Lawrence A. Potempa

Expression, detection and assay of a neoantigen (neo-CRP) associated with a free, human C-reactive protein subunit

It has previously been reported that human C-reactive protein (CRP) can exist in at least two molecular conformations distinguished by antigenic, electrophoretic and ligand-binding reactivities. In the present study we describe the formation, detection and distinctiveness of a conformation expressing a CRP neoantigen (neo-CRP), and report that this form is characteristic in vitro of a free CRP subunit. Soluble native-CRP was found to express neo-CRP antigenicity upon treatment with acid; upon urea-chelation or heating in the absence of calcium; and upon adsorption onto uncoated polystyrene plates. Native-CRP bound by capture ELISA to phosphorylcholine-containing ligand or anti-native-CRP did not express neo-CRP antigenicity, suggesting that PC ligand- or antibody binding is not sufficient to induce expression of the neoantigen. Human CRP which expressed neo-CRP antigenicity had limited solubility and tended to aggregate in buffers of ionic strength 0.15, but remained soluble when the ionic strength was reduced to 0.015. Soluble urea-chelated or acid-treated CRP molecules expressing neo-CRP antigenicity chromatographed and electrophoresed as a single protein with a Mr of approx. 22,000, indicating that the CRP neoantigen can be expressed on free CRP subunits and this expression need not require proteolysis. Further, molecules expressing neo-CRP antigenicity were detected in the plasma of patients with rheumatoid arthritis. The identification and characterization of this CRP neoantigen should serve as a useful marker in studies of CRP subunits and biologically relevant forms of CRP, and should contribute to the elucidation of the role of CRP in the acute inflammatory response.

Antigenic, electrophoretic and binding alterations of human C-reactive protein modified selectively in the absence of calcium

C-Reactive protein (CRP) is a trace component of normal human serum which has a mol. wt of 105,000 and is composed of five apparently identical subunits arranged in cyclic symmetry. The serum concentration of this protein is known to increase dramatically in response to acute inflammatory or necrotic processes. We report here that in the presence of high concentrations of urea significant antigenic, electrophoretic and binding site modifications of CRP occur selectively in the absence of calcium. CRP treated in this way (designated F-CRP) had a pI of 5.4 and alpha-electrophoretic mobility in contrast to the native molecule which had a pI of 6.4 and gamma-mobility. F-CRP retained a partial antigenic identity to native CRP, displayed decreased intrinsic tryptophan fluorescence, and expressed a new antigenic reactivity. A similar neoantigen was expressed by heating CRP selectively in the absence if calcium (63 degrees C, 5 min). Treatment with guanidinium-HCl or deliberate carbamylation did not produce F-CRP or the expression of the F-antigen. The formation of F-CRP in urea or by heat was prevented by the presence of 0.7 mM or more calcium. CRP treated in this way retained identical characteristics to native CRP. F-CRP chromatographed through Sephadex G-150 in the presence or absence of 6M urea as a protein of apparent mol. wt 75, 000 with no evidence for free CRP subunits. The formation of F-CRP from native CRP resulted in a loss of capacity for calcium-dependent binding to the C-polysaccharide despite the persistence of calcium-independent binding reactivity for polycations. These data suggest that in the presence of sufficient calcium CRP can resist urea- or heat-induced structural denaturation, maintaining antigenic, electrophoretic and binding identity to the native molecule. In the absence of calcium, exposure to urea led to increased electrophoretic mobility and exposure of a new antigenic reactivity, and to alterations in the phosphocholine- but not the polycation-binding sites of the native CRP molecule; this new antigenic reactivity may be of value in further studies on the CRP molecule.

Effect of Modified C-Reactive Protein on Complement Activation: A Possible Complement Regulatory Role of Modified or Monomeric C-Reactive Protein in Atherosclerotic Lesions

Objective—The capacity of human C-reactive protein (CRP) to activate/regulate complement may be an important characteristic that links CRP and inflammation with atherosclerosis. Recent advances suggest that in addition to classical pentameric CRP, a conformationally distinct isoform of CRP, termed modified or monomeric CRP (mCRP), may also play an active role in atherosclerosis. Although the complement activation behavior of CRP has been well established, the capacity of mCRP to interact with and activate the complement cascade is unknown. Methods and Results—mCRP bound avidly to purified C1q, and this binding occurred primarily through collagen-like region of C1q. Fluid phase mCRP inhibited the activation of complement cascade via engaging C1q from binding with other complement activators. In contrast, when immobilized or bound to oxidized or enzymatically modified low-density lipoprotein, mCRP could activate classical complement pathway. Low-level generation of sC5b-9 indicated that the activation largely bypassed the terminal sequence of complement, which appears to involve recruitment of Factor H. Conclusions—These results indicate that mCRP can both inhibit and activate the classical complement pathway by binding C1q, depending on whether it is in fluid phase or surface-bound state.

