Ultrasensitive confocal fluorescence microscopy of C-reactive protein interacting with FcgammaRIIa.
BACKGROUND
C-Reactive protein (CRP) is an acute phase protein with a suggested pathogenic role in cardiovascular disease. Previous reports proposed that the low-affinity IgG receptor FcgammaRIIa is the major receptor for CRP. However, these reports were met with criticism because the use of anti-CRP antibodies in the detection of CRP binding to FcgammaRIIa may have caused false-positive results.


RESULTS
To resolve this controversy, we used ultrasensitive fluorescence microscopy to study the association, dissociation, and equilibrium of CRP binding to FcgammaRIIa. CRP indeed binds to FcgammaRIIa, with low association rates and dissociation rates. Anti-CRP antibodies markedly enhance binding, as is evident from the decrease of the equilibrium dissociation coefficient by 2 orders of magnitude.


CONCLUSIONS
Our study demonstrates the virtues of single fluorophore labeling and highlights the pitfalls of immunolabeling in investigating CRP/Fc receptor interactions. Importantly, this article provides the first quantitative characterization of CRP binding to FcgammaRIIa and explains and reconciles the diverse and conflicting data presented in the literature.
Department of Internal Medicine II-Cardiology, University of Ulm, Ulm, Germany.
Dimitar E
DE
Manolov
Carlheinz
C
Röcker
Vinzenz
V
Hombach
G Ulrich
GU
Nienhaus
Jan
J
Torzewski
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D001666:Binding Sites, Antibody; D002097:C-Reactive Protein / Q000276:immunology / Q000378:metabolism*; D019556:COS Cells / Q000737:chemistry / Q000378:metabolism; D002522:Chlorocebus aethiops; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D011485:Protein Binding; D017452:Receptors, IgG / Q000378:metabolism*
D015703:Antigens, CD; C080568:Fc gamma receptor IIA; D005456:Fluorescent Dyes; D017452:Receptors, IgG; D002097:C-Reactive Protein