From the
Department of Medicine, University of
Washington, Seattle, Washington 98195,
Department of Chemistry, Zhejiang University,
Hangzhou 310027, China, and ¶Department of
Pediatrics and ||Department of Cell Biology and
Physiology, Washington University, St. Louis, Missouri 63110
Received for publication, May 6, 2003
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ABSTRACT |
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INTRODUCTION |
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The MMP prodomain contains a highly conserved thiol residue within the consensus sequence PRCXXPD (5). In the inactive state, this conserved thiol binds to the zinc atom of the catalytic domain preventing the latent enzyme from becoming inappropriately active. A pro-MMP can be activated in vitro by proteolytic cleavage of its prodomain, which releases the thiol-zinc interaction and consequently frees the catalytic site to interact with substrate. However, activation of pro-MMPs can be mediated by processes that disrupt the thiol-zinc interaction, and it is likely that proteolysis is not the sole mechanism controlling MMP activity (10).
Reactive oxygen and nitrogen species can regulate MMP activity in
vitro
(1114),
suggesting that the generation of such species by inflammatory cells
(15,
16) controls MMP activation
and inactivation in vivo. Myeloperoxidase, a heme protein secreted by
neutrophils, monocytes, and some populations of macrophages, is one potential
source of reactive oxygen and nitrogen species
(17,
18). This enzyme uses hydrogen
peroxide (H2O2) to generate hypochlorous acid (HOCl)
(19).
![]() | (Eq. 1) |
Pro-MMP-7, pro-collagenase-2, and pro-gelatinase B exposed to increasing concentrations of HOCl initially increase their proteolytic activities (11, 20, 21). We recently demonstrated that HOCl converts the thiol residue of the cysteine switch domain of pro-MMP-7 to the corresponding sulfinic acid (21). Thiol oxidation is associated with autolytic cleavage of pro-MMP-7, strongly suggesting that oxygenation activates the latent enzyme. We have proposed that this pathway plays a critical role in the rupture of atherosclerotic lesions (18, 21, 27). These observations suggest that HOCl provides an oxidative mechanism for activating latent MMPs in vascular disease (28).
The oxidative footprints of myeloperoxidase have been found in human disorders ranging from atherosclerosis to lung disease, indicating that HOCl contributes to tissue damage during inflammation (29). However, hypercholesterolemic mice deficient in myeloperoxidase develop more atherosclerosis than do wild-type mice (30), and MMPs can be inactivated by reactive intermediates (12, 14, 21, 31), suggesting that myeloperoxidase may also restrain pathological tissue injury. In the current studies, we have proposed potential mechanisms for enzyme inactivation and demonstrated that HOCl, but not H2O2, oxidizes methionine and tryptophan residues of active MMP-7. When a specific tryptophan is converted to a novel oxidation product, the active enzyme loses catalytic activity. These observations reveal a molecular mechanism for inactivating MMPs.
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EXPERIMENTAL PROCEDURES |
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Methods
Reaction ConditionsReactions were carried out for 30 min at
37 °C in buffer A (150 mM NaCl, 10 mM HEPES, pH 7.4,
5 mM CaCl2) or buffer B (phosphate-buffered saline, pH
7.4) supplemented with 3.0 µM MMP-7. Reactions were initiated by
adding oxidant and terminated by adding a 10-fold molar excess (relative to
oxidant) of L-methionine. Concentrations of HOCl and
H2O2 were determined spectrophotometrically (
292 = 350 M1
cm1 and
240 = 39.4
M1 cm1)
(35,
36).
Proteolytic DigestionMMP-7 (100 pmol) was incubated overnight at 37 °C with sequencing grade modified trypsin (Promega, Madison, WI) at a ratio of 25:1 (w/w) MMP-7:trypsin in buffer C (10% CH3CN, 50 mM NH4HCO3, pH 8.0). Digestion was halted by freezing the mixture or acidifying (pH 23) it with formic acid or trifluoroacetic acid.
Liquid Chromatography (LC) Electrospray Ionization Mass Spectrometry (MS)LC-MS analyses were performed in the positive ion mode with a Finnigan MAT LCQ ion trap instrument (San Jose, CA) coupled to a Waters 2690 HPLC system (Milford, MA) as described (21). Tryptic peptides were separated at a flow rate of 0.2 ml/min on a reverse-phase column (Vydac C18 MS column; 2.1 x 25 mm) using solvent A (0.2% formic acid in water) and solvent B (0.2% formic acid in 80% CH3CN, 20% water). Peptides were eluted using the following linear gradient: 0 to 15% B over 15 min; 15 to 30% B over 25 min; then 30 to 65% B over 20 min. The electrospray needle was held at 4500 volts. Nitrogen, the sheath gas, was set at 80 units. The collision gas was helium. The temperature of the heated capillary was 220 °C.
