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Annals of Clinical & Laboratory Science 34:287-298 (2004)
© 2004 Association of Clinical Scientists

Review

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

Tetsuo Nakabayashi1, Kazuyoshi Yamauchi1, Mitsutoshi Sugano1, Kenji Sano1, Minoru Tozuka1 and Hiroya Hidaka2
1 Department of Laboratory Medicine, School of Medicine, and 2 Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Nagano, Japan

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.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.

(received 17 February 2004; accepted 8 March 2004)

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


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
High density lipoprotein (HDL) acts in the "reverse cholesterol transport" pathway, whereby cholesterol is transported from peripheral tissues to the liver [1,2]. Apolipoprotein A-I (apo A-I) is the major protein constituent of plasma HDL, and is required for activation of lecithin-cholesterol acyltransferase (LCAT) [3], binding of phospholipid transfer protein to HDL [4,5], and mediating the interaction of HDL with cells [6]. It appears that apoA-I is important in the process by which intracellular cholesterol is translocated to the plasma membrane and then to HDL [7,8].

ApoA-I is a single polypeptide containing 243 amino acids, with a calculated molecular weight of 28,077 Da [9–11]. It consists mainly of eight 22-amino acid repeating segments, typically spaced with helix-breaking proline residues, amphipathic {alpha}-helices, and two 11-amino acid tandem repeats [12,13]. Apo A-I-rich lipoproteins (apo A-I-Lp) can be classified into 2 subfractions based on agarose gel electrophoresis mobility, {alpha} and pre-ß, which are found distributed in the HDL (1.063 < d < 1.21 g/ ml) and VHDL (1.21 < d < 1.25 g/ml) plasma density ranges, respectively [14–17]. {alpha}-HDL may participate in cholesterol efflux from the cell plasma membrane by an aqueous diffusion mechanism [18,19], while pre-ß-HDL particles, which are apoA-I-rich and lipid-poor, are understood to be an efficient initial acceptor of cellular unesterified cholesterol in the first step of reverse cholesterol transport (20–22). The mechanisms that regulate pre-ß-HDL metabolism and control the conversion of pre-ß HDL to {alpha}-HDL are not well understood.

We have identified specific HDL3 binding sites and proteins on human blood monocytes [23]. These monocyte HDL binding proteins resembled the HDL binding proteins from rat and human liver plasma membranes, HB1 and HB2 [24–26]. The HDL binding sites and proteins in monocytes are associated with apoHDL, but not with HDL lipids [23], and may play a role in HDL-mediated cholesterol metabolism.

In the present study, we showed that pre-ß-HDL, in particular the apoA-I component, is degraded when isolated in the absence of protease inhibitors. Then we experimented on the limited proteolysis of a purified apoA-I by various proteases in order to confirm that fragments of protein were specifically formed. Delayed extraction matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with delayed ion extraction (DE-MALDI-TOF MS) was used to analyze the fragments of apo A-I following limited proteolysis with trypsin, chymotrypsin, or S. aureus V8 protease. We also characterized in vitro binding of {alpha}-HDL, pre-ß-HDL, and lyso-pre-ß-HDL to human peripheral blood monocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials.  Sodium chloride (NaCl), sodium bromide (NaBr), potassium bromide (KBr), sodium azide, disodium ethylenediaminetetra-acetic acid (EDTA), benzamidinehydrochloride n-hydrate, 6-amino-n-caproic acid, fluorescein isothiocyanate (FITC), trifluoric acid (TFA), chymotrypsin from bovine pancreas, S. aureus V8 protease, glycerol, and bromphenol blue (BPB) were from WAKO Pure Chemicals (Osaka, Japan). HPLC-grade acetonitrile was from Kanto Chemicals (Tokyo, Japan). TPCK-treated-trypsin (trypsin) was from Worthington Biochem (Lakewood, NJ). Sinapinic acid, ACTH fragment (18–39), and angiotensin I were purchased from Sigma Chemical Co (St. Louis, MO). Lymphoprep and anti-human-apo A-I antibody (goat) were from Daiichi Pure Chemical Co (Tokyo, Japan). Phosphate-buffered saline (PBS) and peroxidase-conjugated anti-goat IgG (rabbit) were from Medical and Biological Laboratories Co, (Tokyo, Japan). PD-10 columns, Heparin-Sepharose CL6B, and DEAE-Sephacel gels were from Pharmacia Biotech (Uppsala, Sweden). The Simultest Control {gamma}1/{gamma}2a-containing mouse IgG1-FITC and IgG2a-PE with gelatin and 0.1% azide were from Becton-Dickinson Immunocytometry Systems (San Jose, CA). Bicinchonic acid (BCA) protein assay reagent kit was from Pierce Chemical Co. (Rockford, IL). Centricon filters were from Amicon (Danvers, MA). Gradient gels (8–16%) containing sodium dodecyl sulfate (SDS) were from Tefco Co (Tokyo, Japan). CHAPS gentamicin, phenylmethyl-sulfonyl fluoride (PMSF), TRIZMA base, and sinapinic acid were from Sigma Chemical Co.

