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

Abstract

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. 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. IntroductionC-reactive protein (CRP) 4The abbreviations used are: CRPpentameric C-reactive proteinmCRPmonomeric C-reactive proteinLDLlow density lipoproteinoxLDLoxidized LDLAβamyloid β protein fragment 1–38PChphosphocholinePnCpneumococcal C-polysaccharideANS1-anilinonaphthalene-8-sulfonic acid. is an evolutionarily conserved protein. CRP is found in all animals, from invertebrates to vertebrates, where it has been sought. Human CRP is a pentameric protein comprised of five identical, non-covalently associated 23,028-Da subunits (reviewed in Refs. 1Singh S.K. Suresh M.V. Voleti B. Agrawal A. Ann. Med. 2008; 40: 110-120Crossref PubMed Scopus (101) Google Scholar, 2Black PubMed Scopus Google Scholar, PubMed Scopus Google At physiological pH, CRP is for its binding to as Med. PubMed Scopus Google CRP a of to PubMed Scopus Google Scholar, PubMed Scopus Google of CRP to proteins, modified low-density and has also been PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, S.K. Suresh M.V. Agrawal A. 2008; PubMed Scopus Google Scholar, B. PubMed Scopus Google Scholar, A. PubMed Scopus Google and sites on of CRP, and the suggest that these sites in the binding of CRP to of its CRP to the form of CRP which from pentameric CRP PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, A. A. PubMed Scopus Google the of CRP in the is in the to PubMed Scopus Google CRP is also found at the sites of in and Med. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Med. Google Scholar, PubMed Scopus Google of CRP at the sites of is of the of For example, CRP is at the of CRP in PubMed Scopus Google Scholar, Med. Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google to the of CRP at the sites of the of CRP at the sites of is not for the of CRP at the sites of is on the and of CRP, which at physiological pH. Because the pH at the sites of acidic PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, B. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, A. Google Scholar, PubMed Scopus Google Scholar, A. Google in this study, we investigated the binding specificities of CRP at acidic pH to the of CRP at the sites of is a complement protein that from complement PubMed Scopus Google also from complement the factor Google Scholar, PubMed Scopus Google We of in the of CRP in of Human CRP from Med. PubMed Scopus Google Scholar, Google Scholar, Google Scholar, M.V. S.K. Agrawal A. PubMed Scopus Google Because bind factor PubMed Scopus Google and CRP also factor conditions PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, A. PubMed Scopus Google it has been that CRP bind to factor and to of CRP M.V. S.K. Agrawal A. PubMed Scopus Google Scholar, A. Suresh M.V. S.K. 2008; PubMed Scopus Google We also of in CRP bind to factor H, it for to the implications of factor in this 2008; PubMed Scopus Google density is an form of to form that to the of which is an inflammatory PubMed Scopus Google CRP is in present in and PubMed Scopus Google Scholar, Med. Google Scholar, PubMed Scopus Google Because the pH of the acidic PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, B. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google in this study, we also investigated the of acidic pH on to factor and we proteins: complement fragment C3b, IgG, amyloid β fragment and to the binding of CRP at acidic pH. We that CRP, at acidic pH, bind to factor H, C3b, IgG, and but not bind to we found that CRP, at acidic pH, bound to all six proteins as CRP a by these proteins that were immobilized to microtiter plates and also exposed to acidic pH at were and were of a in the where the were used to the Acidic pH, CRP to a of to the of protein in which we determined the binding of CRP to immobilized factor H, C3b, IgG, and as a of pH at and in at CRP did not bind to factor at any pH. at CRP bound to factor but the pH was from to and the binding the pH was to were At CRP did not bind to at any pH. At CRP bound to the pH was from to and binding the pH was to in at CRP bound to at pH At CRP bound to the pH was from to and the binding was the pH was to in at CRP did not bind to at any pH. At CRP bound to the pH was from to and binding the pH was to in at CRP bound to at pH At CRP bound to the pH was from to and binding the pH was to We that CRP to at acidic pH S.K. Agrawal A. 