Assay of MMP-7 Proteolytic ActivityMMP-7 activity was
assayed using Invitrogen precast casein zymogram gels or in solution
(21,
37) with the fluorescent Mca
peptide
(7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-Leu--(2,4-dinitrophenylamino)Ala-Ala-Arg-NH2)
as the substrate. MMP-7 (1530 ng) was added to individual wells of a
96-well microtiter plate containing 500 µl of buffer C (0.2 M
NaCl, 50 mM Tris-HCl, pH 7.5, 10 mM CaCl2,
0.02% NaN3, 0.05% w/v Brij35) and 2 µM Mca peptide
and incubated for 20 min at 37 °C. Fluorescence (
ex =
328 nm,
em = 392 nm) was monitored using a microplate
reader (SPECTRAmax GEMINI XS, Molecular Devices, Sunnyvale, CA).
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RESULTS |
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HOCl Oxidizes Specific Residues in MMP-7To investigate the molecular basis for the oxidative inactivation of MMP-7, we first digested unmodified enzyme with trypsin and identified the resulting peptides through LC-MS and MS/MS analysis (Fig. 2). The 11 peptides detected by LC-MS accounted for 92% of the protein sequence. Importantly, they included all of the amino acid residues of MMP-7 known to be highly susceptible to oxidation by HOCl or H2O2 (tryptophan (W) and methionine (M); (Fig. 2, peptides 1, 8, 10, and 11). Because of poor retention on the HPLC column, we were unable to identify small peptides and single amino acid residues.
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To determine whether HOCl modifies MMP-7 at specific sites, we exposed
MMP-7 to a concentration of oxidant (50:1, mol/mol, oxidant/protein) that
resulted in 80% inhibition of the enzyme and then analyzed a tryptic
digest of the enzyme by LC-MS. When the oxidant was
H2O2, the total ion chromatogram of the tryptic digest
was indistinguishable from that of the tryptic digest obtained from the native
protein (compare Fig. 2, A and
B). In striking contrast, peptides 8 and 10 were
selectively absent from the tryptic digest of MMP-7 exposed to HOCl
(Fig. 2C, 8*
and 10*). Loss of the two peptides was associated with the appearance
of one major peak of material (termed peptide A) and four peaks of material
that were poorly seen in the total ion chromatogram. These observations
suggest that HOCl modified specific amino acids of MMP-7 converting two of the
original peptides of the enzyme to five or more new peptides.
HOCl Selectively Oxygenates the Methionine Residue in Peptide 8 To identify the site at which HOCl modifies peptide 8, we exposed MMP-7 to H2O2, HOCl, or H2O2 supplemented with myeloperoxidase for 30 min at 37 °C in buffer A. We then digested the protein with trypsin and analyzed the digest by LC-MS. The reconstructed ion chromatogram of unmodified MMP-7 revealed a peak of material with the anticipated mass to charge ratio (m/z) of singly protonated peptide 8 (Fig. 3A, m/z 819.4, [M + H]+, retention time 27.5 min). MS/MS analysis confirmed the sequence of the peptide, ALNMWGK, which contained methionine and tryptophan residues (underlined letters) potentially susceptible to oxidation. The abundance of this peptide barely changed when MMP-7 was exposed to H2O2 alone. In contrast, when MMP-7 was exposed to HOCl alone or to H2O2 supplemented with myeloperoxidase, peptide 8 was absent from the tryptic digest, and a new peak of material was seen (compare Fig. 3, A and B). The retention time of this singly charged material resembled that of peptide A, and its m/z was 835.4 (Fig. 3C), which is consistent with the addition of 16 atomic mass units to peptide 8 ([M + 16 + 2H]+). MS/MS analysis of the modified peptide indicated that the methionine residue of peptide 8 had gained 16 atomic mass units (Fig. 3D). These observations demonstrate that HOCl generated by the myeloperoxidase-H2O2-Cl system, but not H2O2 alone, converts the alkylated thiol group of the methionine residue in peptide 8 of MMP-7 to the sulfoxide. LC-MS and MS/MS analysis indicated that the tryptophan residue adjacent to this methionine residue remained intact.