Preparation of human lipoproteins.  Human HDL3 (1.125 < d < 1.21 g/ml) and VHDL (1.21 < d < 1.25 g/ml) were isolated from fresh human serum both in the presence and absence of preservatives and antiproteases (0.04% disodium ethylene diaminetetra-acetic acid (EDTA), 0.05% sodium azide, 1 mg/ml gentamicin, 0.3 mg/ml benzamidine, 0.13% {varepsilon}-amino caproic acid, and 1 mM PMSF) by ultracentrifugation according to the method of Havel et al [27]. HDL3 equilibrated by dialysis against 50 mM Tris-HCl buffer (pH 7.4, containing 0.05M NaCl) was applied to a Heparin-Sepharose CL6B column and eluted with the same buffer to obtain HDL3-without-apoE. Protein concentrations of lipoproteins were assayed by a BCA kit.

DEAE-chromatography of VHDL.  VHDL (1.21 < d < 1.25 g/ml) was desalted on a PD-10 column equilibrated in 5 mM Tris-HCl buffer (pH8.0), with or without preservatives and antiproteases as above, and loaded onto a DEAE-Sephacel column equilibrated in Tris-HCl buffer. After the column was washed with the same buffer, lipoproteins were eluted with Tris-HCL buffer containing 60–200 mM NaCl by a stepwise gradient. The lipoproteins were detected by immunoblotting with anti-apoA-I antibody as previously described [23].

Native and SDS-polyacrylamide gel electrophoresis (PAGE).  Sample buffer (0.1 M Tris/HCl buffer, pH 6.8, with 2% glycerol and 0.1% bromophenol blue) without and with 1% SDS (final concentration) was added to the sample and mixed. The mixture was applied to native gels (2–14% polyacrylamide gel) and SDS gels (8–16% polyacrylamide gel), and electrophoresed by the Tefco-system (Tefco, Tokyo, Japan) according to the manufacturer’s procedure. Protein was stained with Coomassie blue. Detection of apoA-I was performed by immuno-blotting, blotting, and immunostaining methods.

Mass analysis.  Molecular weights (MW) of digested peptides were determined in a linear mode with N2 laser (337 nm; step 2300–2700) by DE-MALDI TOF-MS (Voyager STR, PE Biosystems, Framingham, MA) [28]. Samples were desalted by zip tip (C18 spherical silica, 15 mm, Millipore, Bedford, MA) and adjusted to a concentration of 1–10 pmol/ ml with acetonitrile. One µl of sample solution and 1 µl of saturated sinapic acid solution in a 0.25 ml Eppendorff tube were vortex-mixed vigorously. One µl of this mixture was loaded into a well in the sample plate (PE Biosystems), dried, and the plate inserted into the mass spectrometer. A 2-point external calibration was performed using ACTH (amino acids 18–39; MW 2,466.72 Da) and angiotensin I (MW 1,297.51 Da) or bovine insulin (MW 5,734.6) in the positive ion mode. Five-point Savitsky-Golay smoothing was applied to all mass spectra.

