Thein-vitroinfluence of serum amyloid A isoforms on enzymes that regulate the balance between esterified and un-esterified cholesterol

Abstract

The intracellular balance between un-esterified and esterified cholesterol is regulated by two enzyme activities, cholesterol ester hydrolases, which drive the balance in favor of un-esterified cholesterol, and acyl-CoA:cholesterol acyl transferase (ACAT) which acts in the opposite direction. During acute inflammation apo-serum amyloid A (apoSAA) isoforms 1.1 and 2.1 become major constituents of high density lipoprotein and this complex is internalized by macrophages. Mixtures of the two isoforms have been shown to enhance cholesterol esterase activity. Using a purified form of the pancreatic enzyme we have explored the mechanism by which apoSAA may accomplish this stimulation. The pancreatic esterase cleaves cholesteryl-oleate with a Km of 0.255 mM, releasing both cholesterol and oleate. Cholesterol exhibits a product inhibition which is relieved by isoform 2.1 but not 1.1 nor apolipoprotein A-I. The NH2-terminal 16 residues of isoform 2.1 had no effect on the esterase, but the 80 residue peptide constituting its COOH-terminus possessed the stimulatory property. Purified isoforms 1.1, 2.1, 2.2, apolipoprotein A-I, the NH2-terminal 16 residues and COOH-terminal 80 residues of isoform 2.1 were also examined for their effects on macrophage ACAT activity. Isoforms 2.1 and 2.2 produced dose dependent inhibitions of up to 50%, (p<0.001). Isoform 1.1, and apoA-I had no effect on ACAT activity. The NH2-terminal 16 residue peptide of isoform 2.1 reduced the ACAT activity in a dose dependent manner by 74% (p<0.001), whereas the COOH-terminal 80 residues, in contrast to its enhancing effect on the esterase, had no inhibitory effect on ACAT. Such complementary but opposite effects of isoform 2.1 on ACAT and the esterase are consistent with a role for this protein in shifting the balance between unesterified (transportable) and esterified (storage) forms of cholesterol in favor of the latter. They suggest that apoSAA2.1 may mediate cholesterol mobilization at sites of tissue injury.

Peptides derived from serum amyloid A prevent, and reverse, aortic lipid lesions in apoE−/− mice

“…In the prevention mode, six groups of mice were examined, those on 1 ) the high-fat diet; 2 ) standard lab chow (low-fat diet); 3 ) the high-fat diet given mSAA2.1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] ; 4) the high-fat diet given mSAA2.1 74-103 ; 5) the high-fat diet given mSAA2.1 1-20 ϩ mSAA2.1 ; and 6) the high-fat diet given peptide-free liposomes. In each case, the peptides were given as liposomes once every 4 days (four doses), at the termination of which the animals were euthanized and the aortas were stained with Oil Red O.…”

“…Peptides corresponding to mSAA2.1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] , mSAA2.1 , mSAA2.1 51-80 , mSAA2.1 , and human (h)SAA1.1/2.1 (note that the sequence of the human isoforms is identical over the first 50 residues) were prepared as described previously (3). In addition, an R linked to the N terminus of mSAA1.1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] was synthesized.…”

“…Cholesterol efflux was determined by measuring the appearance of [ 3 H]cholesterol in plasma by scintillation spectrometry. To study whether the export of cholesterol from these injected J774 cells to plasma is influenced by mSAA2.1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] , mSAA1.1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] , or hSAA1.1/2.1 , 100 l of liposomes containing 15 g of one of these peptides was injected intravenously, and at various times after this injection, ‫ف‬ 25 l of blood was collected from the tail vein of each animal and the plasma was analyzed by scintillation spectrometry.…”

“…In addition, an R linked to the N terminus of mSAA1.1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] was synthesized. These peptides were obtained from the Protein Synthesis Laboratory at Queen's University or The Hospital for Sick Children (Toronto, Ontario, Canada).…”

“…Our past results suggested that a physiological role of one isoform of a major acute-phase protein synthesized by the liver in response to tissue injury, mouse serum amyloid A 2.1 (mSAA2.1), is the regulation of M cholesterol export (1)(2)(3). Fragmentation of this protein into peptides that span its entire length has revealed that the N-terminal region, mSAA2.1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], is a potent in vitro and in vivo inhibitor of M ACAT (3). A separate region at the C terminus, mSAA2.1 , contains a domain that enhances the in vitro and in vivo activity of neutral cholesteryl ester hydrolase (CEH) (3).…”