Báo cáo khoa học: Selective inhibition of ADAMTS-1, -4 and -5 by catechin gallate esters ppt

Selective inhibition of ADAMTS-1, -4 and -5 by catechin gallate esters
Mireille N. Vankemmelbeke
1
, Gavin C. Jones
1
, Cyprianne Fowles
1
, Mirna Z. Ilic
2
, Christopher J. Handley
2
,
Anthony J. Day
3
, C. Graham Knight
4
, John S. Mort
5
and David J. Buttle
1
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-
trationingreentea(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 IC
50
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 ÔaggrecanasesÕ. Aggrecan degradation products
resulting from aggrecanase action have been found in
in vitro cultures of cartilage treated with proinflammatory
cytokines as well as in synovial fluid of arthritis patients
[13–16]. To date three mammalian ÔaggrecanasesÕ 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)
Eur. J. Biochem. 270, 2394–2403 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03607.x
To 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 (SalIandNotI 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/TOPOÒ vector (Invitrogen
TM
Life Techno-
logies,Paisley,UK).ForADAMTS-4,primerpair
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 FLAG
TM
sequence
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 FLAG
TM
sequence followed by the coding sequence for
ADAMTS-5. This was subcloned into the pMT/BiP/V5-
HisB expression vector (Invitrogen
TM
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/TOPOÒ vector using two primers, 5¢-CC

GATCTCAATATGAAGTTATGC-3¢ (sense) and 5¢-CCT
CTAGATTACTAACATTTCTTCAACAAGCATTG-3¢
(antisense) and the TOPO TA cloningÒ methodology
according to the manufacturer’s recommendations. The
correct coding sequence was confirmed by sequencing in
both directions.
Cell culture
High Five
TM
cells (Invitrogen
TM
Life Technologies, Paisley,
UK) were maintained and propagated in HyQSFXÒ
serum-free insect cell culture medium (Perbio Science UK,
Ltd. Cheshire, UK) containing 10 lgÆmL
)1
gentamycin at
27 °C. The cells were transfected with the recombinant or
empty expression vector using the lipid-based CellFectinÒ
reagent (Invitrogen
TM
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 (Invitrogen
TM
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 4
M
guanidinium (Gn) HCl and dissociative CsCl
2
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 m
M
CaCl
2
, 0.1% w/v Chaps, pH 7.5). The tubes
were incubated at 37 °C 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 37 °C in the assay.

Purification and characterization of recombinant
ADAMTS enzymes
rhADAMTS-1, -4 and -5 were all purified using an
identical protocol. Conditioned High Five
TM
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 l
M
and 10 l
M
, 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 m
M
Tris/
HCl, 0.15
M
NaCl, 0.1% w/v Chaps, pH 7.0). Activity was
eluted using a step-wise gradient (0.15–1
M
NaCl). 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 m
M
Tris/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 m
M
Tris/HCl, 0.1
M
NaCl, 10 m
M
CaCl

2
, 0.1% w/v Chaps, pH 7.5 at 37 °C
for 16 h. Aggrecan fragments were detected by Western
Ó FEBS 2003 Aggrecanase inhibition by catechin gallate esters (Eur. J. Biochem. 270) 2395
blotting 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 Sulfolink

TM
column (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)-NovaSynÒ 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 21 °C, 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-NH
2
[34,35]. Cleavage of the ADAM-10
quenched fluorescence substrate (see above) followed
Michaelis–Menten kinetics with an approximate K
m
of
20 l
M
. Determination of a more accurate K
m
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 l
M
in 100 m
M
Tris/HCl, 0.1
M
NaCl, 10 m
M
CaCl
2
, 0.2% v/v Triton X-100 pH 7.5, or in
50 m
M
Tris/HCl, 100 l
M
ZnCl
2
, 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 Me
2
SO to 2 m

