Large-Scale Expression, Refolding, and Purification of the Catalytic Domain of Human Macrophage Metalloelastase (MMP-12) in Escherichia coli

Parkar, Ashfaq A; Stow, Mark; Smith, Keith; Panicker, A.K.; Guilloteau, J.P.; Jupp, R A; Crowe, Sarah J.

doi:10.1006/prep.2000.1280

Cited by 31 publications

(26 citation statements)

References 32 publications

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“…The lack of structural perturbation by the mutation beyond the side chain itself is evident from comparison of crystal structures (9,30) and from the minimal and localized chemical shift perturbations of NMR spectra (36). Recombinant E219A-inactivated catalytic domain was prepared as described previously (36,58,59 NMR Spectroscopy-NMR spectra and titrations were acquired at 26°C on a Varian Inova 600-MHz spectrometer equipped with a high sensitivity 5-mm cryogenic HCN triple resonance probe with an actively shielded z-gradient coil. NMRPipe (60) was used to process NMR spectra.…”

Section: Methodsmentioning

confidence: 99%

MMP-12 Catalytic Domain Recognizes Triple Helical Peptide Models of Collagen V with Exosites and High Activity

Bhaskaran

Palmier

Lauer‐Fields

et al. 2008

Journal of Biological Chemistry

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Collagens are principal constituents of connective tissues. They are hydrolyzed during development, wound repair, and remodeling of the extracellular matrix. Dysregulated collagenolysis is active in inflammatory diseases such as atherosclerosis, cancer, rheumatoid arthritis, periodontitis, and liver and kidney pathologies. Matrix metalloproteinases (MMPs) 3 play key roles in these processes (1-3). Mutagenesis has established the importance of the V-B loop (joining ␤-strand V and helix B) in collagenolysis by MMP-1 (4). Residues at the C-terminal end of this loop in MMP-8 were also implicated in collagenolysis (5) and triple helical peptidase activity (6).Binding of an inactivated MMP-1 to an intact collagen fibril likely unwinds the triple helix at the cleavage site to provide access for an active MMP-1 catalytic domain to hydrolyze individual chains (7). The direction of collagen triple helices at the active site is unknown, but the orientation can be hypothesized to be the same as in linear peptide substrates that run antiparallel to ␤-strand IV (8, 9). Single chain peptides from the ␣1 chain of type I/III collagens were soaked into crystals of MMP-12 and MMP-8 catalytic domains, and snapshots of bound hydrolysis products were obtained (8).However, neither crystallography nor NMR data representative of interactions with collagen have been reported because of technical limitations. More generally, structural studies of complexes of enzymes with their substrates have been rare presumably because of catalytic turnover and affinities lower than those for transition state analogues. Several biologically active, self-assembling triple helical peptide (THP) mimics of collagens have been developed. These THPs have facilitated numerous biochemical and cell biological studies otherwise infeasible with insoluble, intact collagen fibrils. THPs are successful in reproducing the cleavage sites of MMPs in collagens I, II, III, V, and XI and in reproducing rate dependences on intact triple helical structure (10, 11). Fluorogenic labeling of THPs has facilitated quantitative comparisons of the triple helical peptidase activities of a variety of MMPs in solution and in cell culture assay (6,11,12). The affinities of MMP catalytic domains for triple helices range from 1 to 80 M (6, 11-13). Collagenolysis involves several MMP functions that include initial nonspecific binding and orientation of collagen fibrils (14, 15), manipulation of collagen fibrils with the C-terminal hemopexin domain (16) and catalytic domain (7), unwinding of triple helices within fibrils, and ensuing hydrolysis of single chains from the melted triple helix (7). THPs are not suitable for modeling the initial binding, orientation, and manipulation of full-length collagen assembled into large, insoluble fibrils (6, 11). They are too small to participate in these higher order events (7). Nonetheless THPs have proven themselves to be outstanding substrates for detailed characterization of the triple helical peptidase activities of MMPs and other metalloprotein...

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Section: Methodsmentioning

confidence: 99%

MMP-12 Catalytic Domain Recognizes Triple Helical Peptide Models of Collagen V with Exosites and High Activity

Bhaskaran

Palmier

Lauer‐Fields

et al. 2008

Journal of Biological Chemistry

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“…Expression and Purification of Human Recombinant Metalloelastase (MMP-12) Catalytic Domain-The purification procedure was modified from that of Parkar et al (27). DNA encoding the human rMMP-12 catalytic domain was prepared by PCR cloning from human placenta cDNA using the forward primer 5Ј-GATCTCCATATGTTCAGG-GAAAT-3Ј and the reverse primer 5Ј-GGATCCTTATCCATACAGG-GACTGAAT-3Ј.…”

