Recombinant human serum amyloid A (apoSAAp) binds cholesterol and modulates cholesterol flux

J S Liang,J D Sipe

doi:10.1016/s0022-2275(20)39752-2

Abstract

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.

Highlights

N acute phase response proteins serum amyloid A (apoSAA), and apoSAA? isoforms, was found to exhibit a high affinity for cholesterol
To measure inhibition of apoSAAp adsorption, 100-pl aliquots of Iz5I-labeled apoSAAp (2 pglml) in R P M I medium with varying concentrations of cholesterol, PC, or N H D L were added to 96-well polystyrene plates, incubated at 37OC for 2 h, 10 pl of triplicate aliquots were removed for counting of radioactivity
During the course of a n inflammatory episode, apoSAAl and apoSAA2can transiently constitute as much as 80% of total high density lipoproteins (HDL) proteins, reaching concentrations greater than 1 mg/ml and displacing the primary protein constituent of H D L, apolipoprotein A-I (apoA-I) [11, 13]

Summary

MATERIALS AND METHODS

Recombinant synthetic human apoSAA,, was purchased from Pepro Tech (Rocky Hill, NJ). ApoSAA, is a hybrid molecule corresponding to human apoSAAl, except for the N-terminal methionine and substitution of asparagine for aspartic acid at position 60 and arginine for histidine at position 71; the latter two substituted residues are present in apoSAAnp (Fig. 1). To measure inhibition of apoSAAp adsorption, 100-pl aliquots of Iz5I-labeled apoSAAp (2 pglml) in R P M I medium with varying concentrations of cholesterol, PC, or N H D L were added to 96-well polystyrene plates, incubated at 37OC for 2 h, 10 pl of triplicate aliquots were removed for counting of radioactivity. To measure reversal of adsorption, i.e., the release of bound lZ5I-labeledapoSAAp from microtiter wells by lipid and lipoprotein or antibodies, 100 p1 of cholesterol, PC, or N H D L at varying concentrations in serum-free R P M I medium was added to wells to which 1251-labeled apoSAA, had been adsorbed and, after the removal of the coating medium, washed three times with R P M I lacking apoSAA.

Materials and Methods

Findings

DISCUSSION