M
to
provide the stock solution and diluted to the appropriate
final concentration which ranged from 2 to 2000 n
M
in
enzyme assay buffer. Enzymes were preincubated with the
inhibitors or the appropriate concentration of Me
2
SO as
control at 4 °C 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 Me
2
SO controls.
Log-linear plots of the dose–response curves in combina-
tion with regression analysis allowed us to determine
approximate IC
50
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 n
M
of the inhibitor
for 30 min at 4 °C 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 l
M
to 50 l
M
.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 l
M
final
concentration. The initial steady state of substrate cleavage
(v
o
) was recorded, after which inhibitor or Me
2
SO control
were added to the reaction mix in a minimal volume, and
the new steady state (v
i
) was documented. The percentage
inhibition was calculated as (1 ) v
i
/v
o
) · 100.
2396 M. N. Vankemmelbeke et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Results
Purification 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 1mgmL
)1
protein.
Step Protein (mg) Total Activity (U) Specific activity (UÆmg
)1
) Yield (%) Purification (-fold)
rhADAMTS-1
Medium 44 32 0.70 (100) (1)
Heparin F-F 1.4 47 34 147 49
Mono-Q 0.20 28 140 88 200
rhADAMTS-4
Medium 190 110 0.60 (100) (1)
Heparin F-F 1.1 82 75 75 125
Mono-Q 0.20 44 220 40 367
rhADAMTS-5

Medium 110 720 6.5 (100) (1)
Heparin F-F 2.8 410 150 57 23
Mono-Q 0.10 120 1200 17 185
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 MicroconÒ 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.
Ó FEBS 2003 Aggrecanase inhibition by catechin gallate esters (Eur. J. Biochem. 270) 2397
Antibody 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 ÔaggrecanaseÕ activity
are Glu373 fi Ala in the interglobular domain,
Glu1480 fi Gly between the chondroitin sulfate 1 and 2
attachment regions and Glu1666 fi Gly, Glu1771 fi Ala,
and Glu1871 fi Leu within 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.

2398 M. N. Vankemmelbeke et al. (Eur. J. Biochem. 270) Ó FEBS 2003
rhADAMTS-1, -4 and -5 preparations with and without
100 n
M
TIMP-3. This concentration of TIMP-3 resulted
in almost complete inhibition of the activity of these three
ADAMTS enzymes: 93 ± 0.2%, 100 ± 0.1% and
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 ADAMTS-1, -4 and -
5 in a dose-dependent manner over a 2 n
M
to 2 l
M

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 l
M
(Fig. 4A–C). The
presence of both the catechin and the gallate ester moiety
as separate molecules in the same assay each at 500 n
M
was also not sufficient to inhibit the ADAMTS activities
(data not shown).
IC
50
values for inhibition by EGCG and ECG deduced
from regression analyses of the dose–response curves gave
approximate values of 100–150 n
M
for rhADAMTS-4 and
-5, and 200–250 n
M
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 Zn
2+
-chelating effect since
similar levels of inhibition were achieved when the enzymes
were assayed in the presence of 100 l
M
ZnCl

2
(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 l
M
. Even at the highest concentration tested of 50 l
M
,
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 l
M
.
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 l
M
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
generatedbyrhADAMTS¢. 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
200 V1EVSEPXN 6
G1481RXTXD 6
170 G1667LGSVEL 4
V1EVSEPD 2
A374RGSVIL 1
130 A1772GEGPSGI 8
V1EVSE 3
G1481RXTXD 1
100 L1872GQRPXV 3
G1481RXTXD 1
rhADAMTS-4
280 V1EVSEPDN 13
230 V1EVSEPDN 12

A374XGS 4
200 G1481RGTXD 6
V1EVSEP 1
170 G1667LGSVEL 4
A374RGSV 2
V1EVSEP 1
130 A1772GEGPSGI 20
rhADAMTS-5
280 A374XGSVIL 9
V1EVSEPDN 5
200 G1481RGTIDI 3
A374RGSVXL 1
170 G1667LGSVEL 2
130 A1772GEGPXGI 9
100 L1872GQRPPV 8
Ó FEBS 2003 Aggrecanase inhibition by catechin gallate esters (Eur. J. Biochem. 270) 2399
Discussion
No 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 IC
50
values in the range 86–194 n
M
,
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 l

M
[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 catechins and 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.
2400 M. N. Vankemmelbeke et al. (Eur. J. Biochem. 270) Ó FEBS 2003
integrity. 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 compounds 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 IC
50

values for inhibition of these proteinases of
about 20 l
M
, 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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