This report inves-tigates the ability of purified recombinant human MMP-9 produced in insect cells to degrade native collagen types I and III.. This report therefore investigates the abil
Trang 1collagen types I and III
Heather F Bigg1, Andrew D Rowan1, Michael D Barker2and Tim E Cawston1
1 Musculoskeletal Research Group, Institute of Cellular Medicine, The Medical School, Newcastle University, UK
2 Division of Genomic Medicine, Academic Unit of Pathology, University of Sheffield, Medical School, UK
Collagens are the major structural proteins of
connect-ive tissues such as skin, bone, cartilage and tendon
Interstitial collagen types I, II and III are the most
abundant, and the native triple helical structure of
these molecules makes them highly resistant to
proteo-lysis However, collagenases of the matrix
metallopro-teinase (MMP) family [1] cleave native collagen
types I, II and III at a specific site in all three chains
of the triple helix, approximately three-quarters of the
length from the N-terminus The action of these
col-lagenase enzymes is therefore critical for the initiation
of collagenolysis Once initiated, the cleaved helix
unwinds at physiological temperatures and becomes
susceptible to degradation by other, less-specific
pro-teinases MMP collagenases are active at neutral pH
and play a highly important role in collagen
degrada-tion in vivo The mammalian MMP collagenases cur-rently include the ‘classical’ collagenases, MMP-1, MMP-8 and MMP-13 [2–4] and also the gelatinolytic enzyme, MMP-2 [5–7], and MMP-14 (MT1-MMP) [8],
a member of the membrane-type subclass of MMPs MMP-9 (also known as gelatinase B, 92 kDa gela-tinase or 92 kDa type IV collagenase, EC 3.4.24.35) shares a close structural similarity with MMP-2 [9,10]
It was originally identified as a gelatinolytic enzyme produced by polymorphonuclear leukocytes [11] and subsequent studies have demonstrated secretion in the latent form (proMMP-9) by a variety of cell types It has also been implicated in the pathogenesis of several human diseases, including arthritis [12–15] Unlike other MMPs, MMP-9 and MMP-2 both contain three fibronectin type II repeats inserted into the catalytic
Keywords
arthritis; collagen I; collagen III; collagenase;
matrix metalloproteinase-9
Correspondence
T E Cawston, Musculoskeletal Research
Group, 4th Floor, Catherine Cookson
Building, The Medical School, Framlington
Place, Newcastle University,
Newcastle-upon-Tyne, NE2 4HH, UK
Fax: +44 191 2225455
Tel: +44 191 2225397
E-mail: t.e.cawston@ncl.ac.uk
Website: http://www.ncl.ac.uk/medi/
research/rheumatology/
(Received 3 November 2006, revised 20
December 2006, accepted 22 December
2006)
doi:10.1111/j.1742-4658.2007.05669.x
Interstitial collagen types I, II and III are highly resistant to proteolytic attack, due to their triple helical structure, but can be cleaved by matrix metalloproteinase (MMP) collagenases at a specific site, approximately three-quarters of the length from the N-terminus of each chain MMP-2 and -9 are closely related at the structural level, but MMP-2, and not MMP-9, has been previously described as a collagenase This report inves-tigates the ability of purified recombinant human MMP-9 produced in insect cells to degrade native collagen types I and III Purified MMP-9 was able to cleave the soluble, monomeric forms of native collagen types I and III at 37C and 25 C, respectively Activity against collagens I and III was abolished by metalloproteinase inhibitors and was not present in the concentrated crude medium of mock-transfected cells, demonstrating that
it was MMP-9-derived Mutated, collagenase-resistant type I collagen was not digested by MMP-9, indicating that the three-quarters⁄ one-quarter locus was the site of initial attack Digestion of type III collagen generated
a three-quarter fragment, as shown by comparison with MMP-1-mediated cleavage These data demonstrate that MMP-9, like MMP-2, is able to cleave collagens I and III in their native form and in a manner that is char-acteristic of the unique collagenolytic activity of MMP collagenases
Abbreviations
APMA, p-aminophenylmercuric acetate; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinases.
