Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds.

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl](1/2) at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys(36)-Cys(49) and two disulfide bonds formed by two pair of consecutive cysteines, Cys(22)-Cys(23) and Cys(56)-Cys(57), a unique disulfide structure of polypeptide that has not been documented previously

Identification of Acidic pH-Dependent Ligands of Pentameric C-reactive Protein

C-reactive protein (CRP) is a phylogenetically conserved protein; in humans, it is present in the plasma and at sites of inflammation. At physiological pH, native pentameric CRP exhibits calcium-dependent binding specificity for phosphocholine. In this study, we determined the binding specificities of CRP at acidic pH, a characteristic of inflammatory sites. We investigated the binding of fluid-phase CRP to six immobilized proteins: complement factor H, oxidized low-density lipoprotein, complement C3b, IgG, amyloid β, and BSA immobilized on microtiter plates. At pH 7.0, CRP did not bind to any of these proteins, but, at pH ranging from 5.2 to 4.6, CRP bound to all six proteins. Acidic pH did not monomerize CRP but modified the pentameric structure, as determined by gel filtration, 1-anilinonaphthalene-8-sulfonic acid-binding fluorescence, and phosphocholine-binding assays. Some modifications in CRP were reversible at pH 7.0, for example, the phosphocholine-binding activity of CRP, which was reduced at acidic pH, was restored after pH neutralization. For efficient binding of acidic pH-treated CRP to immobilized proteins, it was necessary that the immobilized proteins, except factor H, were also exposed to acidic pH. Because immobilization of proteins on microtiter plates and exposure of immobilized proteins to acidic pH alter the conformation of immobilized proteins, our findings suggest that conformationally altered proteins form a CRP-ligand in acidic environment, regardless of the identity of the protein. This ligand binding specificity of CRP in its acidic pH-induced pentameric state has implications for toxic conditions involving protein misfolding in acidic environments and favors the conservation of CRP throughout evolution

The Binding of Factor H to a Complex of Physiological Polyanions and C3b on Cells Is Impaired in Atypical Hemolytic Uremic Syndrome

Factor H (fH) is essential for complement homeostasis in fluid-phase and on surfaces. Its two C-terminal domains (CCP 19-20) anchor fH to self surfaces where it prevents C3b amplification in a process requiring its N-terminal four domains. In atypical hemolytic uremic syndrome (aHUS), mutations clustering towards the C-terminus of fH may disrupt interactions with surface-associated C3b or polyanions and thereby diminish the ability of fH to regulate complement. To test this we compared a recombinant protein encompassing CCP 19-20 with sixteen mutants. The mutations had only very limited and localized effects on protein structure. While we found four aHUS-linked fH mutations that decreased binding to C3b and/or to heparin (a model compound for cell-surface polyanionic carbohydrates), we identified five aHUS-associated mutants with increased affinity for either or both ligands. Strikingly, these variable affinities for the individual ligands did not correlate with the extent to which all the aHUS-associated mutants were found to be impaired in a more physiological assay that measured their ability to inhibit cell surface complement functions of full-length fH. Taken together, our data suggest that disruption of a complex fH-self surface recognition process, involving a balance of affinities for protein and physiological carbohydrate ligands, predisposes to aHUS

Factor H–mediated cell surface protection from complement is critical for the survival of PNH erythrocytes

Paroxysmal nocturnal hemoglobinuria (PNH) cells are partially (type II) or completely (type III) deficient in GPI-linked complement regulatory proteins CD59 and CD55. PNH III erythrocytes circulate 6 to 60 days in vivo. Why these cells are not lysed as rapidly by complement as unprotected foreign cells, which normally lyse within minutes, remains undetermined. Factor H plays a key role in the homeostasis of complement in fluid phase and on cell surfaces. We have recently shown that a recombinant protein encompassing the C-terminus of factor H (rH19-20) specifically blocks cell-surface complement regulatory functions of factor H without affecting fluid-phase control of complement. Here we show that PNH II and III cells become highly susceptible to complement-mediated lysis by nonacidified normal human serum in vitro, when the cell surface complement-regulatory functions of factor H are blocked. The results indicate that cells deficient in surface-bound regulators are protected for extended periods of time by factor H