We have now cloned and expressed recombinant human ADAMTS-1, -4 and -5 and report here that the catechin gallate esters found in green tea potently inhibit the aggrecan-degrading activit
Trang 1Selective inhibition of ADAMTS-1, -4 and -5 by catechin gallate esters
Mireille N Vankemmelbeke1, Gavin C Jones1, Cyprianne Fowles1, Mirna Z Ilic2, Christopher J Handley2, Anthony J Day3, C Graham Knight4, John S Mort5and David J Buttle1
1 Division of Genomic Medicine, University of Sheffield Medical School, Sheffield Children’s Hospital, Stephenson Wing, D-Floor, UK;
2 School of Human Biosciences, La Trobe University, Bundoora, Victoria, Australia 3083; 3 MRC Immunochemistry Unit,
Department of Biochemistry, University of Oxford, UK; 4 Department of Biochemistry, University of Cambridge, UK;
5 Joint Diseases Laboratory, Shriners Hospital for Children, Montreal, Quebec, Canada
Three mammalian ADAMTS enzymes, ADAMTS-1, -4
and -5, are known to cleave aggrecan at certain glutamyl
bonds and are considered to be largely responsible for
car-tilage aggrecan catabolism observed during the development
of arthritis We have previously reported that certain
cate-chins, polyphenolic compounds found in highest
concen-tration in green tea (Camellia sinensis), are capable of
inhibiting cartilage aggrecan breakdown in an in vitro model
of cartilage degradation We have now cloned and expressed
recombinant human ADAMTS-1, -4 and -5 and report
here that the catechin gallate esters found in green tea
potently inhibit the aggrecan-degrading activity of these
enzymes, with submicromolar IC50 values Moreover, the
concentration needed for total inhibition of these members
of the ADAMTS group is approximately two orders of magnitude lower than that which is needed to partially inhibit collagenase or ADAM-10 activity Catechin gallate esters therefore provide selective inhibition of certain mem-bers of the ADAMTS group of enzymes and could consti-tute an important nutritional aid in the prevention of arthritis as well as being part of an effective therapy in the treatment of joint disease and other pathologies involving the action of these enzymes
Keywords: ADAMTS; enzyme inhibition; catechin; gallate; aggrecanase
Green tea, made from the leaves of Camellia sinensis,
contains catechins, a group of polyphenolic compounds
with antioxidant properties that have been at the centre of
investigations into the potential medical benefits of
consu-ming green tea The most abundant catechin in green tea
is (–)-epigallocatechin gallate (EGCG) with others such
as (–)-epicatechin (EC), (–)-epigallocatechin (EGC) and
(–)-epicatechin gallate (ECG) also present
Anti-inflamma-tory and anti-mitotic properties have been attributed to
these compounds [1–3] and they have also been reported to
inhibit certain matrixins such as the gelatinases [4–6] The
beneficial effects on a range of clinical conditions including
cancer growth and metastasis [7–11], cardiovascular and liver diseases [12] may therefore be due to one or a combination of these properties
Aggrecan, a large aggregating proteoglycan, is together with type II collagen the major constituent of articular cartilage Degradation of cartilage aggrecan has mainly been attributed to the action of glutamyl endopeptidases, termed aggrecanases Aggrecan degradation products resulting from aggrecanase action have been found in
in vitrocultures of cartilage treated with proinflammatory cytokines as well as in synovial fluid of arthritis patients [13–16] To date three mammalian aggrecanases have been identified: a disintegrin and metalloproteinase with throm-bospondin motifs (ADAMTS)-1, -4 and -5 [17–19] The ADAMTS enzymes belong to a subgroup of metallopep-tidases in Family M12 of Clan MA in the Merops database [20] and are related to the ADAMs and matrixins [21] So far, at least 18 mammalian ADAMTS enzymes have been identified, most of which remain to be fully characterized [21,22]
It has been shown recently that inhibition of ADAMTS-4 and -5 can prevent aggrecan breakdown in osteoarthritic cartilage [23] An in vivo role for ADAMTS-1 in cartilage aggrecan turnover awaits confirmation following the finding that it cleaves aggrecan at glutamyl bonds in vitro [19,24] ADAMTS-1 and -4 have also been shown to cleave other members of the large aggregating proteoglycan family such
as versican and brevican [25–27]
Work in our laboratory has shown that catechin gallate esters are inhibitors of aggrecan and collagen degradation in
an in vitro model of cartilage breakdown [28] The aim of this study was to investigate if catechins and gallate esters directly inhibit the aggrecanases ADAMTS-1, -4 and -5
Correspondence to D J Buttle, Division of Genomic Medicine,
University of Sheffield Medical School, Sheffield Children’s Hospital,
Stephenson Wing, D-Floor, Sheffield S10 2TH, UK.
