出版社：American Society for Biochemistry and Molecular Biology
摘要：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. —Lee, M., P. Uboldi, D. Giudice, A. L. Catapano, and P. T. Kovanen. Identification of domains in apoA-I susceptible to proteolysis by mast cell chymase: implications for HDL function. J. Lipid Res. 41: 975–984.
关键词：α- and preβ-migrating HDL ; monoclonal antibodies ; LCAT ; cellular cholesterol efflux ; proteolysis of apoA-I