Trang 2domain which are thought to mediate the ability to
bind collagen [16,17] However, this domain does not
appear to be essential for the collagenolytic activity of
MMP-2 [7] MMP-9, unlike MMP-2, also contains an
additional 54-amino acid proline-rich insertion,
homol-ogous to the a2 chain of type V collagen [10] To date,
MMP-9 has not been described as a collagenase
Sev-eral previous studies have investigated its ability to
digest native collagen types I, II and III using enzyme
from a variety of sources, both natural and
recombin-ant [5,6,18–23] Three of these demonstrated an
inabil-ity to degrade soluble native collagen I at 22 or 25C
[5,6,18]; a lack of digestion at 37C was additionally
reported by Murphy et al [18] However, another
study [20] has shown digestion of soluble native
colla-gen I at 30 and 37C Four previous studies have
examined the digestion of collagen II and all report no
degradation by MMP-9 [6,18,21,22] However,
investi-gation into the ability to digest native collagen III has
produced disparate results Three reports [6,21,22]
des-cribe degradation at 22, 25 or 27C, whilst two others
[18,20] report digestion at 37 but not 30C [20] or no
digestion at 25 or 37C [18] Furthermore, none of
these previous studies has investigated the initial
MMP-9-mediated cleavage site of collagens I and III
The possibility of contamination with another MMP
collagenase is very difficult to exclude when working
with a natural source However production of
recom-binant protein can also present problems with respect
to correct folding of the enzyme, particularly when
prokaryotic cells are used Both of these issues can be
avoided by expression in insect cells, since these do not appear to produce collagenolytic metalloproteinases This report therefore investigates the ability of purified recombinant human MMP-9 produced in insect cells
to cleave native collagen types I and III; in addition, the initial MMP-9-mediated cleavage site of these sub-strates is investigated for the first time
Results
Characterization of purified, recombinant, human proMMP-9 produced in insect cells
Purified, recombinant, human MMP-9 expressed in insect cells was used to examine the ability of this enzyme to cleave native collagens I and III Recombin-ant human proMMP-9 was purified from the condi-tioned medium of pIB-proMMP-9-transfected insect cells and characterized by silver staining, western blot-ting and zymography (Fig 1) Silver staining under reducing conditions (+ bme) revealed two bands with apparent molecular masses of 85 and 61 kDa, which were identified as MMP-9 by western blotting with two anti-MMP-9 sera (Fig 1A) Human proM-MP-9 from natural sources has a Mr of 92 kDa [10,21,24] and contains both N- and O-linked carbohy-drate [10] The molecular mass of the unglycosylated protein is 81 kDa [10] The 85-kDa species may therefore be a differentially glycosylated full-length form of proMMP-9, but it is also possible that trunca-tion of the polypeptide occurs during expression and
97.4 66.2 45
MAB911 A560/8
Mr
Mr
(kDa)
(kDa) (kDa)
SILVER STAIN
W BLOT
+
Mr
97.4 66.2 45 βme
SILVER STAIN
98 64 50
-A560/8
(kDa)
Mr
βme
W BLOT
148
MAB911
-ZYMOGRAM
APMA -+ +
31
148 98 64 50 36
31
36
βme
Fig 1 Characterization of recombinant human proMMP-9 by silver staining, western blotting and zymography (A,B) Purified recombinant human proMMP-9 (0.5 lg) was run reduced (A, + bme) and nonreduced (B, – bme) on 10% SDS ⁄ PAGE gels followed by silver staining (SILVER STAIN) or transfer to nitrocellulose (W BLOT) as described in Experimental procedures Western blots were probed with a polyclonal sheep anti-(porcine MMP-9 serum) (A560 ⁄ 8, 2 lgÆmL)1) and a monoclonal mouse anti-(human MMP-9) serum (MAB911,
2 lgÆmL)1) M r , the positions of molecular mass markers (kDa) are shown The individual bands present in nonreduced lanes (B) are marked
by arrows (C) Recombinant human proMMP-9 (1.6 ng) was run nonreduced (– bme) on a 10% gelatine zymogram (ZYMOGRAM) either with (+) or without (–) prior activation by 0.67 m M APMA (APMA) for 90 min at 37 C.
