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Hydroxynonenal inactivates cathepsin B by forming Michael adducts with active site residues

¹ Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA
² Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA

Reprint requests to: John W. Crabb, Ph.D., Cole Eye Institute (i31), Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA; e-mail: crabbj{at}ccf.org; fax: (216) 445-3670 or Henry F. Hoff, Ph.D., Department of Cell Biology (NC-10), Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA; e-mail: hoffh{at}ccf.org; fax: (216) 444-9404.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.4400102.

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods References

 
Oxidation of plasma low-density lipoprotein (oxLDL) generates the lipid peroxidation product 4-hydroxy-2 nonenal (HNE) and also reduces proteolytic degradation of oxLDL and other proteins internalized by mouse peritoneal macrophages in culture. This leads to accumulation of undegraded material in lysosomes and formation of ceroid, a component of foam cells in atherosclerotic lesions. To explore the possibility that HNE contributes directly to the inactivation of proteases, structure-function studies of the lysosomal protease cathepsin B have been pursued. We found that treatment of mouse macrophages with HNE reduces degradation of internalized maleyl bovine serine albumin and cathepsin B activity. Purified bovine cathepsin B treated briefly with 15 µM HNE lost 76% of its protease activity and also developed immunoreactivity with antibodies to HNE adducts in Western blot analysis. After stabilization of the potential Michael adducts by sodium borohydride reduction, modified amino acids were localized within the bovine cathepsin B protein structure by mass spectrometric analysis of tryptic peptides. Michael adducts were identified by tandem mass spectrometry at cathepsin B active site residues Cys 29 (mature A chain) and His 150 (mature B chain). Thus, covalent interaction between HNE and critical active site residues inactivates cathepsin B. These results support the hypothesis that the accumulation of undegraded macromolecules in lysosomes after oxidative damage are caused in part by direct protease inactivation by adduct formation with lipid peroxidation products such as HNE.

Keywords: Oxidative damage; hydroxynonenal; cathepsin B; Michael adducts; mass spectrometry

Abbreviations: BHT, butylated hydroxytoluene • BSA, bovine serum albumin • CLN, N-CBZ-l-lysine p-nitrophenyl ester • DTT, dithiothreitol • ESMS, electrospray mass spectrometry • HNE, 4-hydroxy-2-nonenal • LC ESMS, liquid chromatography electrospray mass spectrometry • LDL, low-density lipoprotein • ox-LDL, oxidized LDL • PBS, phosphate-buffered saline • TCA, trichloroacetic acid

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods References

 
Oxidation of plasma low-density lipoprotein (oxLDL) generates lipid oxidation products that have been proposed to play a role in the pathogenesis of atherosclerosis by modulating the expression of inflammatory factors and by creating ligands for macrophage scavenger receptors (Witztum and Steinberg 1991; Podrez et al. 2000). When oxLDL is incubated with macrophages in culture, intracellular degradation appears to be deficient after receptor-mediated endocytosis (Lougheed et al. 1991; Jessup et al. 1992; Hoppe et al. 1994). Long-term incubation of macrophages with oxLDL leads to the accumulation of ceroid, an insoluble protein-lipid product that is a hallmark of foam cell lesions and advanced atherosclerotic plaques (Ball et al. 1986; Yin 1996). In vivo and in vitro inhibition of thiol proteases also can result in the formation of ceroid and lipofuscin-like substances (Ivy et al. 1990). The molecular mechanisms responsible for the poor degradation of oxidized macromolecules in macrophages are not fully understood. One mechanism proposes that cross-linking of substrate proteins by oxidative damage hinders accessibility to proteases (Lougheed et al. 1991; Jessup et al. 1992). Another mechanism proposes that treatment of cells with oxLDL leads to a reduced fusion of endosomes or phagosomes with lysosomes (Hoppe et al. 2001). A third mechanism proposes that specific lysosomal proteases are inactivated by lipid oxidation products generated during the oxidation of LDL (Hoppe et al. 1994; O'Neil et al. 1997). This report shows that the lysosomal protease cathepsin B can be inactivated by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE).