Fax: + 44 114 2755364, Tel.: + 44 114 2717556,
E-mail: d.j.buttle@sheffield.ac.uk
Abbreviations: Abu , L -a-aminobutyryl ((S)-2-amino-butanoyl);
ADAMTS, a disintegrin and metalloproteinase with thrombospondin
motifs; DCI, 3,4-dichloroisocoumarin; E-64, L
-trans-epoxysuccinyl-leucylamido-(4-guanidino)butane; Me 2 SO, dimethylsulfoxide; EC,
(–)-epicatechin; ECG, (–)-epicatechin gallate; EGC,
(–)-epigallocate-chin; EGCG, (–)-epigallocatechin gallate; Gn, guanidinium; HATU,
N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-hexamethylmethanaminium hexafluorophosphate N-oxide; HOAt,
7-hydroxy-1-azatriazole; KLH, keyhole limpet haemocyanin; Mca,
(7-methoxycoumarin-4-yl)acetyl; METH-1, metalloproteinase-1 with
thrombospondin motifs; PG, n-propyl gallate; rh, recombinant
human; sGAG, sulfated glycosaminoglycan; TIMP, tissue inhibitor
of metalloproteinases.
(Received 21 March 2003, accepted 7 April 2003)
Trang 2To this end we have expressed, purified and characterized
recombinant forms of these ADAMTSs
Experimental procedures
Construction of expression vectors
Human ADAMTS-1 (KIAA1346) and ADAMTS-4
(KIAA 0688) clones were kindly provided as inserts in the
pBluescript II SK+vector (SalI and NotI sites) by T Nagase
(Kazusa DNA Research Institute, Kisarazu, Chiba, Japan)
Two sets of primers were designed to subclone the respective
coding sequences including their signal peptides into the
pIB/V5-His/TOPO vector (InvitrogenTM Life
Techno-logies, Paisley, UK) For ADAMTS-4, primer pair
5¢-GCCATGTCCCAGACAGG-3¢ (sense) and 5¢-GGTT
ATTTCCTGCCCGC-3¢ (antisense) and for ADAMTS-1
primer pair 5¢-GACATGGGGAACGCGGAG-3¢ (sense)
and 5¢-CTT AACTGCATTCTGCCATTG-3¢ (antisense)
were used The inserts were sequenced in both directions
The recombinant baculovirus vector pVL 1392
(Pharm-ingen, San Diego, CA, USA) was a kind gift from R Maki
(Neurocrine Biosciences Inc., San Diego, CA, USA) It
contained the coding sequence for human ADAMTS-5
(GenBank accession number AF142099), which had been
modified to contain an N-terminal signal sequence from the
agouti-related protein followed by the FLAGTMsequence
replacing the first 60 nucleotides of the native coding
sequence External primers 5¢-GAAGATCTGACTACAA
GGACGACGATGAC-3¢ (sense) and 5¢-CCTCTAGAT
TACTAACATTTCTTCAACAAGCATTG-3¢ (antisense)
containing a BglII and XbaI restriction site, respectively,
were designed to generate a PCR product which contained
the FLAGTMsequence followed by the coding sequence for
ADAMTS-5 This was subcloned into the
pMT/BiP/V5-HisB expression vector (InvitrogenTM Life Technologies,
Paisley, UK) and its nucleotide sequence was examined by
sequencing in both directions At this stage, two
noncon-servative point mutations were found in the first
thrombo-spondin repeat G1851A and A1855G resulting in the
following amino acid substitutions: G577S and Q579R
These were corrected using overlap extension PCR The
corrected insert including the Drosophila BiP signal
sequence provided by the vector, was then subcloned into
the pIB/V5-His/TOPO vector using two primers, 5¢-CC
GATCTCAATATGAAGTTATGC-3¢ (sense) and 5¢-CCT
CTAGATTACTAACATTTCTTCAACAAGCATTG-3¢
(antisense) and the TOPO TA cloning methodology
according to the manufacturer’s recommendations The
correct coding sequence was confirmed by sequencing in
both directions
Cell culture
High FiveTMcells (InvitrogenTMLife Technologies, Paisley,
UK) were maintained and propagated in HyQSFX
serum-free insect cell culture medium (Perbio Science UK,
Ltd Cheshire, UK) containing 10 lgÆmL)1gentamycin at
27C The cells were transfected with the recombinant or
empty expression vector using the lipid-based CellFectin
reagent (InvitrogenTM Life Technologies, Paisley, UK)
Conditioned cell culture medium was harvested at days 2, 3,
4 and 5 post-transfection and assayed for aggrecanase activity Stably transfected cell lines were generated in the presence of 80 lgÆmL)1 Blasticidin (InvitrogenTM Life Technologies, Paisley, UK) After selection stably trans-fected cells were maintained in the presence of 10 lgÆmL)1 blasticidin Conditioned medium was stored at)40 C Aggrecanase activity assay
