Steinbuch M

Steinbuch M., Audran R. 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. value of 30 to 60 m (5, 6). Binding of CRP to some nuclear proteins, enzymatically modified low-density lipoprotein and phosphoethanolamine-conjugated materials has also been reported (7,C11). The Ca2+- and PCh-binding sites are located on each subunit of CRP, and the available data Ansatrienin A suggest that these sites participate in the binding of CRP to most of its known ligands. Under certain conditions, CRP dissociates to generate the monomeric form of CRP (mCRP), which can be distinguished from pentameric CRP using specific monoclonal antibodies (12,C17). In normal healthy individuals, the median concentration of CRP in the serum is 0.8 g/ml; in acute phase, the concentration increases to 500 g/ml or more (18). CRP is also found at the sites of inflammation, both in humans and experimental animals (19,C23). The deposition of CRP at the sites of inflammation is independent of the circulating concentration of CRP. For example, CRP is deposited at atherosclerotic lesions although the circulating concentration of CRP increases only minimally in atherosclerosis (8, 22,C24). Due to the deposition of CRP at the sites of inflammation, the concentration of CRP at the sites of inflammation is not known. The mechanism proposed for the deposition of CRP at the sites of inflammation is largely based on the PCh-binding and other ligand-binding properties of CRP, which occur at physiological pH. Because the pH at the sites of inflammation may be acidic (25,C35), in this study, we investigated the Ansatrienin A binding specificities of CRP at acidic pH to define the functions of CRP at the sites of inflammation. Factor H is a complement regulatory protein that protects host cells from complement attack (36). Factor H can also protect pathogens from complement attack if the pathogens can recruit factor H (37, 38). We explored CRP-factor H interactions because of their possible involvement in the anti-pneumococcal functions of CRP observed in murine models of pneumococcal infection. Human CRP protects mice from lethality following infection with type 3 (39,C42). Because type 3 pneumococci bind factor H (38) and because CRP also binds factor H under certain conditions (43,C45), it has been proposed that CRP may bind to pneumococci-bound factor H and contribute to anti-pneumococcal function of CRP (46, 47). We explored CRP-factor H interactions also because of their possible involvement in age-related macular degeneration. If CRP could bind to factor H, then it could be important for age-related macular degeneration due to the implications of factor H in this disease (48). Oxidized low density lipoprotein (oxLDL) is an atherogenic form of LDL. It enters macrophages to form foam cells that contribute to the development of atherosclerosis, which is an Ansatrienin A inflammatory disease (49). CRP is localized with LDL in atherosclerotic lesions present in humans and experimental animal models (8, 22, 23). Because the pH of SHCC the atherosclerotic lesions may be acidic (28,C32), in this study, we also investigated the effects of acidic pH on CRP-oxLDL interactions. In addition to factor H and oxLDL, we randomly selected four other proteins: complement component C3 fragment C3b, IgG, amyloid fragment 1C38 (A), and BSA, to explore the binding reactivities of CRP at acidic pH. We expected that CRP, at acidic pH, might bind to factor H, oxLDL, C3b, IgG, and A, but would not bind to BSA. Surprisingly, we found that CRP, at acidic pH, bound to all six proteins including BSA, as if CRP recognized a general pattern created by these proteins that were immobilized to microtiter plates and also exposed to acidic pH at 37 C. EXPERIMENTAL PROCEDURES Purification of CRP, Factor.