Oxidation of LDL leads to multiple changes in the composition of both the lipid and protein constituents of LDL (Esterbauer et al. 1991; Witztum and Steinberg 1991). Free radical–induced formation of hydroperoxides in the unsaturated fatty acids of cholesterol and phospholipid esters (Esterbauer et al. 1991) results in alkoxyl radical formation and subsequent scission to form a variety of reactive lipid oxidation products including HNE. HNE is created by cleavage of oxidized arachidonic and linoleic esters (Fig. 1) and forms Schiff base linkages with primary amino groups and Michael adducts with Lys, His, and Cys residues and also forms fluorescent cross-links (Esterbauer et al. 1991; Xu et al. 2000). HNE modification induces LDL aggregation (Hoff et al. 1992) and impairs proteasome function via cross-linking and other undetermined mechanisms (Friguet and Szweda 1997; Okada et al. 1999). Previously, we reported that a reversal of Michael additions of HNE to lysine may occur at the low pH that exists in lysosomes after receptor-mediated endocytosis of oxLDL (O'Neil et al. 1997). We hypothesized that HNE, still tethered to apo B by a Schiff base linkage also may form Michael additions with particularly vulnerable proteins such as the lysosomal thiol protease cathepsin B (O'Neil et al. 1997). With or without cross-links, Michael additions from HNE may help explain why oxLDL-treated macrophages show reduced activity of cathepsin B but not reduced activity of cathepsin D, an aspartate protease (Hoppe et al. 1994).

View larger version (24K): [in this window] [in a new window]   Fig. 1. HNE Michael adducts. HNE (4-hydroxy-2-nonenal) is generated by oxidative cleavage of linoleic (LA) and arachidonic acid (AA) containing phospholipids and can condense with lysine, cysteine, and histidine residues to form Michael adducts. Reduction with NaBH4 stabilizes Michael adducts. The protein mass addition per adduct is indicated. (PC) Phosphatidyl choline; (Me) methyl.

	Results

TOP Abstract Introduction Results Discussion Materials and methods References

 
HNE reduces the intracellular degradation of maleyl bovine serine albumin and cathepsin B activity in mouse macrophages
We pretreated mouse peritoneal macrophages with HNE and compared the ability of treated and untreated cells to degrade internalized ¹²⁵I-maleyl bovine serine albumin (BSA). Under the 3 h reaction conditions used, HNE and oxLDL were able to inhibit degradation of internalized ¹²⁵I-maleyl BSA by 35% and 65%, respectively, relative to the untreated control cells (Fig. 2a). In our earlier study of macrophages pretreated with oxLDL for 24 h, reduction in degradation of internalized macromolecules was accompanied by 65%–80% reduction in the activity of cathepsin B (Hoppe et al. 1994). Here, we measured cathepsin B activity after HNE treatment of macrophages and found 40% reduction in activity relative to untreated cells (Fig. 2b). In this set of experiments, cells pretreated with oxLDL showed approximately a 56% reduction in cathepsin B activity (Fig. 2b). To rule out the possibility that cytotoxicity of HNE or oxLDL reduced degradation of maleyl BSA by modulating protein synthesis, cells were treated with ³⁵S-methionine during incubation with HNE or oxLDL. No significant reduction in protein synthesis was detected (Fig. 2c). Cells were also pretreated with ¹⁴C-adenine to monitor for cytotoxicity by measuring release into the media from cell membrane rupture. No significant increase in the release of labeled adenine was found in macrophages treated with up to 50 µM HNE or 100 µg/mL oxLDL (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 2. HNE reduces degradation of maleyl BSA and lowers cathepsin B activity in mouse macrophages. Mouse peritoneal macrophages were incubated for 3 h with media containing one of the following: 150 µg/mL oxLDL, 25 µM HNE in 0.5% ethanol, or 0.5% ethanol (control). After this preincubation, the following analyses were performed. (a) Cells were washed three times with fresh media and then incubated with media containing 20 µg/mL of 125I-maleyl BSA. After 2.5 h, the amounts of TCA-soluble label in the conditioned media were determined as described in Materials and Methods. (b) Cathepsin B activity in cell lysates was determined as described in Materials and Methods. (c) Cells were treated with 35S-methionine during the incubation with HNE or oxLDL to evaluate potential cytotoxicity and reduced protein synthesis. No significant cytotoxicity was detected under the conditions used. The data in each panel represent the average and standard deviation of triplicate determinations. (HNE) 4-hydroxy-2 nonenal; oxLDL: oxidation of plasma low-density lipoproteins.

