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Degradation of Pre-ß-High Density Lipoproteins and Their Binding Activity to Human Blood Monocytes

Address correspondence to Hiroya Hidaka, Ph.D., Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto, Nagano Prefecture, 390-8621, Japan; tel 81 263 2368; fax: 81 263 34 5316; e-mail: hiroyan{at}hsp.md.shinshu-u.ac.jp.

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-ß-HDL with a pre-ßmobility on agarose gels is an apoA-I (MW 28 kDa)-rich and a lipid-poor lipoprotein. In the present study, we found that pre-ß-HDL purified by ion-exchange chromatography was susceptible to degradation if isolated in the absence of anti-proteases, resulting in the smaller lyso-pre-ß-HDL. The mass of lyso-pre-ß-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-ß-HDL and lyso-pre-ß-HDL to compete for HDL binding to monocytes was determined using a flow cytometry-based assay. Pre-ß-HDL competed efficiently for binding whereas lyso-pre-ß-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.

Keywords: pre-ß-HDL, apolipoprotein A-I, protease, mass spectrometry, monocytes