibited by CRP. CRP also inhibited membrane localization of activated Rac2, the small G protein regulator of the assembly of the oxidase components in activated neutrophils as well as the cytoskeleton during chemotactic movement. CRP-mediated regulation occurs via the CRP-R because an IgM mouse mAb to the human CRP-R mimicked CRP-induced inhibition of O2- production and chemotaxis. CRP may serve as an antiinflammatory regulator of activities at sites of tissue damage where it selectively accumulates and thus influences neutrophil infiltration and polymorphonuclear neutrophil activities. By contrast, CRP activates cells of the monocyte/macrophage lineage, suggesting differential regulation of these two leukocyte populations at the level of signaling. CRP appears to be a multifunctional protein with the capability of exerting both effector functions for innate host resistance, as well as exerting specific anti-inflammatory effects.

Trypanosoma cruzi: detection of a surface antigen cross-reactive to human C-reactive protein.

C-reactive protein (CRP) is an acute phase protein secreted by liver hepatocytes, and is also found on the surface of lymphocytes and as a membrane-associated protein expressed on rat liver macrophages and human monocytes. C-reactive protein levels increase in the sera of children infected with Trypanosoma cruzi, during the acute phase of Chagas' disease, but its role in the course of this infection is unknown. Experiments designed to detect the binding of CRP to circulating forms of T. cruzi failed to observe it because anti-human CRP antibodies bind to the parasite. The present work intended to further clarify this novel question related to the anti-CRP cross-reactivity with the parasite. Indirect immunofluorescence, immunoenzymatic, flow cytometry, and Western blot assays showed that three different polyclonal anti-human CRP antibody preparations bind to T. cruzi surface. This binding is dose-dependent, saturable, and is inhibited when anti-CRP antibodies from different species were allowed to compete, indicating the specificity of the reactivity. The antibodies recognized a protein band below 23 kDa in Western blot analysis of parasite extracts. The divalent cation chelators EDTA and EGTA impaired the antigen recognition by the antibodies. The binding to parasite surface was also observed with some available monoclonal antibodies raised against human CRP. A polyclonal anti-human CRP presented an inhibitory effect on invasion of heart muscle cells by T. cruzi. Our results indicate that a molecule antigenically related to CRP, a possible CRP-like molecule, is expressed on the surface of T. cruzi.

Effect of human C-reactive protein on chemokine and chemotactic factor-induced neutrophil chemotaxis and signaling.

C-reactive protein (CRP) is a unique serum pentraxin and the prototype acute phase reactant. CRP is a ligand for specific receptors on phagocytic leukocytes, and mediates activation reactions of monocytes/macrophages, but inhibits the respiratory burst of neutrophils (PMN). Since CRP selectively accumulates at inflammatory sites in which IL-8 is also produced, we tested the effects of CRP on the responsiveness of PMN to IL-8 and the bacterial chemotactic peptide, FMLP-phenylalanine (FMLPP). Purified human CRP inhibited the chemotactic response of PMN to IL-8 and FMLPP. A mouse IgM mAb that was generated against the leukocyte CRP receptor (CRP-R) also inhibited the chemotactic response. Incubation of purified CRP with activated PMN generated CRP-derived peptides that also inhibited chemotaxis. A synthetic CRP peptide (residues 27-38) that binds to the CRP-R had weak chemotactic activity, whereas two other CRP synthetic peptides (residues 174-185 and 191-205) inhibited chemotaxis of PMNs to both IL-8 and FMLPP. CRP did not alter receptor-specific binding of IL-8, but exerted its effect at the level of signaling. CRP augmented both IL-8- and FMLPP-induced mitogen-activated protein kinase (extracellular signal-regulated kinase-2) activity. CRP at acute phase levels increased both agonist-induced and noninduced phosphatidylinositol-3 kinase activity. The results suggest a role for CRP as a regulator of leukocyte infiltration at inflammatory sites.

Binding site on human C-reactive protein (CRP) recognized by the leukocyte CRP-receptor.