View larger version (31K): [in this window] [in a new window]   Fig. 3. SDS-PAGE and Western analysis of HNE-modified bovine cathepsin B. Activated bovine cathepsin B was incubated for 1 h with 15 µM HNE and then subjected to SDS-PAGE (1 µg/lane) and Western analysis using a rabbit polyclonal antibody to HNE adducts (Uchida et al. 1993). (a) Coomassie blue-stained gel and (b) Western blot of the HNE-treated cathepsin B preparation used for structural analysis by mass spectrometry. (c) Western blot of HNE-treated and untreated ([C] control) bovine cathepsin B.

View larger version (23K): [in this window] [in a new window]   Fig. 4. LC ESMS of tryptic peptides from HNE-modified cathepsin B. Purified bovine cathepsin B was modified with HNE and digested with trypsin, and LC ESMS was performed with a PE Sciex API 3000 triple quadrupole mass spectrometer as described in Materials and Methods. The elution positions of identified tryptic peptides are indicated in the total ion current and summarized in Table 1. HNE-modified peptides were detected by a mass addition of 158.1. (AT) and (BT) Tryptic peptides from the A and B chains of cathepsin B, respectively.

View larger version (49K): [in this window] [in a new window]   Fig. 5. MS/MS spectra of the HNE-modified active site cysteine peptide. Nanoelectrospray MS/MS spectra are shown for chymotryptic peptides generated from HNE-modified tryptic peptide AT4 (DQGSCGSC*WAFGAVEAISDR): (A) singly charged precursor m/z 1157.7 from DQGSCGSC*W; (B) doubly charged precursor m/z 568.6 from AFGAVEAISDR. A Michael adduct was found only on the active site cysteine ([asterisk] A-chain Cys 29).

View larger version (49K): [in this window] [in a new window]   Fig. 6. MS/MS spectra of the HNE-modified active site histidine peptide. Nanoelectrospray MS/MS spectra are shown for V8 protease peptides generated from HNE-modified tryptic peptide BT10 (SGVYQHVSGEIMGGH*AIR): (A) doubly charged precursor m/z 532.2 from IMGGH*AIR; (B) doubly charged precursor and m/z 507.4 from SGVYQHVSGE. A Michael adduct was found only on the active site histidine ([asterisk] B-chain His 150).

	Discussion

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View larger version (50K): [in this window] [in a new window]   Fig. 7. The three-dimensional structure of cathepsin B. The structure of bovine spleen procathepsin B (Yamamoto et al. 2000) is shown with the active site cysteine and histidine residues that are modified by HNE shown in green and yellow, respectively. Also highlighted in red is the occluding loop (residues 105–125) associated with cathepsin B carboxypeptidase activity (Illy et al. 1997). The structure was drawn using Insight II software (Biosym Technologies), and the PDB coordinates 1QDQ for bovine cathepsin B were obtained from the Structure database, NCBI.

In summary, HNE or oxLDL treatment of macrophages inhibits intracellular degradation of macromolecues and reduces cathepsin B activity. HNE treatment of purified bovine cathepsin B results in selective modification of active site residues Cys 29 (A chain) and His 150 (B chain), with the generation of Michael adducts and significantly reduced enzyme activity. Notably, others have established the presence of HNE Michael adducts in oxLDL (Esterbauer et al. 1991; Bolgar et al. 1996). Overall, these results support the hypothesis that specific lysosomal proteases are inactivated by HNE generated during oxidation of LDL.

	Materials and methods

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Materials
Bovine spleen cathepsin B (EC 3.4.22.1), N-CBZ-L-lysine p-nitrophenyl ester, fatty acid–free bovine serum albumin, butylated hydroxytoluene, trichloroacetic acid, and dithiothreitol were purchased from Sigma Chemical Co. Carrier-free Na¹²⁵I and ¹⁴C-adenine were from ICN, Roswell Park Memorial Institute media (RPMI-1640) was from Whittaker Bioproducts Inc., and fetal calf serum was from Gibco Laboratories. Tissue culture plates were obtained from Costar; C57BL/6 mice (16–20 weeks of age) from Charles River Laboratory; and polyvinylidene difluoride (PVDF) membrane was from Amersham-Pharmacia Biotech. HNE and rabbit polyclonal antiserum to HNE were kind gifts from Drs. Lawrence M. Sayre and Luke I. Szweda, respectively, Case Western Reserve University, Cleveland, OH.