Aggrecan, purified from bovine nasal cartilage by extraction with 4M guanidinium (Gn) HCl and dissociative CsCl2 density gradient ultracentrifugation [29] was entrapped in polyacrylamide and used as a substrate to determine aggrecan-degrading activity as previously described [29–31] Aliquots of the aggrecan/polyacrylamide particles (4 mg) were incubated with the respective enzymes in a total volume of 500 lL (assay buffer: 0.1 M Tris/HCl, 0.1 M NaCl, 10 mMCaCl2, 0.1% w/v Chaps, pH 7.5) The tubes were incubated at 37C for up to 3 h At the end of the incubation the particles were subjected to brief centrifuga-tion and the sulfated glycosaminoglycan (sGAG) content
in the supernatant was measured using dimethylmethylene blue [32] One unit of enzyme activity was defined as that which released 5 lg sGAGs per h at 37C in the assay
Purification and characterization of recombinant ADAMTS enzymes
rhADAMTS-1, -4 and -5 were all purified using an identical protocol Conditioned High FiveTM cell culture medium was thawed and the proteinase inhibitors 3,4-dichloroisocoumarin (DCI) and L -trans-epoxysuccinyl-leucylamido-(4-guanidino)butane (E-64) were added to final concentrations of 50 lM and 10 lM, respectively After an initial buffer exchange on a preparative Sephadex G-25 column (Amersham Pharmacia Biotech, Bucking-hamshire UK), samples were assayed for aggrecanase activity (see above) and applied to a heparin-Sepharose Fast-Flow column (Amersham Pharmacia Biotech, Buck-inghamshire UK) equilibrated with buffer A (50 mMTris/ HCl, 0.15MNaCl, 0.1% w/v Chaps, pH 7.0) Activity was eluted using a step-wise gradient (0.15–1MNaCl) Active fractions were pooled, desalted and loaded onto a Mono Q
HR 5/5 (Amersham Pharmacia Biotech, Buckinghamshire UK) column, equilibrated with buffer B (50 mMTris/HCl, 0.1% w/v Chaps, pH 7.5) Active fractions were eluted between 0.2 M and 0.45 M NaCl on a salt gradient and were pooled The purity of the enzyme preparations was assessed by SDS/PAGE followed by silver staining of the gels and via Western blot analysis using ADAMTS-specific antibodies (see below)
We assayed conditioned medium from mock-transfected insect cells for aggrecanolytic activity using the assay described above Proteinase contamination of the rhADAMTS enzyme preparations was examined in two ways Firstly, the enzyme preparations were incubated with purified aggrecan monomer Briefly, 10 units of rhADAMTS-1, and 5 units of rhADAMTS-4 or -5 were incubated with 5 mg purified aggrecan monomer (see above) in enzyme assay buffer: 50 mM Tris/HCl, 0.1 M NaCl, 10 mM CaCl2, 0.1% w/v Chaps, pH 7.5 at 37C for 16 h Aggrecan fragments were detected by Western
Trang 3blotting using the monoclonal antibody 5/6/3-B-3 (ICN
Flow) which recognizes terminal unsaturated chondroitin
6-sulfate disaccharides The fragments were then isolated
and subjected to N-terminal sequence analysis as previously
described [31] An additional control consisted of analysing
the final enzyme preparations for potential contaminating
MMP activity using a quenched fluorescence substrate
which is cleaved by all MMPs, but which is not cleaved by
aggrecanases (see below)
Antibodies to ADAMTS enzymes
Antibodies were raised to peptides prepared by standard
solid-phase methods and purified by reverse-phase HPLC
The identity of the peptides was confirmed by MS Peptides
were coupled to KLH using N-succinimidyl bromoacetate
[33] or to ovalbumin The ADAMTS-1 antibody MV-8 was
raised in rats against KLH-coupled
DPLKKPKHFID-Abu-C (human ADAMTS-1 amino acids 932–942) The
ADAMTS-4 antibody was raised in rabbits using a mixture
of two peptides VMAHVDPEEPGGC and CGGYNHR
TDLFKSFPGP (human ADAMTS-4 amino acids 394–403
and 590–603, respectively) ovalbumin conjugates The
rabbit ADAMTS-5 antibody (3235) was raised against
ovalbumin-conjugated ILTSIDASKPGGC and CGGKN
GYQSDAKGVKTFV (human ADAMTS-5 amino acids
442–451 and 636–650) and affinity-purified
immuno-globulin was prepared using a SulfolinkTMcolumn (Pierce