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HOCl Converts Peptide 10 to Four ProductsPeptide 10 (VVWGTADIMIGFAR) also contains tryptophan and methionine residues that might be vulnerable to HOCl. We therefore used LC-MS and MS/MS to determine the relative abundance of peptide 10 and its potential oxidation products in tryptic digests of untreated MMP-7 and MMP-7 exposed to oxidant. MS/MS analysis confirmed the sequence of doubly protonated peptide 10 in the digest of unmodified MMP-7 (Fig. 4A). The relative abundance of this peptide (Fig. 2, A and B) changed little when MMP-7 was exposed to H2O2 alone. In contrast, when MMP-7 was exposed to HOCl or to H2O2 supplemented with myeloperoxidase, peptide 10 disappeared and several small peaks of material appeared in the total ion chromatogram of the tryptic digest (Fig. 2C). LC-MS analysis with reconstructed ion chromatograms showed that the tryptic digest contained four new peaks of material that appeared to derive from peptide 10 (data not shown): two major products of m/z 766.6 ([M 4 + 2H]+2) and m/z 774.6 ([M + 12 + 2H]+2); and two minor products of 776.6 ([M + 16 + 2H]+2) and 784.6 ([M + 32 + 2H]+2).
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HOCl Oxygenates the Tryptophan Residue in Peptide 10 We used MS/MS to determine which amino acid residues had been modified in the two minor oxidation products of peptide 10 (Fig. 4, B and C). This approach indicated that when peptide 10 gained 16 atomic mass units ([M + 16 + 2H]+2) the tryptophan residue was oxygenated. When peptide 10 gained 32 atomic mass units, both the methionine residue and the tryptophan residue became oxygenated ([M + 32 + 2H]+2). These observations suggest that the tryptophan residue in peptide 10 is more readily oxygenated than the methionine residue. These observations indicate that tryptophan and methionine residues in different regions of MMP-7 are differentially susceptible to oxidation by HOCl, perhaps because of differences in flanking sequences or the secondary or tertiary structure in the two regions of the enzyme.
HOCl Converts the Tryptophan Residue of Peptide 10 to a Novel Oxidation ProductWe next characterized the two major products that we had detected in the tryptic digest of MMP-7 exposed to HOCl. MS/MS analysis of the peptide of m/z 766.6 ([M 4 + 2H]+2) (Fig. 4D) confirmed that this material was derived from peptide 10; it also revealed two important features: (i) loss of the y12 (Trp) and y11 ions (Gly) found in native peptide 10 and (ii) major ions at y10 (m/z 1094.4), and y12 4 (m/z 1333.5). These observations suggest that peptide 10 is modified by a reaction that removes 4 atomic mass units from WG. Detection of an ion of m/z 240.1 ([WG 4 + 2H]+) by MS/MS (Fig. 4D) supported this interpretation.
MS/MS analysis of the material of m/z 774.6 ([M + 12 + 2H]+2) demonstrated a ladder of y ions (Fig. 4E). This result was similar to that observed in the material of m/z 766.6 ([M 4 + 2H]+2) except that the methionine residue had gained 16 atomic mass units ([M + 16 4 + 2H]+2). These observations indicate that the tryptophan residue in peptide 10 was more susceptible than that peptide's methionine residue was to oxidation by HOCl. They also strongly suggest that HOCl oxidized WG by removing 4 atomic mass units.
HOCl Generated by Myeloperoxidase Converts WG to WG4 in MMP-7To determine whether HOCl generated by myeloperoxidase also oxidizes WG of peptide 10, we exposed MMP-7 to H2O2 alone, HOCl alone, or H2O2 supplemented with myeloperoxidase (H2O2 + MPO) for 30 min at 37 °C in buffer A. Reconstructed ion chromatograms were used to monitor the ions derived from peptide 10 in the tryptic digest of the enzyme. The doubly charged peptide 10 (m/z 768.6, [M + 2H]+2) was present in the digests of the native enzyme and of MMP-7 exposed to H2O2 alone (Fig. 5A). When the enzyme was exposed to HOCl or H2O2 supplemented with myeloperoxidase, peptide 10 disappeared almost completely. Its disappearance was associated with the appearance of product peptides (Fig. 5, B and C) that had either lost 4 atomic mass units (m/z 766.6, [M 4 + 2H]+2) or had both lost 4 atomic mass units and gained 16 atomic mass units (m/z 774.6, [M 4 + 16 + 2H]+2). These results indicate that HOCl generated by the myeloperoxidase-H2O2-Cl system, but not H2O2 alone, can convert WG in peptide 10 of MMP-7 to a product that has lost 4 atomic mass units.
