The in-vitro influence 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 esterrfied cholesterol is regulated by two enzyme activities, cholesterol ester hydrolases, which drive the balance in favor of un-esterrfied cholesterol, and acyl-CoA: cholesterol acyl transferase (ACAT) which acts in the opposite direction. During acute inflammation upo-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 purlfed 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 ole-ate. 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 esteruse, 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 mac-rophage ACATactivity. 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 A CA T activity. The NH-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 A CA T 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.

Key Words:

• Serum amyloid A
• cholesterol
• macrophages
• inflammation
• esterlfication
• de-estertfication
• acylCoA
• cholesterol acyl trans-ferase
• neutral cholesterol esterase