Detection of chymase-digested C-terminally truncated apolipoprotein A-I in normal human serum.

Abstract In atherosclerotic artery walls, mast cells, an inflammatory cell, are activated and secrete some proteases including chymase. Chymase, a chymotrypsin-like protease, cleaves the C-terminus of apolipoprotein A-I (apoA-I) at Phe225. This cleavage reduces the ability of apoA-I to promote the efflux of cellular cholesterol. The aim of this study is to detect C-terminally truncated apoA-I in normal human serum. For this purpose, we generated a monoclonal antibody that specifically recognizes C-terminally truncated apoA-I by immunizing mice with a peptide that corresponds to human apoA-I amino acid residues 216–225. The monoclonal antibody, termed 16–4 mAb, selectively reacted with recombinant C-terminally truncated apoA-I, but not recombinant full-length apoA-I. A two-dimensional electrophoresis analysis also indicated that only two out of six spots that contained apoA-I fragments and had a molecular mass of 26 kDa after chymase digestion reacted with the 16–4 mAb. We detected an extremely small amount of C-terminally truncated apoA-I in normal human serum by concentrating the serum through affinity chromatography using a 16–4 mAb-conjugated resin, and then performing Western blot analysis. The 16–4 mAb could be useful to examine whether C-terminally truncated apoA-I is associated with the progression of atherosclerosis.