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WG Is a Site-specific Target for Modification by HOClBoth peptide 8 and peptide 10 contained methionine and tryptophan-glycine residues, but only the tryptophan residue in peptide 10 was oxidized when MMP-7 was exposed to HOCl. The selective oxidation of tryptophan in this peptide might reflect a site-specific reaction. To investigate this possibility, we synthesized a synthetic peptide, VVWGTA, which duplicates the region of peptide 10 that contains the vulnerable tryptophan residue. However, the peptide presumably lacks the secondary and tertiary structure of that part of the enzyme. After exposing the synthetic peptide to HOCl for 30 min at 37 °C in buffer A, the reaction mixture was analyzed by LC-MS and MS/MS. The total ion chromatogram of the reaction mixture revealed almost complete loss of the precursor peptide (Fig. 6A, peak M) and evidence of the appearance of one major and four minor product peptides (peaks 15). The molecular mass (m/z 632.2, [M + H]+) and sequence of the unmodified peptide were confirmed by LC-MS and MS/MS analysis (Fig. 6, B and C). LC-MS analysis of the major peak of new material (peak 5) demonstrated a peptide of m/z 628.2 (Fig. 6D), indicating that the precursor peptide had lost 4 atomic mass units ([M 4 + H]+). MS/MS analysis of peak 5 revealed a series of ions consistent with loss of 4 atomic mass units from the precursor peptide, including VWG 4, b5 4, b4 4, and y4 4 (Fig. 6E). Importantly, MS/MS analysis of peak 5 showed a prominent ion of m/z 240.1, which is consistent with loss of 4 atomic mass units from WG (compare Fig. 6, C and E). LC-MS analysis of the minor products of the reaction (peaks 14) revealed two ions of m/z 648 and two ions of m/z 664, suggesting that the precursor peptide (m/z 632.2) had been converted to two different products by addition of 1 and 2 oxygen atoms, respectively. Collectively, these observations strongly suggest that WG in peptide 10 is susceptible to site-specific modification by HOCl and that the major product lacks 4 atomic mass units (WG4). They also suggest that this peptide can be oxygenated into products that contain either one or two oxygen atoms.
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When MMP-7 Is Exposed to HOCl, Oxidation of Peptide 10 Is Associated
with Loss of Enzymatic ActivityTo determine whether oxidation of
specific amino acid residues in peptide 8 or peptide 10 might inactivate
MMP-7, we examined the relationship between the disappearance of each peptide
and loss of proteolytic activity. Because enzymes exposed to high
concentrations of oxidant might undergo stochastic oxidative modifications, we
focused our studies on MMP-7 that was 90% inactivated. Under these
conditions, there should be a linear relationship between loss of proteolytic
activity and peptide modification if oxidation played a causal role in
regulating enzyme activity.
Peptide 8 disappeared completely from the tryptic digest of MMP-7 exposed to HOCl, but there was little correlation between its loss and the proteolytic activity of the enzyme (Fig. 7). In contrast, loss of peptide 10 was strongly associated with loss of proteolytic activity. These observations indicate that peptide 8 is more sensitive to oxidation than peptide 10 but that its modification does not diminish enzyme activity. In contrast, oxidative modification of peptide 10 is associated with loss of MMP-7 activity and therefore is likely involved in enzyme inactivation.
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DISCUSSION |
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Importantly, our observations indicate that, in peptide 10 of MMP-7, HOCl generates a major product of unknown structure involving tryptophan and an adjacent glycine residue. MS/MS analysis demonstrated that tryptophan-glycine was oxidized to an intermediate that had lost 4 atomic mass units (WG4). Moreover, LC-MS and MS/MS studies of the synthetic peptide VVWGTA, which mimics this region of MMP-7, confirmed that HOCl specifically targeted the tryptophan-glycine site to generate WG4. Thus, our observations indicate that HOCl converts tryptophan-glycine to a novel oxidation product, WG4.