C-reactive protein (CRP), the prototypical inflammatory acute phase reactant in humans, interacts with monocytes and neutrophils via a specific receptor. To map the site on CRP recognized by the CRP receptor (CRP-R), synthetic peptides corresponding to the surface region on each of the five identical subunits were tested as competitors vs. [125I]-CRP for cell binding. A peptide of residues 27-38 (TKPLKAFTVCLH) efficiently inhibited CRP binding when compared to other nonoverlapping peptides. This peptide was termed the cell-binding peptide (CB-Pep). The F(ab')2 of an IgG Ab to the CB-Pep specifically inhibited CRP binding upon reacting with the ligand. Competitive binding studies with synthetic peptides truncated from either the NH2- or COOH-terminus of the CB-Pep revealed that the minimum length recognized by the CRP-R consisted of residues 31-36: KAFTVC. Conservative substitutions of residues within the CB-Pep indicated that the four residues AFTV were critical for CRP-R binding. The CB-Pep also inhibited induced superoxide generation by HL-60 granulocytes. The minimum length required for the inhibition was also KAFTVC; however, only Phe-33 and Leu-37 were critical residues in this assay. Anti-CB-Pep IgG Ab reacted more extensively with heat-modified CRP, suggesting that an altered conformation of CRP is preferentially recognized by the CRP-R. The results suggest that this contiguous sequence on a beta-strand on one face of each of five subunits of the CRP pentamer serves as a unique recognition motif for inflammatory leukocytes.

Acute phase levels of C-reactive protein enhance IL-1 beta and IL-1ra production by human blood monocytes but inhibit IL-1 beta and IL-1ra production by alveolar macrophages.

C-reactive protein (CRP), the major acute phase protein in humans, was purified free of endotoxin (LPS) (< 10 pg of LPS/mg of purified CRP) and evaluated for its ability to modulate LPS-induced production of IL-1 beta and IL-1 receptor antagonist (IL-1ra) from human PBMC and lung macrophages. PBMC (5 x 10(6)/ml) released low levels of IL-1 beta in response to either CRP (250 micrograms/ml) or LPS (100 ng/ml) for 18 h (0.3 +/- 0.1 and 1.5 +/- 0.7 ng/ml, respectively). However, when CRP (250 micrograms/ml) and LPS (100 ng/ml) were combined, PBMC released 9.7 +/- 2.9 ng/ml (p < 0.001 vs LPS alone). This synergy was removed by immunodepletion of CRP before stimulation. With respect to IL-1ra, although CRP induced IL-1ra production from PBMC (0.8 +/- 0.3 ng/ml control, 2.6 +/- 1.3 ng/ml with CRP), CRP did not synergize with LPS for IL-1ra production (15.0 +/- 0.7 ng/ml LPS alone vs 15.4 +/- 1.4 ng/ml LPS and CRP). In contrast, lung macrophages responded to CRP quite differently than PBMC. Macrophages (10(6)/ml) were not stimulated to produce IL-1 beta or IL-1ra by CRP alone. When combined with LPS, CRP inhibited IL-1 beta and IL-1ra release induced by LPS (for IL-1 beta release, LPS induced 3.0 +/- 1.7 ng/ml vs 1.1 +/- 0.4 for combined LPS and CRP; for IL-1ra release, LPS induced 12.9 +/- 2.3 ng/ml vs 7.6 +/- 2.3 ng/ml for combined LPS and CRP). These data suggest that acute phase levels of CRP may have divergent effects depending on the target population. CRP may be largely proinflammatory to blood monocytes responding to LPS since IL-1 beta production is augmented over IL-1ra production. However, in tissue compartments the effects of CRP may be largely immunosuppressive to LPS-induced tissue macrophage IL-1 beta production.

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.

Calcium ion binding regions in C-reactive protein: location and regulation of conformational changes.

