Abstract
When stimulated, rat serosal mast cells degranulate and secrete a cytoplasmic neutral protease, chymase. We studied the fragmentation of apolipoprotein (apo) A-I during proteolysis of HDL3 by chymase, and examined how chymase-dependent proteolysis interfered with the binding of eight murine monoclonal antibodies (Mabs) against functional domains of apoA-I. Size exclusion chromatography of HDL3 revealed that proteolysis for up to 24 h did not alter the integrity of the α-migrating HDL, whereas a minor peak containing particles of smaller size with preβ mobility disappeared after as little as 15 min of incubation. At the same time, generation of a large (26 kDa) polypeptide containing the N-terminus of apoA-I was detected. This large fragment and other medium-sized fragments of apoA-I produced after prolonged treatment with chymase were found to be associated with the αHDL; meanwhile, small lipid-free peptides were rapidly produced. Incubation of HDL3 with chymase inhibited binding of Mab A-I-9 (specific for preβ1HDL) most rapidly (within 15 min) of the eight studied Mabs. This rapid loss of binding was paralleled by a similar reduction in the ability of HDL3 to induce high-affinity efflux of cholesterol from macrophage foam cells, indicating that proteolysis had destroyed an epitope that is critical for this function. In sharp contrast, prolonged degradation of HDL3 by chymase failed to reduce the ability of HDL3 to activate LCAT, even though it led to modification of three epitopes in the central region of apoA-I that are involved in lecithin cholesterol acyltransferase (LCAT) activation.This differential sensitivity of the two key functions of HDL3 to the proteolytic action of mast cell chymase is compatible with the notion that, in reverse cholesterol transport, intactness of apoA-I is essential for preβ1HDL to promote the high-affinity efflux of cellular cholesterol, but not for the α-migrating HDL particles to activate LCAT.
Highlights
When stimulated, rat serosal mast cells degranulate and secrete a cytoplasmic neutral protease, chymase
The various HDL particles are in a Abbreviations: apoA-I, apolipoprotein A-I; Mab, monoclonal antibody; LCAT, lecithin cholesterol acyltransferase; HDL3, high density lipoproteins3; PBS, phosphate buffered saline; BSA, bovine serum albumin; FFA, free fatty acid; DMEM, Dulbecco’s modified Eagle’s medium; TLC, thin-layer chromatography; BTEE, N-benzoyl-L-tyrosine ethyl ester; AP, alkaline phosphatase; pNPP, p-nitrophenyl phosphate; TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; 2D-PAGGE, two dimensional polyacrylamide gradient gel electrophoresis; LpA-I, lipoproteins containing apoA-I; ELISA, enzyme linked inmunosorbent assay; LCAT, lecithin cholesterol acyltransferase
To gain further insight into the specific degradation of apoA-I in HDL3 by chymase, we examined this apolipoprotein for fragmentation and loss of specific epitopes when subjected to proteolysis by granule remnants, and related the observed changes to two functions of apoA-I in HDL, namely the induction of cholesterol efflux from macrophage foam cells and the activation of LCAT
Summary
Rat serosal mast cells degranulate and secrete a cytoplasmic neutral protease, chymase. Incubation of HDL3 with chymase inhibited binding of Mab A-I-9 (specific for pre1HDL) most rapidly (within 15 min) of the eight studied Mabs This rapid loss of binding was paralleled by a similar reduction in the ability of HDL3 to induce high-affinity efflux of cholesterol from macrophage foam cells, indicating that proteolysis had destroyed an epitope that is critical for this function. In sharp contrast, prolonged degradation of HDL3 by chymase failed to reduce the ability of HDL3 to activate LCAT, even though it led to modification of three epitopes in the central region of apoA-I that are involved in lecithin cholesterol acyltransferase (LCAT) activation This differential sensitivity of the two key functions of HDL3 to the proteolytic action of mast cell chymase is compatible with the notion that, in reverse cholesterol transport, intactness of apoA-I is essential for pre1HDL to promote the high-affinity efflux of cellular cholesterol, but not for the ␣-migrating HDL particles to activate LCAT.—Lee, M., P. After cholesterol has been taken up by these acceptors, the activation of LCAT plays an important role in the clearance of excess cholesterol from peripheral cells to the liver, as this enzyme allows channeling of cholesterol from the initial acceptors to the spherical ␣-migrating HDL, leading to maturation of the HDL particles
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