Rockford, IL) substituted with the peptide CGGKN
GYQSDAKGVKTFV Animals were immunized by
fort-nighly injections of carrier-conjugated peptides emulsified in
Freund’s adjuvant Test bleed titres were determined by
ELISA Briefly, plates were coated with immunizing peptide
in carbonate buffer, pH 9.0, and blocked in 1% BSA, rinsed
and treated with the primary antibody After 1.5 h, the
plates were washed and incubated with alkaline
phospha-tase conjugated secondary antibody, washed three times,
and substrate was added (p-nitrophenyl phosphate tablets;
Sigma-Aldrich) Absorbance was measured at 405 nm on a
plate reader The specificity of the antisera was determined
via comparison with nonimmune control serum
Cross-reactivity of the antisera with the other ADAMTS enzymes
was also examined
Synthesis of the ADAM-10 substrate
Mca-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Dpa-Arg-OH
This was made on Fmoc-Arg(Pbf)-NovaSyn TGA resin
(0.1 mmol) using standard Fmoc protocols in a PerSeptive
Biosystems 9050 Plus PepSynthesiser Briefly, Fmoc-amino
acids (0.4 mmol) were activated with HATU (0.4 mmol) in
the presence of diisopropylethylamine (0.8 mmol) HOAt
(0.4 mmol) was added when coupling Gln Fmoc
depro-tection was with a mixture of 2% (v/v) piperidine and 2%
(v/v) 1,8-diazabicyclo[5,4,0]undec-7-ene in
dimethylforma-mide For the coupling of 7-methoxycoumarin-4-acetic acid
(0.4 mmol), the resin was gently shaken with HOAt
(0.4 mmol) and diisopropylcarbodiimide (0.5 mmol) in a
minimal volume of dichloromethane containing 10% (v/v)
N,N-dimethylpropyleneurea and the reaction was allowed
to proceed to completion overnight The peptide was
released by treatment with trifluoroacetic
acid/water/triiso-propylsilane (92.5 : 5 : 2.5, v/v) for 2 h at 21C, applied to
a column of Vydac 218TPB1520 and eluted with a gradient
of 5–50% acetonitrile in 0.1% trifluoroacetic acid Fractions containing homogeneous product were identified by ana-lytical HPLC, pooled and freeze-dried The identity of the purified peptide was confirmed by MALDI-TOF (expected mass 1542.6 Da, observed mass 1542.5 ± 0.7 Da)
Assays for matrixin and ADAM-10 activity rhADAM-10, expressed as a soluble enzyme, was provided
by Procter & Gamble, Cincinnati, OH, USA Purified human collagenase-1 (EC 3.4.24.7) and collagenase-3 were both from Biogenesis Ltd, Poole, UK The substrate used for the assay of the matrixins was Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 [34,35] Cleavage of the ADAM-10 quenched fluorescence substrate (see above) followed Michaelis–Menten kinetics with an approximate Km of
20 lM Determination of a more accurate Km value was not possible due to the insolubility of the substrate The methods for assaying the matrixins and ADAM-10 were essentially the same using a Perkin Elmer LS 50B lumin-escence spectrometer (excitation 328 nm, emission 393 nm) controlled by the Flusys software package [36] Peptide substrates were used at 5 lM in 100 mMTris/HCl, 0.1 M NaCl, 10 mMCaCl2, 0.2% v/v Triton X-100 pH 7.5, or in
50 mMTris/HCl, 100 lMZnCl2, 10% v/v MeOH, pH 7.5 for the matrixins and ADAM-10, respectively In all assays substrate hydrolysis never exceeded 10% of total
Determination of enzyme inhibition For rhADAMTS assays, EC, ECG, EGC, EGCG and
PG (all‡ 95% pure from Sigma-Aldrich Company Ltd, Poole, Dorset, UK) were dissolved in Me2SO to 2 mMto provide the stock solution and diluted to the appropriate final concentration which ranged from 2 to 2000 nM in enzyme assay buffer Enzymes were preincubated with the inhibitors or the appropriate concentration of Me2SO as control at 4C for 30 min prior to assaying their activity using the aggrecanase assay described above Percentage inhibition was calculated by comparing the levels of activity with those of the enzyme and Me2SO controls Log-linear plots of the dose–response curves in combina-tion with regression analysis allowed us to determine approximate IC50 values for the inhibitory catechins In the case of rhTIMP-3 (the full-length inhibitor was kindly provided by Immunex Corp., Seattle, WA, USA), the enzymes were preincubated with 100 nM of the inhibitor for 30 min at 4C before assaying