C-reactive protein (CRP) is a pentameric acute phase serum protein composed of identical 206 amino acid subunits that associate by non-covalent bonds. The biological activities ascribed to CRP are initiated by binding ligands via the single PC-binding site within each subunit. CRP binding to PC requires a conformational change in the intact pentraxin triggered by the binding of two free Ca2+ ions per subunit. Residues 134-148 of each subunit were previously implicated by indirect measures as one of the Ca(2+)-binding sites. In this study, 45Ca2+ autoradiography revealed that fragments of CRP of 6.5 and 16 kDa generated by proteolysis between residues 146 and 147 bind Ca2+ indicating that a second Ca(2+)-binding site is located within the C-terminal 60 amino acids. Synthetic peptides corresponding to residues 134-148 and 152-176 both bound 45Ca2+ in equilibrium dialysis experiments with a Kd = 5.2 x 10(-4) and 1.7 x 10(-4) M, respectively. The addition of Ca2+ to peptide 152-176 induced a shift in the CD-spectra between 210 and 230 nm. Rabbit anti-peptide 152-176 antibody (Ab) inhibited the availability of an epitope within the PC-binding site of CRP recognized by mAb EA4-1. Reactivity of CRP with both anti-peptide 134-148 mAb and anti-peptide 152-176 Ab enhanced the expression of the PC-binding site epitope. The results suggest that the two distinct Ca(2+)-binding sites within each CRP subunit are composed of residues 134-148 and 152-176 and that these two nearly adjacent sites cooperate to exert an allosteric change in conformation allowing access to the PC-binding site.

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.

Cell attachment peptide of C-reactive protein: critical amino acids and minimum length.

Human C-reactive protein (CRP) is an acute phase blood component that accumulates at sites of tissue damage and necrosis and is degraded by neutrophils to biologically active peptides. A dodecapeptide composed of amino acids 27-38 of CRP mediates cell attachment in vitro. This peptide was designated the cell-binding peptide (CB-Pep) of CRP. Characterization of the interaction between fibroblasts and modified synthetic peptides with sequential deletions from either the N-terminus or C-terminus revealed that the minimal sequence for cell attachment or inhibition of cell attachment to the CB-Pep was Phe-Thr-Val-Cys-Leu, which corresponds to residues 33-37 within each of the five 206 amino acid subunits of CRP. The pentapeptide by itself mediated cell attachment. Substitutions for each residue within the CB-Pep indicated that the critical residues for activity were Phe-33 and Thr-34. This cell-binding pentapeptide represents a recognition motif for cell adhesion not found in other proteins.

Cloning and tissue-specific expression of the gene for mouse C-reactive protein.

C-reactive protein is a serum acute-phase reactant that increases several thousand-fold in concentration during inflammation in most mammals. However, mouse C-reactive protein is considered to be a minor acute-phase reactant, since its blood level increases only from approx. 0.1 to 1-2 micrograms/ml. A mouse genomic clone of approximately 5 kb was obtained to determine the molecular basis for the regulation of the expression of mouse C-reactive protein. Several cis-acting elements in the 5' flanking region that potentially regulate transcription were identified: two glucocorticoid-responsive elements, two CCAAT-enhancer-binding protein C (C/EBP) consensus elements that are required for the interleukin-1 responsiveness of some acute-phase reactant genes, an interleukin-6-responsive element, two hepatocyte nuclear factor-1 (HNF-1) elements and a single heat-shock element. Transfection of the hepatoma cell line Hep 3B.2 with a pCAT expression vector containing the 5' flanking sequence from -1083 to -3 bp from the transcriptional start site, and truncations of this sequence, localized elements that control the tissue-specific expression of mouse C-reactive protein to the two HNF-1 elements and a C/EBP, interleukin-1-responsive element located between -220 and -153, and -90 and -50 bp from the transcriptional start site. A constitutive nuclear protein from mouse-liver hepatocytes specifically binds to the HNF-1 elements. These findings explain the tissue-specific expression of the gene, as well as its limited expression during the acute-phase response.

The mouse C-reactive protein (CRP) gene is expressed in response to IL-1 but not IL-6.

C-Reactive protein (CRP) is a minor acute phase reactant (APR) in the mouse, whereas CRP is the prototypical and one of the major positive APRs in all other mammals. MoCRP gene expression was tissue specific for the liver and induced by culture supernatants of LPS-activated macrophages. MoCRP gene expression by isolated hepatocytes in culture increased c, 3-fold in response to interleukin (IL)-1, but not IL-6. IL-6 is the most potent inflammatory cytokine for the induction of human CRP and many other APRs. By contrast, gene expression of the major APR of the mouse, serum amyloid P-component (SAP), a structural homologue of CRP, increased in response to either IL-1 or IL-6 under the same conditions. The region containing two potentially IL-1 responsive C/EBP elements in the moCRP gene failed to respond to IL-1 when a pCAT construct containing the elements was transfected into Hep 3B2 hepatoma cells. Therefore, IL-1 may influence the expression of the moCRP gene at the post-transcriptional rather than at the transcriptional level. The findings suggest that moCRP may be a minor APR because of the limited response of the gene to inflammatory cytokine signals.