Purification of apo A-1.  Human HDL3 (1.125 < d < 1.21 g/ml) was prepared from fresh normal human serum by ultracentrifugation (Optima L-70K, Beckman Instruments, Palo Alto, CA). HDL3 was dialyzed overnight in 5 mM Tris HCl buffer (pH 7.4), then delipidated using ethanol and ether (3/2 v/v), and the apo HDL3 was dissolved in 5 mM Tris HCl buffer (pH 7.4). Apo A-I was purified from apo HDL3 by gel filtration chromatography using Sephacryl S-200 (Pharmacia, Uppsala, Sweden). Purified apo A-1 was concentrated to 1 mg/ml protein concentration, and stored at –40°C until use.

Proteolysis of Apo A-I.  Purified apo A-I (8 µg) was incubated for 5 min at room temperature with each protease at various ratios in 10 ml of 50 mM Tris HCl buffer (pH 8.0). The enzyme-to-substrate ratios (E:S ratio of mass) for the trypsin incubations were 5,000:1 (1.6 ng trypsin), 50,000:1 (0.16 ng), and 200,000:1 (0.04 ng). The E:S ratios for chymotrypsin were 10,000:1 (0.8 ng chymotrypsin) and 50,000:1 (0.16 ng). The E:S ratios for S. aureus V8 protease were 50,000:1 (0.16 ng protease) and 500,000:1 (0.016 ng). The proteolytic products were immediately analyzed using DE-MALDI TOF-MS.

Amino acid sequence analysia.  The proteolytic products were analyzed by sodium dodecyl sulfate-8–16% gradient polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the peptide bands in the gels were electrophoretically transferred to a nitrocellulose memebrane. The membranes were stained with Coomassie blue, and the bands were cut from the membrane and subjected to automated sequence analysis on a pulsed-liquid sequencer (Applied Biosystems, Foster City, CA) equipped with a 120A Applied Biosystems PTH-analyzer.

FITC-labeled HDL.  FITC-labeled HDL3 (FITC-HDL3) was prepared using a modification of the method of Riggs et al [29]. FITC (1 mg) in 0.1 M carbonate buffer (pH 9.6) was added to 10 mg of HDL3 protein previously adjusted to pH 9.6. The mixture was gently rocked for 1 hr at room temperature and applied to a PD-10 column equilibrated with PBS to separate free FITC from conjugated FITC-HDL3. The FITC-HDL3 was then equilibrated with PBS and concentrated using Centricon filters. For analysis, an aliquot of FITC-HDL was mixed with glycerol and BPB and electrophoresed on a native gradient gel, while a second aliquot was mixed with 5% SDS containing glycerol and BPB and analyzed by SDS-PAGE.

Preparation of mononuclear cells from human blood.  Human mononuclear cells were prepared according to a modified method of Ting et al [30]. Fresh human blood (5 ml) collected in tubes containing heparin or ethylene diaminetetra-acetic acid dipotassium salt (Terumo, Tokyo, Japan) was diluted 2-fold with PBS, then layered gently onto 2 ml of Lymphoprep (d = 1.077 g/ml). After centrifugation at 400 x g for 30 min at 20°C, the mononuclear cells collected from the intermediate phase were washed twice with PBS at 4°C, and then resuspended in 1 ml of PBS. Mononuclear cells were counted using a blood cell counter (NE-7000, Sysmex, Tokyo, Japan).

