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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.

We previously identified a binding site for high density lipoprotein-3 (HDL3) on the surface of human peripheral blood monocytes. Here we describe the purification and measurement of HDL-binding proteins present on these cells. Purification of HDL-binding proteins was achieved by chromatography using DEAE ion-exchange, wheat germ lectin, and apoHDL3 affinity columns. Subsequent use of SDS-PAGE and ligand blotting lead to the identification of two major proteins with apparent molecular masses of 100 and 120 kDa, plus several smaller proteins that appeared to be degradation products. Analysis of purified HDL-binding proteins by reverse-phase HPLC showed that the two main proteins at 100 and 120 kDa were eluted in 65 and 70% acetonitrile, respectively, indicating the proteins are strongly hydrophobic. To measure the amount of HDL-binding protein present in CHAPS-solubilized human mononuclear cells, a 96-well plate assay was developed that was based on the same principles as the purification method. The present study of HDL-binding proteins in mononuclear cells advances our understanding of the physiological roles and fate of HDL.

Hypertriglyceridemia is an independent risk factor for atherosclerosis. This risk is most likely due to accumulation of circulating triglyceride rich lipoproteins with heterogeneous particles. The identification and characterization of these triglyceride rich lipoproteins is important to detect abnormality of triglyceride metabolism. In the present study, we developed a new method that combines ultracentrifugation and agarose gel electrophoresis with triglyceride- and cholesterol-staining. We investigated 40 subjects with hypertriglyceridemia. Triglyceride rich lipoproteins with very light density were recovered in the aqueous fraction after ultracentrifugation (17,000 x g, 15 min). The lipoproteins recovered in the aqueous fraction contained chylomicrons, if present, their remnants, and light-VLDL (d1.000 g/ml) containing apoB-100, but not normal VLDL (d1.006 g/ml) and IDL. Triglyceride rich lipoproteins in the aqueous fraction were characterized by electrophoresis patterns of triglyceride- and cholesterol-staining. Forty patients with hypertriglyceridemia were separated into 8 groups according to their electrophoretic patterns. In lipoproteins recovered in the aqueous fraction from each group, the triglyceride level was correlated with the respective cholesterol level. In summary, a system using ultracentrifugation and agarose gel electrophoresis with triglyceride- and cholesterol-staining is useful for characterization of triglyceride rich lipoproteins and their remnants.

Apolipoprotein (apo) J, abundant in cerebrospinal fluid (CSF), is known to play a role in the pathogenesis of Alzheimer's disease (AD); however, the mechanism remains obscure. To characterize the apoJ-containing lipoproteins in CSF, we compared the distribution of apoJ in CSF lipoprotein partides with those of apoE and apoAI. CSF lipoproteins (fractionated by ultracentrifugation, gel-filtration chromatography, and agarose-gel electrophoresis) were characterized by immunoblot analysis using anti-apoJ, anti-apoE, and anti-apoAI antibodies. Immunoprecipitation and immunoabsorption were used to clarify the combinations in which these apolipoproteins exist. All of the apoJ in CSF was in the fraction with density ofor = 1.250 g/ml after ultracentrifugation; relatively little apoE and apoAI was in that fraction. In gel-filtration chromatography, the main peak of apoJ-containing lipoprotein particles was clearly distinguishable from those of apoE- and apoAI-containing lipoproteins. Immunoabsorption and agarose-gel electrophoresis indicated that the dominant apoJ-containing lipoprotein partides did not contain apoE. These findings indicate that a significant fraction of the apoJ present in CSF does not co-exist with apoE or apoAI within the same particles. Immunoprecipitation revealed two types of particles: one that contains no apoAl but apoE and another that contains no apoE but apoAI. These results show that several subfractions of lipoprotein particles exist in CSF, differing from each other in their combinations of apoE, apoJ, and apoAI. We concluded that there are at least 9 forms or combinations (including free apolipoproteins) of apoJ, apoE, and apoAl in the CSF.