Degradation of pre-beta-high density lipoproteins and their binding activity to human blood monocytes.
We have previously reported that high density lipoprotein3 (HDL3), apolipoprotein A-I (apoA-I) rich lipoprotein, binds specifically to the surface of human blood monocytes. Pre-beta-HDL with a pre-beta mobility on agarose gels is an apoA-I (MW 28 kDa)-rich and a lipid-poor lipoprotein. In the present study, we found that pre-beta-HDL purified by ion-exchange chromatography was susceptible to degradation if isolated in the absence of anti-proteases, resulting in the smaller lyso-pre-beta-HDL. The mass of lyso-pre-beta-HDL was confirmed using a delayed extraction matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (DE-MALDI-TOF MS), which showed a fragment of approximately 22,378.9 Da. We further investigated limited proteolysis of apo A-I purified from human plasma HDL with various proteases, and cleavage appeared to be limited to the C-terminal end of apo A-I (amino acids 188-223). The ability of pre-beta-HDL and lyso-pre-beta-HDL to compete for HDL binding to monocytes was determined using a flow cytometry-based assay. Pre-beta-HDL competed efficiently for binding whereas lyso-pre-beta-HDL was significantly less effective. The data may indicate that the binding sites on monocytes specifically recognize apoA-I. We suggest that limited proteolysis around amino acids 188-223 of apo A-I may affect lipid binding, which may in turn affect HDL structure and function.
Department of Laboratory Medicine, School of Medicine, Shinshu University, Matsumoto, Nagano, Japan.
Tetsuo
T
Nakabayashi
Kazuyoshi
K
Yamauchi
Mitsutoshi
M
Sugano
Kenji
K
Sano
Minoru
M
Tozuka
Hiroya
H
Hidaka
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000378:metabolism*; D001665:Binding Sites; D005434:Flow Cytometry; D053442:High-Density Lipoproteins, Pre-beta; D006801:Humans; D008075:Lipoproteins, HDL / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009000:Monocytes / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship
D016632:Apolipoprotein A-I; D053442:High-Density Lipoproteins, Pre-beta; D008075:Lipoproteins, HDL