Flow cytometry of mononuclear cells.  Flow cytometric analysis of mononuclear cells was performed using the FACSort (Becton-Dickinson, Sunnyvale, CA) following the manufacturer’s instructions. In brief, mononuclear cells (approximately 1 x 105 in 1 ml of PBS) were incubated with 1 mg of FITC-HDL3 protein for 1 hr at 37°C. After washing with 20 x PBS (v/v), the cells were centrifuged at 3000 rpm for 3 min at 4°C, suspended in 0.5 ml of PBS, and analyzed by FACSort. In some experiments, mononuclear cells previously incubated with FITC-HDL3 at 37°C for 1 hr were treated with trypsin at 37°C for 15 min. The cells were then washed and resuspended in PBS and analyzed by the FACSort system. The peak channel of the logarithmic fluorescence histogram was used as an index of the amount of fluorescence bound to each particle. The cytometer electronically processes the electronic signals from each cell, creating numeric values for 3 parameters; forward scatter (FSC), side scatter (SSC), and fluorescence-1 (FL1). It assigns each value a channel number, from 0 to 1023. These numbers are measurements of the relative light intensity scattered or emitted by a cell when it passes through the laser beam.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Agarose gel electrophoresis of VHDL DEAE column fractions.  To separate pre-ß-HDL from {alpha}-HDL, VHDL was added to a DEAE-Sephacel column and the lipoproteins were eluted using NaCl. Column fractions were analyzed by agarose gel electrophoresis. When isolated in the presence of preservatives and antiproteases, pre-ß-HDL was eluted with 80 mM NaCl, and {alpha}-HDL with 100 mM NaCl (Fig. 1Go). In contrast, if the elution buffer did not contain preservatives and antiproteases, pre-ß-HDL was eluted with 100 mM NaCl, and {alpha}-HDL with 150 mM NaCl. ß-HDL and {alpha}-HDL were both present in fractions eluted with >150 mM NaCl.



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  		Fig. 1. Agarose gel electrophoresis analysis of VHDL in DEAE ion-exchange chromatography fractions. VHDL (1.21 < d < 1.25 g/ml) was prepared from human serum by ultracentrifugation, equilibrated with Tris-HCl buffer (5 mM, pH8.0), and applied to a DEAE-Sephacel column. Bound lipoproteins were eluted with various concentrations of NaCl (0 – 0.2 M), electrophoresed on agarose gels, and blotted onto nitrocellulose membranes. The membranes were sequentially incubated with an anti-apoA-I antibody, a peroxidase-conjugated secondary antibody and 4-chloro-1-naphtol and H2O2. Panel A shows DEAE column fractions of VHDL isolated in the presence of preservatives and antiproteases, while Panel B shows fractions of VHDL isolated in the absence of preservatives and antiproteases.

 
HDL and apoA-I in DEAE column fractions.  HDL present in DEAE column fractions was analyzed by native-PAGE and SDS-PAGE to determine the particle sizes. Pre-ß-HDL isolated in the presence of preservatives and antiproteases was found to be approximately 250–300 kDa, the same size as {alpha}-HDL (Fig. 2AGo). However, pre-ß-HDL isolated in the absence of preservatives and antiproteases was smaller and more variable in size, being between 180–230 kDa (Fig. 2BGo). In contrast, {alpha}-HDL in 150–200 mM NaCl fractions was the same size regardless of whether preservatives and antiproteases were present. Thus, although the size of {alpha}-HDL was unaffected by the presence of preservatives and antiproteases, the particle size of pre-ß-HDL was larger in their presence. The small pre-ß-HDL was termed the lyso-pre-ß-HDL.



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  		Fig. 2. Native- and SDS-PAGE of VHDL in DEAE column fractions. VHDL were isolated and chromatographed on DEAE columns in either the presence (Panels A and C) or absence (Panels B and D) of preservatives and antiproteases. Eluted fractions were electrophoresed on 2–14% native gels (Panels A and C), or 8–16% SDS-PAGE (Panels B and D), which were then stained with Coomassie blue R-250. Lane 1 in panels A and B: electrophoresis calibration kit for MW determination of high molecular weight proteins (Pharmacia). Lane 1 in panels C and D: prestained SDS-PAGE standard (low range) marker (Bio-Rad). Panels A and C: lane 2–10, fractions 1, 3, 4, 5, 6, 8, 10, 12, and 14 in Fig. 1-AGo, respectively. Panels B and D: lane 2–10, fractions 1, 2, 3, 6, 8, 10, 12, 15, and 18 in Fig. 1-BGo, respectively

 
SDS-PAGE analysis of the HDL subfractions showed that in pre-ß-HDL isolated with preservatives and antiproteases, the molecular size apo-A-I was approximately 28 kDa, which is the same size as apo-A-I present in {alpha}-HDL (Fig. 2CGo). However, analysis of apo-A-1 from pre-ß-HDL isolated without preservatives and antiproteases showed that 2 major protein bands were present, with molecular masses of approximately 23 kDa and 5 kDa (Fig. 2DGo). It appears these 2 smaller apo-A-I bands are the result of degradation of the 28 kDa protein.

