Elucidation of a protease-sensitive site involved in the binding of calcium to C-reactive protein.
C-reactive protein (CRP) is a Ca2+-binding protein composed of five identical 23-kDa subunits arranged as a cyclic pentamer, present in greatly elevated concentration in the blood during the acute phase of processes involving tissue injury and necrosis. In the present work, it was found that treatment of human CRP with Pronase or Nagarse protease produces two major fragments which remain associated in physiological buffers but are separable under denaturing conditions. To localize the cleavage site(s), the fragments were characterized according to molecular mass, amino acid composition, partial amino acid sequence, and reactivity with monoclonal antibodies specific for the fragments and for defined CRP epitopes including residues 147-152 and 199-206. Nagarse protease cleaves the CRP subunit between residues 145 and 146, producing two fragments, 16 and 6.5 kDa (calculated molecular mass). Pronase cleaves the CRP subunit between residues 146 and 147, producing a 16-kDa fragment (A1) and a 6.5-kDa fragment (B); an additional fragment (A2) approximately 1 kDa smaller than fragment A1 is also apparently produced due to a secondary cleavage site in fragment A1. Cleavage appears to be completely inhibited in the presence of 1 mM CaCl2. Ca2+ does not protect cleaved CRP from heat-induced aggregation (i.e., precipitation) as it does the intact protein. Protease-cleaved CRP loses the ability to bind to the Ca2+-dependent ligand phosphorylcholine but remains the ability to bind to the Ca2+-independent ligand arginine-rich histone. Equilibrium dialysis indicates that intact CRP binds 2 mol of Ca2+/mol of subunit with a Kd of 6 X 10(-5) M.(ABSTRACT TRUNCATED AT 250 WORDS)
Kinoshita
C M
CM
Department of Immunology/Microbiology, Rush Medical College, Chicago, Illinois 60612.
Ying
S C
SC
Hugli
T E
TE
Siegel
J N
JN
Potempa
L A
LA
Jiang
H
H
Houghten
R A
RA
Gewurz
H
H
D000595:Amino Acid Sequence; D001202:Ascitic Fluid; D002097:C-Reactive Protein / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002850:Chromatography, Gel; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects
D002097:C-Reactive Protein; D010447:Peptide Hydrolases; D002118:Calcium