For the determination of inhibition of collagenases and ADAM-10, quenched fluorescence substrate assays were employed (above) The catechins and PG were used at concentrations ranging from 0.2 lM to 50 lM The hydroxamate inhibitor BB-94, a broad-spectrum MMP and ADAM inhibitor (provided by A Galloway, British Biotech Ltd), was used as a positive control at 10 lMfinal concentration The initial steady state of substrate cleavage (vo) was recorded, after which inhibitor or Me2SO control were added to the reaction mix in a minimal volume, and the new steady state (vi) was documented The percentage inhibition was calculated as (1) v/v)· 100
Trang 4Purification and characterization of rhADAMTS enzymes
Conditioned medium from mock-transfected insect cells
contained no detectable aggrecanolytic activity (results not
shown) rhADAMTS-1, -4 and -5 were partially purified by
affinity chromatography on heparin-Sepharose followed by
anion-exchange chromatography using a Mono Q column
at pH 7.5 The purification process was monitored using the
aggrecanase assay described in Experimental procedures As
shown in Table 1, each chromatography step was associated
with an increase in specific activity of the respective
enzymes, with final purification factors ranging from
190- to 370-fold An increase in total rhADAMTS-1 activity
following affinity purification may be indicative of
separ-ation from an inhibitor
Silver staining of SDS/PAGE gels of the partially purified enzyme preparations revealed that a substantial number of protein bands remained (Fig 1, lanes 1, 3 and 5) Western blots using the MV-8 antibody, directed against the C-terminus of the rhADAMTS-1, revealed an intense band of approximately 89 kDa (Fig 1, lane 2) This band probably corresponds to the active, mature enzyme (theoretical molecular mass 78.8 kDa) A band of similar size (p87) has previously been reported for mature ADAMTS-1 [37] C-Terminal processing of recombinantly expressed ADAMTS-1 has been described previously [37,38] The MV-8 antibody, directed against the C-terminus of the enzyme also detected a faint band of about 59 kDa (Fig 1, lane 2), which could represent the C-terminus of a truncated form of ADAMTS-1 Antibody MV-8 did not cross-react with ADAMTS-4 or -5 (data not shown)
Table 1 Purification of rhADAMTS-1, -4 and -5 from conditioned insect cell medium Conditioned medium was applied to a heparin-Sepharose Fast-Flow column after an initial buffer exchange on a preparative Sephadex G-25 column equilibrated with 50 m M Tris/HCl, 0.1 M NaCl, 0.1% w/v Chaps, pH 7.0 Proteins were eluted using a step-wise gradient up to 1 M NaCl, and fractions were assayed as described in the Experimental Procedures Section Active fractions were pooled, desalted and loaded onto a Mono Q HR 5/5 column, equilibrated with 50 m M Tris/HCl, 0.1% w/v Chaps, pH 7.5 Active fractions were eluted using a linear (0–1 M NaCl) gradient and pooled Total protein amounts were estimated via absorption at 280 nm, assuming A 280,cm of 1.0 ¼ 1 mg mL)1protein.
Step Protein (mg) Total Activity (U) Specific activity (UÆmg)1) Yield (%) Purification (-fold) rhADAMTS-1
rhADAMTS-4
rhADAMTS-5
Fig 1 SDS PAGE and Western blots of partially purified rhADAMTS-1, -4 and -5 Panels A, B and C represent preparations of rhADAMTS-1, -4 and -5, respectively Mono-Q peaks of aggrecanase activity were concentrated 10- to 20-fold using Microcon YM-30 devices (Millipore Cor-poration, Bedford, USA.) and about 2 lg total protein was loaded on to 7.5% polyacrylamide SDS-gels run under reducing conditions and subjected to silver staining (lanes 1, 3 and 5) For Western blots 2.5 lg total protein was used in each case The blot of rhADAMTS-1 with antibody MV-8 (1 : 500) is shown in lane 2, of rhADAMTS-4 with antibody 3170 (1 : 500) in lane 4 and of rhADAMTS-5 with antibody 3235 (1 : 500)
in lane 6 The arrows indicate possible forms of rhADAMTS-1, -4 and -5 corresponding to those identified by Western blotting.