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We that binding of CRP to immobilized proteins at acidic pH was and and CRP, the binding not by the of in the binding of CRP to all six immobilized proteins at pH that binding of CRP to immobilized proteins at acidic pH was a of CRP and not a at acidic of the binding of and CRP to immobilized proteins at acidic pH. were factor H, C3b, IgG, and sites in the were CRP and in pH 4.6, were to the and at for CRP was as in the to was by a and of the was at and on the as of of the binding of CRP to immobilized proteins by at acidic pH. of a protein were factor H, C3b, IgG, and sites in the were CRP in pH 4.6, and was to the and at for CRP was as in the to binding of CRP to immobilized protein in the of was as as of pH CRP but the the of acidic pH on the of CRP by gel and gel filtration, we determined CRP pentameric at pH for at in the of CRP at pH and were in the at pH was the in the at pH the of CRP the at pH was the of CRP at pH of CRP, which was at pH for at and the pH was was also to the of CRP at pH that CRP pentameric at pH we determined the pentameric of CRP was modified at pH CRP, at pH was was binding of to CRP at pH was that at pH and as by the in the pH of CRP which were at pH for at was the binding of to CRP was not that at pH and was we the binding of to CRP at pH in the of and in the of that the in and was on the acidic pH and was not to the of from CRP at acidic of acidic pH on the pentameric of of CRP at pH and CRP at pH CRP at pH at for after CRP at pH at for the pH was CRP at pH at for after CRP at pH at for the pH was CRP at pH in the of CRP at pH CRP at pH CRP at pH at for CRP at pH at for CRP at pH at for CRP at pH at for CRP at pH was at for an the pH was the pH was to and CRP was at for an the pH was This was of pH. as of as in was found to binding of CRP to immobilized proteins, we determined the acidic pH-induced in CRP in the of CRP at pH and was by the of CRP and and that CRP was to acidic pH and that the acidic pH-induced of the pentameric of CRP was for the binding of CRP to immobilized proteins. the of the acidic pH-induced in the of CRP, we the pH of a CRP and the of CRP did not CRP was at pH and the CRP was at pH from gel and at acidic pH, CRP was not CRP was not the pentameric of CRP was and at modifications were Acidic pH the of determined the of acidic pH on the of CRP a in the activity a in the of We used the to At pH CRP bound at to at pH the activity of CRP was At of CRP was to binding of CRP to the acidic pH of CRP was to the activity of CRP was that acidic pH modified the of CRP, but the was in the activity of CRP at acidic pH. of a were sites in the were CRP was to the and at for CRP was a and and of the was at and on the CRP was in pH CRP was in pH 4.6, and at for to the as in except that the pH was CRP to the determined CRP pentameric it was bound to at pH 4.6, by and native pentameric CRP PubMed Scopus Google not native pentameric CRP the of is in the of the it modified and of CRP PubMed Scopus Google Scholar, PubMed Scopus Google At pH CRP by but not that the native pentameric of CRP was after binding to at pH CRP by but not At pH 4.6, the was and was of CRP after binding to at pH the acidic pH of CRP was the of CRP was and the was that CRP, after binding to at pH 4.6, was pentameric but the was also that acidic in the of CRP was for H, the of to Acidic pH for CRP to we the of pH on the protein specificity of CRP CRP, at pH 7.0, did not bind to any of the six immobilized proteins. 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This also that the acidic pH was for CRP to of binding to factor H, but, the acidic pH was not for factor to a protein. the pH of the CRP which was and for at was and the binding was at pH 7.0, the binding of CRP to C3b, IgG, and BSA was that exposure of CRP and these immobilized proteins to acidic pH was for the binding of protein activity of CRP at acidic pH. were factor H, C3b, IgG, and sites in the were CRP was to the and at for CRP was as in the to CRP was in pH CRP was in pH 4.6, and at for to the as in except that the pH was CRP to the as of protein assays. and of CRP after to at pH as in we found that CRP a after binding to at pH 4.6, we determined CRP also the after binding to immobilized protein the of a protein in which we the binding of CRP to factor H, C3b, IgG, and BSA at as a of pH by and was used as a the the were to that in CRP bound to all immobilized proteins. did not CRP bound to immobilized proteins. the not to and to in the that CRP, after binding to immobilized proteins at acidic pH, was pentameric but the was modified in a to CRP in the to in CRP after binding to immobilized proteins at acidic pH. were factor H, C3b, IgG, and CRP in pH was to the and at for CRP was a of the of the was at and on the of CRP to of the of the binding on CRP for immobilized proteins, we investigated the of for the binding of CRP to immobilized proteins. 