Analysis of the binding of C-reactive protein to histones and chromatin.
C-reactive protein (CRP) is an acute phase serum protein in man which binds to phosphocholine (PC) in a calcium-dependent manner. CRP has been shown to bind to chromatin and nucleosome core particles. However, CRP does not bind to DNA and there is conflicting evidence regarding the binding of CRP to histones. In the present study, binding of CRP to chromatin was confirmed by ELISA using chromatin bound to microtiter wells. When chromatin depleted of histone H1 was used in the same assay, no CRP binding was detected. Similar results were observed using a competitive inhibition ELISA. These results indicate an important role for H1 in the binding of CRP to chromatin. Further studies were done to characterize the binding of CRP to purified individual histones. CRP binding to histones was demonstrated first by blotting. Calf thymus histones were separated on a 15% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with 125I-CRP. CRP bound to H1 and H2A and to a lesser extent to H2B. Non-specific binding to H3 was seen and no binding to H4 was observed. CRP binding to purified individual histones was tested by ELISA. Essentially identical results were seen to those obtained by blotting. CRP binding to the H2A-H2B complex was observed as well as reactivity with trypsin-resistant fragments of H2A, H2B, and H3. By blotting and by ELISA all CRP reactions were blocked by PC and EDTA indicating binding through the calcium-dependent PC-binding site on CRP. These studies further characterize the nature of the binding of CRP to chromatin and histones and show that the presence of H1 on chromatin is required for CRP binding.
Du Clos
T W
TW
VA Medical Center, Albuquerque, NM.
Zlock
L T
LT
Rubin
R L
RL
D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D015153:Blotting, Western; D002097:C-Reactive Protein / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D004347:Drug Interactions; D004797:Enzyme-Linked Immunosorbent Assay; D006657:Histones / Q000378:metabolism*; D006801:Humans; D010767:Phosphorylcholine / Q000494:pharmacology; D014357:Trypsin
D002843:Chromatin; D006657:Histones; D010767:Phosphorylcholine; D002097:C-Reactive Protein; D014357:Trypsin