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

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

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-

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

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

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

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

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

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

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

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.

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

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

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

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

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