When we exposed MMP-7 to HOCl, oxidation of peptide 10, but not of peptide 8, was associated strongly with loss of proteolytic activity. LC-MS and MS/MS analyses of peptide 10 revealed that WG4 was a major product, but we also detected oxygenated methionine and tryptophan residues. These observations suggest that formation of WG4 (or other oxidation products derived from tryptophan and methionine) inactivates MMP-7. Consistent with this mechanism, H2O2 failed to inactivate MMP-7, and LC-MS analysis demonstrated that neither peptide 8 nor peptide 10 was modified by this oxidant. The methionine in peptide 8 was more sensitive to oxidation by HOCl than was WG in peptide 10, suggesting that the methionine residue could act as an endogenous antioxidant in the proteinase (38). It is unclear how oxidation of peptide 10 inactivates MMP-7. The crystal structure of the enzyme reveals that peptide 10 lies in a region that is far from the active site of the enzyme (39). In future studies, it will be important to determine the structure of WG4 and to explore how this novel modified dipeptide and other oxidized amino acids in peptide 10 affect MMP-7 activity.
HOCl produced by myeloperoxidase might not be the only oxidant capable of inactivating MMPs (17, 18, 21). A related phagocyte enzyme, eosinophil peroxidase, converts bromide ion to hypobromous acid, which modifies tryptophan residues in lipoproteins more efficiently than HOCl (40, 41). Other possible candidates include brominating oxidants produced by myeloperoxidase (42, 43) and peroxynitrite, nitrogen dioxide radical, and hypothiocyanate (16, 17, 21, 44).
We showed previously that HOCl oxygenates the thiol residue of the cysteine switch domain activating latent MMP-7 (21). Tandem MS analysis revealed that the cysteine of pro-MMP-7 is the primary target for oxidation at low concentrations of HOCl. However, when pro-MMP-7 was exposed to higher concentrations of HOCl, we also detected WG4 (data not shown). These observations suggest that the thiol of the cysteine switch domain is the preferred site of oxidation in pro-MMP-7. However, both pro-MMP-7 and MMP-7 become targets for the formation of WG4 at higher concentrations of HOCl. Thus, oxidants generated by phagocytes in vivo might contribute to both activation and inactivation of MMPs (Scheme 1). Which action predominates might depend on the local concentration of HOCl at a particular time. Because phagocytes store MMPs and myeloperoxidase in secretory compartments, degranulation of these components could create high local concentrations of both enzymes near the cell surface (4, 17, 18). Because the phagocyte NADPH oxidase is associated with the plasma membrane, production of H2O2 is also localized to the cell surface (45). Moreover, because pro-MMP-7, pro-collagenase-2, and pro-gelatinase B are activated by low concentrations of HOCl but disabled by higher concentrations of oxidant (11, 20, 21, 31), traditional enzyme kinetics cannot fully explain the regulation of MMP activity by reactive oxygen species in tissue or extracellular fluids (4). Instead, regulation is likely to be highly localized both temporally and spatially by rapid local changes in proteinase and oxidant concentrations. By analogy, it is noteworthy that neutrophils produce evanescent "quantum bursts" of pericellular proteolytic activity when activated in vitro (4). Therefore, regulation of proteolytic activity might depend critically on local concentrations of oxidants and MMPs near the phagocyte surface.
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Our suggestion that HOCl inactivates MMP-7 by oxidizing specific amino acid residues provides an alternative pathway to the tissue inhibitor of metalloproteinases mechanism traditionally associated with MMP inactivation (5). This new mechanism implicates phagocytes, the cellular hallmark of inflammation, in protecting matrix from degradation by MMPs. Moreover, HOCl converts latent pro-MMP-7 to MMP-7, suggesting that post-translational oxidative modifications control both activation and deactivation of enzyme activity (11, 12, 21, 26, 31). Generation of oxidants by myeloperoxidase in vivo could confine MMP activation to bursts of pericellular proteolysis that are highly regulated in space and time. Because dysregulation of matrix degradation has been implicated in many pathological disorders, our findings could have broad implications for understanding and potentially preventing the degradation of healthy tissue.
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FOOTNOTES |
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** To whom correspondence should be addressed: Metabolism, Endocrinology and Nutrition, Box 356426, University of Washington, Seattle, WA 98195. E-mail: heinecke{at}u.washington.edu.
1 The abbreviations used are: MMP, matrix metalloproteinase; MMP-7,
matrilysin; LC, liquid chromatography; HPLC, high performance liquid
chromatography; M, peptide; MS, mass spectrometry; MS/MS, tandem mass
spectroscopy; m/z, mass to charge ratio; WG4,
tryptophan-glycine oxidation product lacking 4 atomic mass units; Mca peptide,
7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-Leu--(2,4-dinitrophenylamino)Ala-Ala-Arg-NH2.
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REFERENCES |
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