Mass spectrometric analysis of apoA-I in HDL.  In addition to SDS-PAGE analysis, the masses of apoA-I proteins were also estimated by MALDI-TOF mass spectrometry. Apolipoproteins were pre-treated with a matrix reagent as described in the Materials and Methods. The data in Fig. 3Go indicate that apo A-I in {alpha}-HDL had a molecular mass of approximately 28,122.6 Da, while the apo A-I fragment isolated in the absence of preservatives and antiproteases was approximately 22,378.9 Da.



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  		Fig. 3. Molecular mass determination of apoA-I by MALDI-TOF mass spectrometry. Lyso-pre-ß-HDL and {alpha}-HDL were delipidated and the apolipoproteins resuspended in 0.1% TFA. The protein samples were mixed with sinapinic acid and the molecular masses determined by MALDI-TOF MS. Panel A: apo A-I in {alpha}-HDL isolated in the presence of preservatives and antiproteases (mature ApoAI), Panel B: apo A-I fragment isolated in the absence of preservatives and antiproteases.

 
MALDI-TOF mass spectrometry of apo A-I.  To study whether purified apoA-I is proteolytically cleaved upon incubation with proteases, we purified apoA-I from apo HDL3 as described in the Methods section. DE MALDI-TOF mass spectrometric analysis of apo A-I showed 2 major peaks at molecular weights 28,116.0 and 14,057.0 Da (Fig. 4Go), which are consistent with protonated and doubly protonated apo A-I (apo A-I2+), respectively. It is likely that two minor peaks at 4,521.2 and 5,375.3 Da are contaminants.



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  		Fig. 4. Positive ion MALDI TOF mass spectrum of purified apo A-I. After desalting by zip tip, purified apo A-I (approximately 100 pmol) was mixed with saturated sinapic acid solution and the MW determined in a linear mode with N2 laser (337 nm; step: 2,300–2,700) by DE-MALDI TOF-MS. The range of the mass spectrum of purified apo A-1 was 3,000–30,000 Da.

 
Limited proteolysis by trypsin.  Purified apo A-I (8 µg) was incubated with various concentrations of trypsin (1.6, 0.16, and 0.04 ng), and 1 µl of the reaction mixture plus matrix reagent was injected onto the sample plate and analyzed using MALDI-TOF MS. Treatment with the lowest concentration of trypsin (E: S ratio of mass 1:200,000) resulted in a profile similar to that seen with untreated apo AI (compare Fig. 4Go and Fig. 5Go, panel-C profile). Increasing the concentration of trypsin resulted in a decrease in the 28,116.9 ± 13.8 Da (mean ± SD) peak, and an increase in the peaks representing a long fragment (21,931.1 ± 2.3 Da) and shorter fragments (6,192.0 ± 2.9, 5,378.5 ± 2.2, 4,180.2 ± 2.7, and 3,183.8 ± 0.9 Da) (Fig. 5Go). At the highest trypsin concentration (E:S ratio 1:5000), fragments at 15,412.5 and 6,545.6 Da were also detected (Fig. 5AGo). It appeared that the 4,524.2 Da fragment was a contaminating peptide since it did not change with increasing trypsin concentration. The 21,931.1 ± 12.0 and 15,412.5 Da fragments were apo A-I (1–188) and apo A-I (1–131) peptides containing the apo A-I N-terminus, as determined by N-terminal sequencing (Table 1Go). The short fragments of 6,192.0 ± 2.9, 5,378.5, ± 2.2, 4,180.2 ± 2.6, and 3,183.8 ± 0.9 Da corresponded to apo A-I (189–243), (196–243), (207–243), and (216–243) respectively. The 6,545.6 Da fragment may be apo A-I (132–188). In mass spectrometry of trypsin, alone, no peak was detected between 3,000 and 30,000 Da (Fig. 5DGo).