Trang 5Antibody 3170 detected three bands of 81 kDa,
76 kDa and 55 kDa in our partially purified
rh-ADAMTS-4 (Fig 1, lane 4) The 81- and 76-kDa bands
are likely to represent different forms of mature ADAMTS-4
(theoretical molecular mass 68.3 kDa) The 75- and
55-kDa forms of rhADAMTS-4 (the 55-kDa form shows
higher aggrecanase activity) have been described as the
mature and secondarily processed forms of the enzyme upon
expression in a human chondrosarcoma cell line [39] More
recently Flannery et al described two autocatalytic
process-ing events for recombinantly expressed ADAMTS-4 [40] A
cleavage in the spacer region generated a 53-kDa form of the
enzyme, in agreement with our data We did not detect a
40-kDa form generated by cleavage in the cysteine-rich
region of the enzyme This could be due to the difference in
expression systems used Comparison of Western blots with
the corresponding silver-stained gels (Fig 1, lane 3, arrows)
suggests three potential ADAMTS-4 bands
Western blot analysis of partially purified rhADAMTS-5
with antibody 3235 revealed two main bands of 64
and 37 kDa The 64-kDa band is likely to represent a
C-terminally processed form of mature ADAMTS-5 as its
theoretical molecular mass (not accounting for
carbo-hydrate) is 73.7 kDa ADAMTS-5 purified from
carti-lage-conditioned medium has revealed multiple bands in
the range of 40–65 kDa [17] In addition, similarly
processed forms of the enzyme have been detected in
synovium-conditioned medium from arthritis patients [31]
and in a GnHCl extract from cartilage of an arthritic
patient [23]
Aggrecanolytic activity of rhADAMTS enzymes
Inhibitors of cysteine and serine proteinases were added to
conditioned culture medium to abolish activity of any such
contaminating proteinases We analysed the partially
puri-fied enzymes for matrixin activity by use of a quenched
fluorescence substrate known to be cleaved by these
enzymes [35] No such activity was detected in any of the three rhADAMTS preparations (data not shown) ADAMTS-1, -4 and -5 are unusual in that they have strict specificity for cleavage between glutamatic acid residues and uncharged aliphatic amino acids in the core proteins of large aggregating proteoglycans This specificity
is unique among mammalian proteinases The reported cleavage sites in aggrecan generated by aggrecanase activity are Glu373fi Ala in the interglobular domain, Glu1480fi Gly between the chondroitin sulfate 1 and 2 attachment regions and Glu1666fi Gly, Glu1771 fi Ala, and Glu1871fi Leuwithin the chondroitin sulfate 2 attachment region [13] We digested purified aggrecan monomer with the rhADAMTS enzyme preparations and isolated the fragments for N-terminal sequence analysis
as described in Experimental procedures The digestions resulted in all cases in at least five aggrecan core protein fragments, as determined by staining with colloidal Coo-massie blue (Fig 2, lanes 2, 4 and 6) and Western blot analysis with antibody 5/6/3-B-3 (lanes 3, 5 and 7) [41] The fragments generated by the three different rhADAMTS enzyme preparations were very similar N-Terminal sequence analysis of these fragments (Table 2 and Fig 3) showed that they resulted from typical aggrecanase cleav-ages in poorly glycosylated regions of the aggrecan core protein These cleavage sites have been reported for both ADAMTS-4 and -5 [17,31,42] We describe here for the first time the N-terminal sequences of the major aggrecan fragments generated by ADAMTS-1 This enzyme was initially reported to cleave aggrecan only at the C-terminus [19] Recently however, the use of cleavage site-specific antibodies has demonstrated that ADAMTS-1 is capable
of generating similar aggrecan fragments to those produced
by ADAMTS-4 and -5 [24] The finding in a different laboratory that ADAMTS-1 failed to cleave aggrecan is not
in line with this larger body of evidence [43]
TIMP-3 has been shown to be a potent inhibitor of ADAMTS-4 and -5 [44,45] We therefore assayed our
Fig 2 SDS/PAGE and Western blotting of aggrecan core protein fragments generated by rhADAMTS-1, -4 and -5 rhADAMTS-1 (10 units) and rhADAMTS-4 and -5 (5 units) were incubated with 5 mg of aggrecan monomer followed by deglycosylation of the generated fragments with chondroitin ABC lyase and electrophoresis on 4–10% gradient polyacrylamide gels Lane 1 is undigested aggrecan monomer treated with chondroitin ABC lyase, 95 kDa Panels A, B and C represent aggrecan digests produced by rhADAMTS-1, -4 and -5, respectively Lanes 3, 5, and 7 are Western blots of aggrecan core protein fragments detected with antibody 5/6/3-B-3; lanes 2, 4 and 6 are the Coomassie blue-stained gels of these fragments See Table 2 for the N-terminal sequences of the main aggrecan fragments.