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.

Internalization and degradation of receptor bound C-reactive protein by U-937 cells: induction of H2O2 production and tumoricidal activity.

The presence of a membrane receptor for C-reactive protein (CRP-R) on the human monocytic cell line U-937 was the basis for determining the metabolic fate of the receptor-bound ligand and the functional response of the cells to CRP. Internalized [125I]CRP was measured by removing cell surface-bound [125I]CRP with pronase. Warming cells to 37 degrees C resulted in the internalization of approx. 50% of the receptor-bound [125I]CRP or receptor-bound [125I]CRP-PC-KLH complexes. U-937 cells degraded about 25% of the internalized [125I]CRP into TCA-soluble radiolabeled products. The lysosomotrophic agents (chloroquine, NH4Cl) greatly decreased the extent of CRP degradation without altering binding or internalization. In addition, a pH less than 4.0 resulted in dissociation of receptor-bound [125I]CRP. Treatment of U-937 cell with monensin, a carboxylic ionophore which prevents receptor recycling, resulted in accumulation of internalized [125I]CRP. Therefore, it appears that the CRP-R complex is internalized into an endosomal compartment where the CRP is uncoupled from its receptor and subsequently degraded. CRP initiated the differentiation of the U-937 cells so that they acquired the ability to produce H2O2 and also display in vitro tumoricidal activity. The results support the concept that internalization and degradation of CRP leads to the activation of monocytes during inflammation.

Monoclonal antibodies to the calcium-binding region peptide of human C-reactive protein alter its conformation.

Five mouse mAb were generated against a synthetic peptide corresponding to the proposed Ca(2+)-binding region of human C-reactive protein (CRP). The peptide consists of amino acids 134 to 148 and possesses a calmodulin Ca(2+)-binding sequence. The mAb reacted with a surface epitope(s) on native, intact CRP as well as the closely related pentraxin protein, serum amyloid P-component. Three of the 5 mAb inhibited the Ca(2+)-dependent phosphorylcholine-(PC) binding activity of CRP, but did not bind to the PC-binding region itself. Four of the five mAb also inhibited the recognition of an epitope in the PC-binding site of CRP. Four of the mAb partially, or completely, protected CRP from selective cleavage by pronase between residues 146 and 147. The findings suggest that the Ca(2+)-binding region is on the surface of CRP, has substantial flexibility, and is probably responsible for the allosteric effects of Ca2+ ions on CRP.

A synthetic peptide corresponding to the phosphorylcholine (PC)-binding region of human C-reactive protein possesses the TEPC-15 myeloma PC-idiotype.

C-reactive protein (CRP) is a major acute phase reactant in most mammalian species. CRP molecules from all species display Ca2(+)-dependent binding to phosphorylcholine (PC). The conserved PC-binding region of CRP corresponds to amino acids 51-66 within the human CRP sequence. A synthetic peptide composed of residues 47-63 of human CRP was previously shown to possess PC binding activity. The charged amino acids at positions 57, 58, 60, and 62 of this synthetic peptide were critical for PC-binding based on lower binding activity of synthetic peptides containing uncharged residues at these positions. The PC-binding peptide was used to generate mouse mAb that were tested for reactivity with intact CRP and with the TEPC-15 (T-15) mouse myeloma protein that also binds PC. The PC-binding peptide of CRP was recognized by two mAb specific for the T-15 Id. One of the mAb generated against the PC-binding peptide of CRP (IID6.2) recognized an epitope on the T-15 protein that was also recognized by the near-binding site-specific mAb (F6) to the T-15 PC-Id. Binding of IID6.2 to T-15 myeloma protein was not inhibited by PC and did not require Ca2+; however, binding was inhibited by the synthetic PC-binding peptide itself. Recognition of synthetic peptides containing uncharged amino acid substitutions by mAb F6 and IID6.2 was greatly reduced indicating that the shared epitope on T-15 and CRP was composed of similar charged residues. Therefore, CRP displays the same idiotope as an antibody that shares its specificity for the hapten, PC.