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  		Fig. 5. Products from limited proteolysis of apo A-I by trypsin. Apo A-I (8 mg) was incubated with various amounts of trypsin (0.04, 0.4, and 1.6 ng) giving substrate-to-enzyme ratios of 5,000:1 (panel a), 50,000:1 (panel b), 200,000:1 (panel c), and only enzyme (panel d). Molecular weights of the proteolytic fragments were determined using DE-MALDI TOF-MS. The range of the mass spectrum of digested peptides was 3,000–30,000 Da. The signal spectrum was expanded in the high molecular weight range (10,000–30,000 Da) when analyzing fragments from the 5,000:1 incubation.

 

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  		Table 1. Molecular mass values of apolipoproteins and peptides digested by trypsin

 
Limited proteolysis by chymotrypsin and S. aureus V8 protease.  Purified apo A-I was incubated with various concentrations of chymotrypsin or S. aureus V8 protease, and the molecular weights of the apo A-I fragments were determined by MALDI-TOF MS as described above. Treatment of apo A-I by chymotrypsin produced fragments of 5,717.6 and 22,401.5 Da (Fig. 6AGo). These data suggest apo A-I was cleaved at only one position, since the fragments correspond in size to amino acids 1–192 and 193–243 (Table 2Go). Peaks at 4,527.3 and 5,380.8 Da appear to be contaminants, as they did not significantly increase in size with increase in protease concentration. S. aureus V8 protease treatment produced fragments of 2,337.1 ± 1.4 and 25,786.1 Da (Fig. 6BGo). These data suggest apo A-I was cleaved at only one position, since the fragments correspond in size to amino acids 1–223 and 224–243 (Table 2Go). Again, peaks at 4,527.4 and 5,381.8 Da appear to be contaminants, as they did not significantly increase in size at increasing protease concentrations.



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  		Fig. 6. Products from limited proteolysis of apo A-I by chymotrypsin and S. aureus V8 protease. Purified apo A-1 (8mg) was incubated with various amounts of chymotrypsin (0.8 and 0.16 ng) giving substrate-to-enzyme ratios of 10,000:1 (panel A-a) and 50,000:1 (panel A-b), and only enzyme (panel A-c). S. aureus V8 protease (0.016 and 0.16 ng) was added to give substrate-to-enzyme ratios of 50,000:1 (panel B-a) and 500,000:1 (panel B-b), and only enzyme (panel B-c). The molecular weights of the proteolytic fragments were determined using DE-MALDI TOF-MS. The range of the mass spectrum of digested peptide was 3,000–30,000 Da.

 

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  		Table 2. Molecular mass values of apolipoproteins andpeptides digested by chymotrypsin or S.aitretts V8 protease

 
Cleavage points of the limited proteolysis.  Fig. 7Go shows the carboxy terminal sequence of apo A-I from amino acids 166–243. The potential cleavage points of the proteases, as well as the actual cleavage points observed in the present study, are illustrated, and it is clear that only limited proteolysis occurred under the conditions used. Cleavage was restricted to the polypeptide units defined by helix 8, helix 9, and the ß-region structure (closed characters in Fig. 7Go).



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  		Fig. 7. Protease cleavage sites in apo A-I (166–243). The amino acid sequence of the apo A-I polypeptide is divided into separate units representing helix structure 7 (amino acids 166–186), helix structure 8 (aa 187–208), helix structure 9 (aa 209–219), the ß-region (aa 220–227), and helix structure 10 (aa 228–243). Open circles, triangles, and squares show sites potentially sensitive to trypsin, chymotrypsin, and S. aureus V8 protease cleavage, respectively. Closed circles, triangles, and squares show sites actually cleaved in the current study by trypsin, chymotrypsin, and S. aureus V8 protease, respectively.