Complement Activation by C-Reactive Protein on the HEp-2 Cell Substrate

The complement (C) activation by C-reactive protein (CRP) in acute-phase sera is routinely tested in our laboratory by means of an indirect immunofluorescence method (C3-IFT) on rat kidney sections. This C3-IFT assay is based on the binding of CRP to the renal tissue followed by the fixation of C4 and C3 components to distinct vessel-associated medullary structures as a result of CRP-mediated C activation in vitro. While the activation cascade leading to the deposition of C4 and C3 could previously be deduced experimentally, we were unable as yet to visualize CRP and the components of the C1 complex on kidney sections when testing patients sera by indirect immunofluorescence. In an attempt to analyze the mechanisms of unexpectedly negative C3-IFT results (e.g. bacterial endocarditis) we employed monolayers of fixed HEp-2 cells which have previously been shown to be a suitable substrate for CRP binding. By incubating purified native CRP supplemented with normal human serum as a source of C we detected the C components Clq, Clr, Cls, C4 and C3 in the same speckled immunofluorescent pattern on HEp-2 cell nuclei as described characteristically for CRP binding. The serial activation steps from CRP up to C3 could also be followed on HEp-2 cells using C3-IFT-positive acute-phase sera. However, certain C3-IFT-negative acute-phase sera showed an arrest between Cls and C4 of the CRP-mediated C activation cascade. HEp-2 cells can thus be used to monitor the process of autologous C activation initiated by endogeneous CRP in patients sera. In contrast to native CRP, urea-modified CRP (mCRP) did not bind to HEp-2 cell nuclei, but was detected in association with distinct filamentous cytoplasmic structures. Unlike its native counterpart, binding of mCRP was not followed by a deposition of C components.

Evidence that serum amyloid P component binds to mannose-terminated sequences of polysaccharides and glycoproteins.

Serum amyloid P component (SAP) is a normal human serum protein with pentraxin structure that has morphological and immunochemical identity to the amyloid P component found in normal tissue and amyloid deposits. In the presence of calcium, SAP binds to certain complex polysaccharides, including agarose and zymosan. While the binding of SAP to agarose involves interaction with a galactose pyruvate acetal, the ligand in zymosan has not been defined. In the present study we determined that SAP binds to ligand(s) in a soluble extract of zymosan prepared by alkaline hydrolysis, which contains the mannose oligosaccharide sequences alpha DMan1----3DMan and alpha DMan1----6DMan. SAP did not bind to the alkali-insoluble fraction of zymosan, which is predominantly a glucan polymer, and its binding to zymosan extract which had been absorbed with concanavalin A was markedly reduced, suggesting that mannose residues are involved in the binding of SAP to zymosan. We also demonstrated that SAP binds to the glycoproteins ovalbumin, thyroglobulin, beta-glucuronidase and C3bi, which contain mannose-terminated sequences, while it did not bind to native and desialized preparations of ovomucoid, alpha 1-acid glycoprotein and glycophorin, which lack terminal mannose residues. SAP did not bind to pneumococcal C polysaccharide or to N-acetylglucosamine oligosaccharides covalently linked to a protein carrier. The binding of SAP to ligand(s) in zymosan extract or ovalbumin was inhibited by the preincubation of SAP with either zymosan extract or ovalbumin glycopeptides, both of which share similar mannose oligosaccharide sequences. All of the SAP binding reactions required calcium, were maximal at approximately 1 mM calcium, and gave similar results whether purified SAP or SAP in serum was used. These findings indicate that mannose-terminated oligosaccharides of polysaccharides and glycoproteins represent a new class of ligands for SAP and suggest that SAP may function as a mannose-binding protein.

Deposition of Modified or Native C-Reactive Protein in Atherosclerotic Arteries?