Section: Methodsmentioning

confidence: 99%

Cathepsin V, a Novel and Potent Elastolytic Activity Expressed in Activated Macrophages

Yasuda

Greenbaum

et al. 2004

Journal of Biological Chemistry

176

138

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Atherosclerosis is characterized by a thickening and loss of elasticity of the arterial wall. Loss of elasticity has been attributed to the degradation of the arterial elastin matrix. Cathepsins K and S are papain-like cysteine proteases with known elastolytic activities, and both enzymes have been identified in macrophages present in plaque areas of diseased blood vessels. Here we demonstrate that macrophages express a third elastolytic cysteine protease, cathepsin V, which exhibits the most potent elastase activity yet described among human proteases and that cathepsin V is present in atherosclerotic plaque specimens. Approximately 60% of the total elastolytic activity of macrophages can be attributed to cysteine proteases with cathepsins V, K, and S contributing equally. From this 60%, two-thirds occur extracellularly and one-third intracellularly with the latter credited to cathepsin V. Ubiquitously expressed glycosaminoglycans (GAGs) such as chondroitin sulfate specifically inhibit the elastolytic activities of cathepsins V and K via the formation of specific cathepsin-GAG complexes. In contrast, cathepsin S, which does not form complexes with chondroitin sulfate is not inhibited; thus suggesting a specific regulation of elastolytic activities of cathepsins by GAGs. Because the GAG content is reduced in atherosclerotic plaques, an increase of cathepsins V and K activities may accelerate the destruction of the elastin matrix in diseased arteries.Atherosclerosis is characterized by arterial intimal enlargement and subsequent lipid deposition leading to the formation of blood stream obstructing plaques. The infiltration of monocyte-derived macrophages (MDMs) 1 and smooth muscle cells (SMCs) into the intima of the inflamed artery contributes to the formation of atherosclerotic lesions. Atherosclerotic lesions resident MDMs and SMCs produce a large number of extracellular matrix-degrading enzymes (1), such as cysteine proteases (2-5) and matrix metalloproteinases (MMPs) (6 -8).Elastin and collagen are the two major extracellular matrix components that provide elasticity and tensile strength to the arterial wall. The destruction of elastin and collagen causes a weakening and rupture of blood vessels (9, 10). MMPs, serine, and cysteine proteases have been identified as major elastolytic proteases in arteries (11, 12). Among cysteine proteases, cathepsins S and K have been considered as the most potent elastolytic activities with cathepsin K exhibiting a slightly higher activity than cathepsin S (13, 14). However, cathepsin K-deficient human macrophages derived from patients with pycnodysostosis were shown to retain their high cysteine proteasedependent elastolytic activities (15). This finding implied that additional cathepsins may contribute to the overall elastolytic activity in human macrophages.We and others (16 -18) have identified and partially characterized a novel human cysteine protease closely related to cathepsin L that was named cathepsin V (also known as cathepsin L2). Cathepsin V shares 80% protein s...

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“…All hrMMPs were expressed in E. coli. Recombinant human MMP-12 CD and recombinant human MMP-9 CD without fibronectin type II inserts (expressed in E. coli as described [51,52] ) were a kind gift from AstraZeneca R&D (Lund & Moelndal, Sweden). TIMP-1 from human neutrophil granulocytes was from Calbiochem (La Jolla, CA, USA).…”

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confidence: 99%

Design of Peptide Hydroxamate‐Based Photoreactive Activity‐Based Probes of Zinc‐Dependent Metalloproteases

et al. 2010

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Keywords: Metalloenzymes / Activity-based profiling / Photoaffinity labeling / Diazirine / Alkylation / Diastereoselectivity Metalloproteases (ADAMs, MMPs) are multidomain proteins that play key roles in extracellular matrix remodelling and degradation, in cell-cell and cell-matrix interactions and in the proteolytic liberation of membrane-anchored proforms of cytokines and growth factors, the so-called ectodomain shedding. In this work we describe the development of photoactivatable activity-based probes with which active

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Large-Scale Expression, Refolding, and Purification of the Catalytic Domain of Human Macrophage Metalloelastase (MMP-12) in Escherichia coli

Cited by 31 publications

References 32 publications

MMP-12 Catalytic Domain Recognizes Triple Helical Peptide Models of Collagen V with Exosites and High Activity

MMP-12 Catalytic Domain Recognizes Triple Helical Peptide Models of Collagen V with Exosites and High Activity

Cathepsin V, a Novel and Potent Elastolytic Activity Expressed in Activated Macrophages

Design of Peptide Hydroxamate‐Based Photoreactive Activity‐Based Probes of Zinc‐Dependent Metalloproteases

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