 
Binding study.  The association of pre-ß- and {alpha}-HDL with mononuclear cells was measured by flow cytometry. One µg of FITC-HDL3 was incubated with mononuclear cells (approximately 1 x 105 in 1ml of PBS) for 1 hr at 37°C, and binding was competed by a 20-fold excess of unlabeled pre-ß- or {alpha}-HDL, which had been isolated with or without preservatives and antiproteases. Specific activities of HDL-binding proteins in monocyte cells were measured by FACScan as described in the Methods. The competition binding data are represented graphically in Fig. 8Go, which shows that significant (about 60%) inhibition of binding was observed following addition of unlabeled pre-ß-HDL prepared with preservatives and antiproteases. A similar level of competition was seen after addition of unlabeled {alpha}-HDL, whether or not it was prepared with preservatives and antiproteases. By contrast, unlabeled pre-ß-HDL prepared without preservatives and antiproteases competed only minimally.



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  		Fig. 8. Competitive binding of HDL subfractions to monocytes. In competitive binding studies, human mononuclear cells (approximately 1 x 10 5/ml) were incubated for 1 hr at 37°C with FITC-labeled HDL3 and various unlabeled HDL subfractions, and FITC-HDL3 binding was measured using FACSort. The data are the means of duplicate points and are representative of two similar experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we present evidence that human blood monocytes possess a specific binding site for pre-ß-HDL, which is the same site as that which binds {alpha}-HDL. Pre-ß-HDL prepared in the absence of preservatives and antiproteases is degraded to a smaller particle that contains hydrolysed apo-A-I of approximately 23kDa and is termed lyso-pre-ß-HDL. The limited proteolysis of human plasma apo A-I by trypsin, chymotrypsin, and S. aureus V8 protease appeared to occur only at the C-terminal end of apoA-I, within amino acids 188–223. The binding activity of lyso-pre-ß-HDL appeared to be significantly less than that of pre-ß-HDL. Limited proteolysis of apoA-I may regulate HDL structure and function, since the C-terminal domain of apo A-I is crucial for self-association and lipid binding.

We previously identified specific HDLß binding sites on human blood monocytes [23]. The present study indicates that pre-ß-HDL was bound to human blood monocytes at the same site as {alpha}-HDL. Pre-ß-HDL is a discoid, lipid-poor, apo A-I-rich particle. Previous studies have indicated that HDL particles bind to cells via the apo A-I and A-II proteins, rather than via the lipid [23,24,31]. Fidge et al [23] suggested that HDL-binding proteins from rat liver plasma membrane mediate HDL uptake into liver cells. HDL-binding sites on human blood monocytes show similar binding parameters as HDL-binding proteins from rat liver plasma membrane. The present study showed that degradation of pre-ß-HDL significantly decreased its binding to monocytes, probably due to the protease cleavage of apoA-I, which is likely to be the predominant ligand recognized by HDL-binding proteins on the surface of monocytes. Our data suggest that the carboxyl-terminal region of apoA-I associates with HDL-binding sites on the surface of monocytes. Morrison et al [32,33] and Allan et al [34] reported that the carboxyl-terminal region of apoA-I, in particular the amino acid sequences 205–220 and 230–243, contains a binding domain that mediates the specific interaction of HDL3 with liver plasma membranes. The results of these studies indicated that the carboxy terminal region of apo A-I was particularly sensitive to proteolysis. This region of apo A-I is involved in self-association and lipid binding [35,36], suggesting that mild proteo-lytic conditions may affect these properties of apo A-I, which in turn may affect HDL structure and functions. The present data suggest that pre-ß-HDL acts as a ligand of binding to the HDL-binding proteins, but not lyso-pre-ß-HDL.