To the Editor: nnAccumulating evidence suggests a predictive role of elevated serum concentrations of C-reactive protein (CRP) for atherosclerosis and its thrombotic complications.1 These findings apparently reflect an inflammatory component of the multifactorial atherosclerotic process. Furthermore, it is increasingly recognized that CRP may not merely represent an indicator of inflammation but may also, because of its known functional properties, be actively involved in the initiation or perpetuation of local inflammatory reactions.2 3 A direct approach in the study of the latter hypothesis is the search for CRP in atherosclerotic lesions. [banner]nnIn this context, the recent publication of Torzewski et al4 in Arteriosclerosis, Thrombosis, and Vascular Biology is of actual relevance. By means of immunohistochemistry, the authors clearly demonstrate deposits of CRP beside terminal complement proteins in the arterial wall of patients with early atherosclerotic lesions. Because ligand-bound CRP activates the classical pathway of complement, the authors suggest that the colocalization of terminal complement proteins with CRP might reflect complement activation by CRP in situ. Even if we share the hypothesis that …

Identification of Multiple Forms of the P Component of Amyloid Isolated from Human Serum

We examined isolated serum amyloid P component (SAP), the circulating counterpart of the amyloid P component, and established a previously unreported heterogeneity for SAP by immunological and biochemical methods. Highly purified SAP had a gel filtration Mr of 255,000, a Stokes radius of 57 A, a calculated frictional coefficient of 1.4, and 10 subunits of Mr of approximately 25,000. We present evidence suggestive of varying affinities of SAP for agarose, to which SAP is known to adsorb in the presence of calcium, by fused rocket immunoelectrophoresis. We resolved SAP subunits by isoelectric focusing into multiple species with isoelectric points of 6.08 (two forms), 6.02, and 5.95; three of these forms were delineated by high resolution two-dimensional SDS gel electrophoresis to have an Mr = 25,500, while a fourth (pI = 6.08) had an Mr = 24,500. The observed isoelectric charge heterogeneity could not be eliminated by neuraminidase treatment event though the electrophoretic migration of native desialized SAP was impeded (alpha 1 to beta) when examined by crossed immunoelectrophoresis, being consistent with the removal of anionic charges. Further, an additional neuraminidase-generated component was detected at the beta-position which could be removed by concanavalin A affinity. These data suggest SAP may exist in microheterologous or allotypic forms, the significance of which is under investigation.

C-REACTIVE PROTEIN [CRP) REACTIVITY WITH POLYCATIONS: MODULATORY ROLES FOR CALCIUM, PHOSPHOCHOLINE (PC) AND HEPARIN

In the presence of C-reactive protein (CRP), certain polycationic polymers have been shown to initiate complement (C] pathway activation and deplete early acting C-components..2 Selective precipitation between CRP and the polycationic polymers was demonstrated under serum-free using fluidphase nephelometric meth~dology.~ Furthermore, CRP-polycation activity was inhibited by tetra-L-lysine (TLL), a small, nonprecipitating analogue of the active polymers tested, while the calcium-dependent binding of C-polysaccharide (CPSI6 to CRP was not. In the present study we have further defined the relationship between the sites involved in reactivity with polycations and CPS. CRP was isolated from pleural fluids by affinity chromatography on PCsubstituted sepharose essentially as described Various polycations, heparin and PC-ligands were obtained commercially. Nephelometric analyses were performed in 1 ml of either 5 mM Imidazole-saline (pH 7.3) or Veranol-buffered saline (pH 7.3) using a Beckman Immunochemistry analyzer, with reactions monitored at room temperature in a rapidly stirred cell. Selective CRP-polycation precipitation was shown to be dependent on polycation size and ratio to CRP, with small (2700-6000 dalton) poly-L-lysine (PLL) polymers, 13,900 dalton poly-L-arginine (PLA) and 6000 dalton protamine the most active molecules in~estigated.3.~ Optimal reactions were observed at equivalence ratios of 2-4 molecules of polycation for each CRP molecule, and resulted in particulate complexes that formed quickly and remained stable over 30 min. The addition of calcium above 0.1 mM significantly depressed the precipitation response whether added before or after complex formation. None of the additional divalent cations tested (manganese, magnesium, copper and zinc) were similarly inhibitory. The depressed CRP-polycation precipitation reactivity, even at calcium concentrations above 1.0 mM, was completely reversed in the presence of a molar excess of PC but not a-glycerol PC. Heparin also reversed the inhibitory effect of calcium and contributed additional regulation to the CRPpolycation interaction. CRP-polycation complexes formed near equivalence were rapidly dissociated by all concentrations of heparin tested (from 0.1 to 5 times (w:w) the polycation concentration]. In polycation excess, however, small amounts of heparin were found to augment the precipitation response observed with CRP and polycation alone. The interaction of polyanion, polycation and CRP has been reported to result in C-activation. The effects of PC and related molecules, polyanions and calcium may be of importance in triggering or regulating local inflammatory responses in the presence of CRP and polycations.