Trang 6rhADAMTS-1, -4 and -5 preparations with and without
100 nM TIMP-3 This concentration of TIMP-3 resulted
in almost complete inhibition of the activity of these three
95 ± 1.7% for ADAMTS-1, -4 and -5, respectively
In summary, the addition of type-specific covalent
inactivators of serine and cysteine proteinases, the lack of
cleavage of a quenched fluorescence substrate sensitive to hydrolysis by matrixins, the detection of aggrecan fragments generated exclusively by cleavage of glutamyl bonds and inhibited by TIMP-3, and the lack of aggrecanolytic activity
in medium from mock-transfected cells, are consistent with the presence of recombinant aggrecanase activities in our enzyme preparations, with no contaminating proteolytic activities being detected
Inhibition of ADAMTS activity by catechin gallate esters Work in our laboratory has previously shown that catechin gallate esters, found in abundance in green tea effusions, inhibit cartilage aggrecan breakdown in an
in vitro model [28] We therefore analysed these com-pounds for inhibition of the aggrecan-degrading activity
of ADAMTS-1, -4 and -5 The catechin gallate esters EGCG and ECG potently inhibited ADAMTS1, 4 and
-5 in a dose-dependent manner over a 2 nM to 2 lM concentration range, whereas catechins lacking the gallate moiety, EC and EGC, and the gallate group in isolation represented by PG, showed very little inhibition even at the highest concentration tested of 2 lM(Fig 4A–C) The presence of both the catechin and the gallate ester moiety
as separate molecules in the same assay each at 500 nM was also not sufficient to inhibit the ADAMTS activities (data not shown)
IC50values for inhibition by EGCG and ECG deduced from regression analyses of the dose–response curves gave approximate values of 100–150 nMfor rhADAMTS-4 and -5, and 200–250 nM for rhADAMTS-1 The correlation coefficients for the regression analyses were all equal to or larger than 0.9 The inhibition observed with the catechin gallate esters was not due to a Zn2+-chelating effect since similar levels of inhibition were achieved when the enzymes were assayed in the presence of 100 lM ZnCl2 (data not shown) The inhibition was reversible, as removal of the catechin gallates by buffer exchange (dialysis or gel filtration) produced a reappearance of enzyme activity (data not shown)
Inhibition of collagenase and ADAM-10 activity
by catechin gallate esters Catechins and gallates were also analysed for inhibitory potential against collagenase-1 (MMP-1) and collagenase-3 (MMP-13), as well as ADAM-10 as a representative of the ADAM group of metalloproteinases Statistically significant inhibition of the collagenases by any of the catechins or gallates required a concentration of at least
20 lM Even at the highest concentration tested of 50 lM, the maximum inhibition observed was still below 50% (EGCG; 29 ± 4% inhibition of collagenase-1 activity and
30 ± 9% inhibition of collagenase-3: ECG; 14 ± 5% inhibition of collagenase-1 and 20 ± 7% inhibition of collagenase-3) The inhibition of ADAM-10 was equally poor, less than 20% inhibition being achieved by any of the catechins or gallates at the highest concentration of 50 lM
As a positive control, the hydroxamate inhibitor BB-94, a broad-spectrum matrixin and ADAM inhibitor, completely inhibited collagenase-1 and -3 and ADAM-10 at 10 lM concentration
Fig 3 Schematic representation of aggrecanase cleavage sites in the
aggrecan core protein G1, G2 and G3 represent the three globular
domains of the aggrecan core protein; KS, CS1 and CS2 represent the
keratan sulfate, chondroitin sulfate 1 and chondroitin sulfate 2
attachment regions, respectively.
Table 2 N-terminal sequences of aggrecan core protein fragments
generated by rhADAMTS¢ Fragments were generated, separated and
sequenced as described under Experimental procedures, and their
positions on 4–10% polyacrylamide gels are shown in Fig 2 Amino
acid numbering is according to the published sequence of bovine
aggrecan [66].