Degradation of maleyl bovine serum albumin by macrophages in culture
LDL was isolated from fresh plasma obtained from the Cleveland Clinic Blood Bank and oxidized with CuSO₄ as described elsewhere (Hoppe et al. 1994). BSA was subjected to maleylation (Haberland and Fogelman 1985), labeled with Na¹²⁵I, and used as a ligand for receptor-mediated endocytosis. Mouse peritoneal macrophages were harvested 2–3 days after thioglycollate stimulation of female C57BL/6 mice by peritoneal lavage with ice-cold phosphate-buffered saline (PBS). Primary cultures were prepared at a density of 10⁶ cells/16-mm-diameter well in RPMI-1640 containing 10% fetal calf serum and used 48 h after plating (Hoppe at al. 1994). Cells were treated with HNE or oxLDL for 3 h, washed two times with PBS to remove oxLDL or HNE, then incubated with ¹²⁵I-maleyl BSA and media assayed for trichloroacetic acid–soluble, noniodine degradation products essentially as described elsewhere (Hoppe et al. 1994). Cells were washed three times with PBS, dissolved in 0.1 N NaOH, and assayed for protein content using the bicinchoninic acid assay and BSA as a standard (Pierce Chemicals). Cytotoxicity was determined by measuring the incorporation of ³⁵S-methionine into proteins, and by determining the release of ¹⁴C-adenine from cells (Coffey et al. 1995). In the latter technique, macrophages were first incubated with media containing ¹⁴C-adenine (0.2 µ Ci/mL) for 12 h, washed with media, and then incubated with HNE or oxLDL for 3 h at 37°C. The amount of radiolabel present in the conditioned media as a result of release from cells then was measured.

Preparation of macrophage cell extracts
Mouse peritoneal macrophages were assayed for cathepsin B activity as previously described (Hoppe et al. 1994). Briefly, after incubation with HNE or oxLDL, macrophage monolayers were washed twice with PBS, scraped with a rubber policeman, pelleted by centrifugation at 2000g for 10 min and resuspended in 1 mL of buffer. The cells then were ruptured by passing 10 times through a 30-gauge needle, followed by 10 cycles of freezing and thawing, and finally by two 15-sec bursts of sonication (Hoppe et al. 1994). The homogenates were spun at 5000g to remove particulate matter, and supernatant fractions were collected and defined as cell extracts.

Enzyme assay for cathepsin B activity
The thiol protease activity of cell extracts and purified cathepsin B preparations was assayed by the N-CBZ-L-lysine p-nitrophenyl ester (CLN) procedure (Hoppe et al. 1994). Briefly, bovine cathepsin B was activated by incubating the proenzyme for 15 min at 37°C in 0.2 M Na acetate at pH 4.5 containing 2 mM DTT-EDTA. The activated enzyme was treated with 15 µM HNE for periods of up to 60 min. An aliquot of the mixture of activated enzyme with or without HNE was adjusted to 1 mL with the same buffer, and the enzyme reaction was initiated by addition of CLN to a final concentration of 0.5 mM. Cathepsin B activity at 37°C was expressed as change in absorbance at 326 nm wavelength per min because of hydrolysis of CLN.

