Recombinant human serum amyloid A (apoSAAp) binds cholesterol and modulates cholesterol flux.
During acute inflammation, the serum amyloid A (apoSAA) proteins apoSAA1 and apoSAA2 are transiently associated with high density lipoproteins (HDL) in concentrations of as much as 1000-fold more than their concentrations during homeostasis; however, their effect on HDL function is unclear. Recombinant apoSAAp, a hybrid of the closely related human apoSAA1 and apoSAA2 isoforms, was found to exhibit a high affinity for cholesterol. The adsorption of apoSAAp to polystyrene microtiter wells at physiological pH, temperature, and salt concentration was inhibited and reversed by cholesterol. ApoSAAp, to a greater extent than apoA-I, albumin, or fetal bovine serum, enhanced diffusion of cholesterol from HDL across a membrane that retained molecules > 3.5 kDa. Cholesterol from 25 nM to 125 microM inhibited binding of [3H]cholesterol to 167 nM apoSAAp. A cholesterol binding assay was developed to determine the dissociation constant for binding of [3H]cholesterol to apoSAAp; Kd = 1.7 +/- 0.3 x 10(-7) M and the maximum binding capacity (Bmax) is 1.1 +/- 0.1 mol/mol. After binding cholesterol, the apparent size of apoSAAp as determined by gel filtration on Sephacryl S-100 was increased from 12 to 23 kDa. ApoSAAp enhanced free [14C]cholesterol uptake from tissue culture medium by HepG2 cells, an effect that was dose dependent and blocked by polyclonal antibodies to human apoSAA1 and apoSAA2. ApoSAAp, unlike apoA-I, was taken up from serum-free medium by HepG2 cells and appeared to be degraded by cell-associated enzymes. Unlike peritoneal exudate cells, human HepG2 hepatoma cells do not secrete an enzyme that degrades apoSAAp. These results suggest that apoSAA can potentially serve as a transient cholesterol-binding protein.
Liang
J S
JS
Department of Biochemistry, Boston University School of Medicine, MA 02118.
Sipe
J D
JD
D000327:Adsorption; D000595:Amino Acid Sequence; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism* / Q000494:pharmacology; D008076:Cholesterol, HDL / Q000378:metabolism; D002850:Chromatography, Gel; D006801:Humans; D007457:Iodine Radioisotopes; D008113:Liver Neoplasms / Q000378:metabolism; D008969:Molecular Sequence Data; D011137:Polystyrenes; D011994:Recombinant Proteins / Q000378:metabolism; D000685:Serum Amyloid A Protein / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D014316:Tritium; D014407:Tumor Cells, Cultured
D008076:Cholesterol, HDL; D007457:Iodine Radioisotopes; D011137:Polystyrenes; D011994:Recombinant Proteins; D000685:Serum Amyloid A Protein; D014316:Tritium; D002784:Cholesterol