Molecular mass (kDa) N-terminal sequence Yield (pmol)
rhADAMTS-1
280–230 V1EVSEPDN 45
G1481RXTXD 6
V1EVSEPD 2 A374RGSVIL 1
130 A1772GEGPSGI 8
G1481RXTXD 1
G1481RXTXD 1 rhADAMTS-4
A374RGSV 2
130 A1772GEGPSGI 20
rhADAMTS-5
V1EVSEPDN 5
A374RGSVXL 1
130 A1772GEGPXGI 9
Trang 7No aggrecanolytic activity was detected in mock-transfected
insect cell-conditioned medium The expression of human
ADAMTS-1, -4 and -5 using a constitutive system in insect
cells produced relatively low amounts of recombinant
protein Partial purification led to increases in specific
activity of between 190- and 370-fold, but SDS gel
electrophoresis still demonstrated a number of
contamin-ating proteins Despite the lack of purity of our enzyme
preparations, we were unable to detect any contaminating
proteolytic activity Possible serine and cysteine proteinase activity was abolished by the addition of enzyme inactiva-tors, and no matrixin-like activity was detected by use of a broad-spectrum quenched fluorescence substrate In addi-tion, only fragments of aggrecan produced by the action of glutamyl endopeptidase activity were found following hydrolysis of the aggrecan core protein
ADAMTS-1, -4 and -5 are thought to be the proteinases responsible for the breakdown of cartilage aggrecan, which
is one of the events leading to joint failure in the arthritic diseases [46] As such, they are candidate targets for novel therapeutic intervention strategies The use of broad-spec-trum matrixin inhibitors in clinical trials has so far proved unsuccessful due to unacceptable side-effects [47–49], and it
is to be expected that more selective proteinase inhibitors will be required for successful chondroprotective inter-vention A recent report has described a series of inhibitors that show good aggrecanase vs matrixin selectivity, but no information for inhibition of ADAMs was given [50] We present here the surprising finding that catechin gallate esters, abundant components of green tea effusions, provide selective inhibition of aggrecanases, even when compared to phylogenetically related proteinases such as an ADAM and two collagenases, with a difference in potency of approxi-mately two orders of magnitude The poor inhibition by catechins lacking the gallate ester group (EC and EGC), or
by the gallate group in isolation (PG) and the fact that both modules as separate molecules in the same assay did not inhibit the rhADAMTS enzymes indicates a co-operative effect between the catechin and gallate moieties We have previously demonstrated the inhibition of cartilage aggrecan breakdown by catechin gallate esters in a tissue culture model [28], and the data presented in this paper suggest that this is due to a direct inhibitory effect of these compounds
on the activity of ADAMTS-1, -4 and -5 We have not attempted to define the mechanism of inhibition other than
to demonstrate that inhibition was reversible and was not due to Zn sequestration We hypothesize that these relatively small compounds are competing with substrate for the active site of the aggrecanases However, other possibilities exist, such as allosteric inhibition, or even direct binding to substrate
We have previously reported the inhibition by catechin gallates of type II collagen breakdown in an in vitro model
of cartilage breakdown [28] Our finding that two collagen-ases implicated in cartilage collagen hydrolysis, collagenase-1 and collagenase-3 [51–54] were not potently inhibited by EGCG and ECG, is evidence that this is not via direct inhibition of these collagen-degrading enzymes Other mechanisms are possible, including effects on proinflam-matory signalling pathways For instance, it has been reported that EGCG inhibits the chymotrypsin-like activity
of the proteasome, with IC50values in the range 86–194 nM, and subsequent accumulation of IjB-a [55] This would result in inhibition of NF-jB activation and in downstream inhibition of NF-jB-regulated genes such as the matrixins [56,57]
The catechin gallate esters are bioavailable following the consumption of green tea, with reported plasma concentra-tions in the range 0.1–5 lM [58], and a half-life of a few hours [59,60] It is therefore possible that the drinking of green tea will have a prophylactic effect on cartilage
Fig 4 Inhibition of rhADAMTS-1, -4 and -5 by catechinsand gallates.
rhADAMTS-1 (A), -4 (B), and -5 (C) were assayed using
aggrecan-containing polyacrylamide particles in the absence and presence of
catechins and gallates over the concentration range 2 n M to 2 l M EC,
s; ECG, h; EGC, m; EGCG, r; PG, X The lines represent linear
regression analyses All assays were performed at least twice.
Trang 8integrity Indeed, it has been reported that mice fed on a
polyphenolic fraction of green tea have reduced signs of
collagen-induced arthritis [61] Alternatively these
com-pounds could serve as lead comcom-pounds in the design of
more potent inhibitors that will halt cartilage breakdown
There are many reports in the literature of beneficial
effects of green tea consumption, some of which relate to
pathologies in which turnover of extracellular matrix
proteins is a major component, such as stroke and cerebral
haemorrhage [62] and cancer [63,64] The anticancer effects
of polyphenolic compounds from green tea have been
attributed, at least in part, to their direct inhibition of
matrixins such as the gelatinases [4–6] However, the
reported IC50values for inhibition of these proteinases of
about 20 lM, similar to our findings reported here for two
collagenases and ADAM-10, are beyond the concentration
attainable following green tea consumption It is plausible
that at least some of the beneficial effects are provided
instead by direct inhibition of ADAMTS enzymes, some of
which have been implicated in cancer [65], perhaps in
combination with down-regulation of other proteinases at
the mRNA level
Acknowledgements
We wish to thank the Arthritis Research Campaign, UK for funding
this research This work was also supported by the Wellcome Trust
UK, Australian National Health and Research Council and the
Arthritis Foundation of Australia.
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