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CRP a of the which of CRP also the that CRP has been conserved throughout for and by protein misfolding and conformationally altered proteins in acidic IntroductionC-reactive protein (CRP) 4The abbreviations used are: CRPpentameric C-reactive proteinmCRPmonomeric C-reactive proteinLDLlow density lipoproteinoxLDLoxidized LDLAβamyloid β protein fragment 1–38PChphosphocholinePnCpneumococcal C-polysaccharideANS1-anilinonaphthalene-8-sulfonic acid. is an evolutionarily conserved protein. CRP is found in all animals, from invertebrates to vertebrates, where it has been sought. Human CRP is a pentameric protein comprised of five identical, non-covalently associated 23,028-Da subunits (reviewed in Refs. 1Singh S.K. Suresh M.V. Voleti B. Agrawal A. Ann. Med. 2008; 40: 110-120Crossref PubMed Scopus (101) Google Scholar, 2Black PubMed Scopus Google Scholar, PubMed Scopus Google At physiological pH, CRP is for its binding to as Med. PubMed Scopus Google CRP a of to PubMed Scopus Google Scholar, PubMed Scopus Google of CRP to proteins, modified low-density and has also been PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, S.K. Suresh M.V. Agrawal A. 2008; PubMed Scopus Google Scholar, B. PubMed Scopus Google Scholar, A. PubMed Scopus Google and sites on of CRP, and the suggest that these sites in the binding of CRP to of its CRP to the form of CRP which from pentameric CRP PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, A. A. PubMed Scopus Google the of CRP in the is in the to PubMed Scopus Google CRP is also found at the sites of in and Med. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Med. Google Scholar, PubMed Scopus Google of CRP at the sites of is of the of For example, CRP is at the of CRP in PubMed Scopus Google Scholar, Med. Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google to the of CRP at the sites of the of CRP at the sites of is not for the of CRP at the sites of is on the and of CRP, which at physiological pH. Because the pH at the sites of acidic PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, B. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, A. Google Scholar, PubMed Scopus Google Scholar, A. Google in this study, we investigated the binding specificities of CRP at acidic pH to the of CRP at the sites of is a complement protein that from complement PubMed Scopus Google also from complement the factor Google Scholar, PubMed Scopus Google We of in the of CRP in of Human CRP from Med. PubMed Scopus Google Scholar, Google Scholar, Google Scholar, M.V. S.K. Agrawal A. PubMed Scopus Google Because bind factor PubMed Scopus Google and CRP also factor conditions PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, A. PubMed Scopus Google it has been that CRP bind to factor and to of CRP M.V. S.K. Agrawal A. PubMed Scopus Google Scholar, A. Suresh M.V. S.K. 2008; PubMed Scopus Google We also of in CRP bind to factor H, it for to the implications of factor in this 2008; PubMed Scopus Google density is an form of to form that to the of which is an inflammatory PubMed Scopus Google CRP is in present in and PubMed Scopus Google Scholar, Med. Google Scholar, PubMed Scopus Google Because the pH of the acidic PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, B. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google in this study, we also investigated the of acidic pH on to factor and we proteins: complement fragment C3b, IgG, amyloid β fragment and to the binding of CRP at acidic pH. We that CRP, at acidic pH, bind to factor H, C3b, IgG, and but not bind to we found that CRP, at acidic pH, bound to all six proteins as CRP a by these proteins that were immobilized to microtiter plates and also exposed to acidic pH at