HNE modification of bovine cathepsin B, sodium borohydride reduction, alkylation, and proteolytic digestions
Purified and activated bovine cathepsin B (8 nmole based on vendor quantification) in 250 µL of 100 mM sodium acetate at pH 4.5 and 1 mM DTT-EDTA was added to 1.6 µmole HNE dissolved in 25 µL of ethanol. After an 8-min incubation at 37°C, 8 M urea in 200 mM ammonium bicarbonate at pH 8.0 (1 mL) was added to stop autoproteolysis, and the preparation was frozen. To stabilize Michael adducts and reduce disulfide bonds, we added 1 M sodium borohydride in 0.1 M NaOH to the HNE-modified protein to a final concentration of 50 mM, and the EDTA concentration was adjusted to 1 mM. After 30 min incubation at room temperature in a fume hood, hydrogen gas bubbling was stopped and 2.8-fold molar excess of iodoacetamide was added relative to the cysteine content. After a 15-min incubation under argon, the alkylation reaction was stopped by the addition of 1 µmole DTT. The modified cathepsin B was dialyzed exhaustively into 15 mM N-ethylmorpholine acetate at pH 8.0, 40% acetonitrile using 1000-MW cutoff Spectrapor dialysis tubing. After dialysis, the modified cathepsin B was quantified by amino acid analysis and recovery determined to be 109 µg (4 nmole). HNE-modified and S-carboxyamidomethylated cathepsin B (2.1 nmole) was digested with 1.8 µg of trypsin (Promega) in 15 mM N-ethylmorpholine acetate at pH 8.0, 40% acetonitrile at 37°C for 16 h. Subfragmentation of tryptic peptides with V8 protease or chymotrypsin (0.1 µg protease) was in 10 mM ammonium bicarbonate containing 20% acetonitrile at 25°C for 12 h (Crabb et al. 1986).

Amino acid analysis, electrophoresis, and Western blot analysis
Phenylthiocarbamyl amino acid analysis was performed using an Applied Biosystems model 420H/130/920 automated analysis system (Crabb et al. 1998a). SDS-PAGE was performed on 12% acrylamide gels using a Mini-Protein II slab gel system (Bio-Rad) and visualized by Coomassie blue staining. For Western analyses, the gels were blotted to PVDF membranes and probed with rabbit polyclonal antibodies to Michael adducts (Uchida et al. 1993). Immunoreactivity was detected by chemiluminescence (Amersham-Pharmacia Biotech).

Mass spectrometry
ESMS and liquid chromatography ESMS (LC ESMS) were performed with a Perkin-Elmer Sciex API 3000 triple quadrupole mass spectrometer equipped with an ionspray source (Crabb et al. 1998b). Nitrogen was used as the nebulization gas (at 40 p.s.i.) and curtain gas and was supplied from a nitrogen dewar. For LC ESMS, the cathepsin B tryptic digest was diluted twofold with 0.1% trifluoroacetic acid and 1 nmole (27 µg) chromatographed on a 5 µm Vydac C18 column (1.0 x 150 mm) using an Applied Biosystems model 140D HPLC system, aqueous acetonitrile/propanol/trifluoroacetic acid solvents and a linear gradient of 2%– 80% acetonitrile over 60 min at a flow rate of 40 µL/min. The column eluant was split with 25% directed to the mass spectrometer. The remainder of the eluant was collected manually in fractions by monitoring the total ion current. A scan range of m/z 350–2200 in the positive ion mode was used with 0.2-a.m.u. steps, 0.5-msec dwell time per step, 70-V orifice potential, and 4900-V ionspray.

Nanoelectrospray MS/MS was performed with a nanoelectrospray interface (MDS Protana) attached to the Perkin-Elmer Sciex API 3000 triple quadrupole mass spectrometer. Select chromatography fractions from LC ESMS (5 µL) were placed in gold-coated glass capillaries (4-µm outlet; New Objectives, Inc.), and the samples were infused into the mass spectrometer with 1000–1500 V applied to the capillary and orifice potential set at 40 V. Precursor ions were selected in the first quadrupole (Q1) and collision-induced dissociation effected with nitrogen gas in the second quadrupole (Q2). Product ion spectra were obtained by scanning the third quadrupole (Q3) using a step size of 0.2 Dalton and 0.5 msec dwell time per step in the positive ion mode. The Q1 resolution was lowered to allow transmission of M⁺¹ precursor ions into Q2; Q3 was kept at unit mass resolution.

	Acknowledgments

 
This work was supported in part by NIH grants EY06603 and HL53315, a Research Center Grant from the Foundation Fighting Blindness, Hunt Valley, MD, and funds from The Cleveland Clinic Foundation. We thank Xiaorong Gu and Drs. Sanjoy Bhattacharya and Barnali Neel Chaudhuri for assistance with illustrations and Drs. G. Hoppe, M. Kinter, and L. Sayre for reviewing the manuscript before publication.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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