Lyso-pre-ß-HDL contained apo-AI fragments of approximately 23kDa and 5kD, as identified by SDS-PAGE. It appears that these fragments were due to proteolysis of mature 28 kDa apoA-I during preparation, since such fragments were not evident in the presence of preservatives and antiproteases. It appears that the antiproteases in plasma were removed during ion-exchange chromatography or blocked by preservatives and antiproteases. Kunitake et al [37] have reported that pre-ß-migrating HDL contains 2 polypeptides with Mr of approximately 26 and 14 kDa, which were both recognized by anti-apoA-I polyclonal antibody. Interestingly, {alpha}-HDL was not degraded during chromatography regardless of the presence of preservatives and antiproteases. The data suggest that in pre-ß-HDL, an apoA-I aa-sequence sensitive to proteases is exposed, whereas in {alpha}-HDL, it is not susceptible to protease attack.

MALDI-TOF mass spectroscopy analysis of apo A-I in pre-ß-HDL showed that the mass of the major fragment was approximately 22,378.4 Da. This corresponds to an N-terminal apoA-I fragment encompassing amino acids 1–192. The limited proteolysis of apo A-I by either chymotrypsin or S. aureus V8 protease resulted in only one cleavage, Tyr (192)-His (193) and Glu (223)-Ser (224) respectively, and the short fragments were detected with almost equal signal strength as those produced by trypsin. Ji et al [38] identified these proteolytic fragments of apo-AI in solution and also in reconstituted HDL prepared with apoA-I and dipalmitoyl phospatidylcholine; they were produced by proteolytic reagents, chymotrypsin, trypsin, elastase, and subtilisn. The molecular mass of the N-terminal proteolytic fragment was 22,384 Da, as determined by electrospray ionization mass spectrometry [38]. Using MALDI-TOF mass spectroscopy, Safi et al [36] reported that the molecular weight of a major enteropeptidase-cleavage fragment of apoA-I was 21,912 Da, which corresponds to an N-terminal apoA-I fragment encompassing amino acids 1–188. These studies suggest that the region around amino acid 190 in apoA-I is sensitive to protease cleavage.

In limited proteolysis by trypsin, chymotrypsin, and S. aureus V8 protease, the specific cleavage sites were variously located in each polypeptide unit, helices 8 and 9, and ß-region structure units. Apo A-I consists of 11- and 22-amino acid repeating segments, typically spaced with helix-breaking proline residues [39]. These helix structure units exist mainly between amino acids 44–243. The amino acids 188–223 of apoA-I polypeptide encompass helix 8 (amino acids 187–208), 9 (aa 209–219), and ß-region (aa 220–227) [21]. Although there are some sites to receive specifically an attack of protease in the amino acids 188–223, a few points in apo A-I polypeptide were cleaved by the limited proteolysis. The sites of cleavage by trypsin were –Lys-X- (X: any kind of amino acid) or –Arg-X- (Lys and Arg); the cleavage site by chymotrypsin was - Aaa-X- (Aaa: Tyr, Phe, Trp, and Leu); cleavage sites by S. aureus V8 were –Glu-X- or –Asp-X (Glu and Asp). It was reported that helices 8–10 are largely unstructured in solution and become more helical when bound to lipid [40–43]. In the hypothetical structure-based lipid binding model for apo A-I, helices 9 and 10 are associated with the lipid layer. It may be that cleavage in the region of amino acids 188–223 alters the ability of apo A-I to bind lipid. The sensitive site for protease in apo A-I may be exposed out of the molecular chain in the 3-dimensional structure, because these units are flexible structures in the lipid-binding step of apo A-I. Cleavage at the carboxy terminus of apo A-I may have significant physiological consequences.

The short fragments of apo A-I were produced following limited proteolysis by trypsin, chymotrypsin, or S. aureus V8 protease, and the cleavage sites were located in the C-terminal end within amino acids 188–223. Limited proteolysis of apoA-I may decrease self-association or lipid binding, and may have significant effects on key functions of HDL in processes such as reverse cholesterol transport.

We conclude that, like {alpha}-HDL, pre-ß-HDL is a member of the HDL family that can bind to monocytes. Degradation of pre-ß-HDL occurs during purification in the absence of preservatives and antiproteases. Although it is unclear what happens in vivo, the degradation of pre-ß-HDL apoA-I may regulate pre-ß-HDL catabolism.


   References
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 Abstract
 Introduction
 Materials and Methods
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 Discussion
 References
 

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