title: 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.
authors.affiliation: Analytical Laboratory Chemistry, Graduate School of Health Care Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Usami
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Matsuda
authors.forename: Mitsutoshi
authors.initials: M
authors.lastname: Sugano
authors.forename: Nau
authors.initials: N
authors.lastname: Ishimine
authors.forename: Yuriko
authors.initials: Y
authors.lastname: Kurihara
authors.forename: Tamaki
authors.initials: T
authors.lastname: Sumida
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Yamauchi
authors.forename: Minoru
authors.initials: M
authors.lastname: Tozuka
mesh_terms: D000328:Adult; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology; D016632:Apolipoprotein A-I / Q000097:blood* / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods*; D053818:Chymases / Q000378:metabolism*; D006801:Humans
chemical_list: D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D053818:Chymases