Trang 3purification The 61-kDa species is clearly a truncated
MMP-9 fragment, on account of its lower molecular
mass and immunoreactivity to two anti-MMP-9 sera
(Fig 1A)
Silver staining and western blotting under
nonreduc-ing conditions (– bme) (Fig 1B) revealed additional
higher molecular mass bands, which therefore appear
to be disulphide-bonded MMP-9 complexes In
addi-tion, the 85-kDa species displayed heterogeneity when
run nonreduced, since it migrated as three separate
bands; this may result from differential disulphide
bond formation Zymography (nonreducing
condi-tions, – bme) (Fig 1C) revealed a similar pattern of
gelatinolytic bands, except that a single band only was
seen for the 85-kDa species This indicates that the
additional nonreduced forms of this species lack
gela-tinolytic activity, which may be due to incorrect
disul-phide bond formation The 61-kDa form appears to be
an active site-containing fragment, as it has
gelatinoly-tic activity (Fig 1C) Activation of the proMMP-9
with p-aminophenylmercuric acetate (APMA) prior to
zymography increased the migration of all species,
including the 61-kDa fragment, therefore
demonstra-ting that all species were proenzyme forms (Fig 1C)
Purified, recombinant, human MMP-9 cleaves
soluble, native type I collagen
The ability of purified recombinant human MMP-9
produced in insect cells to cleave type I collagen was
tested using soluble substrate at 37C (Fig 2) The
collagen retained its native, triple helical structure
under these assay conditions, as it remained resistant
to trypsin (Figs 2A.T) The activity of the trypsin was
confirmed by total lysis of denatured substrate
(Fig 2A, T,denat) Importantly, preparation of the
type I collagen did not include pepsin digestion, as this
may result in increased susceptibility to gelatinolysis at
37C Furthermore, trypsin sensitivity is a reliable
indicator of whether the collagen is susceptible to a
gelatinolytic attack, as progressive heat denaturation
of the collagen at increasing temperatures shows that
resistance to trypsin is lost under the same conditions
as resistance to gelatinolysis (data not shown)
Extensive digestion of the type I collagen b and a
chains was seen in the presence of either MMP-1 or
proMMP-9 when combined with APMA
MMP-1-mediated cleavage did not generate the characteristic
three-quarter length fragments seen at lower incubation
temperatures, because at 37C these cleavage products
spontaneously denature and are susceptible to further
MMP-1-mediated gelatinolytic degradation For the
same reason, no fragments at all were detected in the
presence of MMP-9, as this enzyme is a potent gelatinase No collagen digestion was seen without APMA activation of the proMMP-9 (Fig 2A, – APMA, + proMMP-9) However, some conversion of
b12 dimers to a1 and a2 monomers is apparent in this lane, to give an increased level of both the a chains and
a slightly increased mobility of the a2chain This indi-cates the presence of a2 chain telopeptidase activity, resulting from a low level of spontaneous proMMP-9 activation during the assay Digestion of the a2 N-ter-minal telopeptides of native type I collagen by MMP-9 has been reported previously [21] MMP-9-mediated digestion (both collagenolytic and telopeptidase) was
- + - +
- + MAB911
proMMP-9 + APMA MMP-1 MMP-13
β 11
β 12
α 1
α2
buff er M
P-1
T Tde
t
- + + + + + + +
APMA
E T
seri
ne/
+ + + -+
+ + +
-TIM
P-2
EtO H
1,10 m k
β 11
β12
α 1
α2
cyst ne
+
-moc k
A
B
Fig 2 Recombinant human MMP-9 cleaves soluble, triple helical type I collagen (A) Soluble type I collagen from bovine skin (27 lgÆ lane)1) was digested for 72 h at 37 C with buffer alone (buffer), 0.3 lg MMP-1 (MMP-1), 0.2 lg trypsin (T), 0.5 lg recombinant human proMMP-9 (proMMP-9) or 24 lg of protein from the concen-trated crude culture medium of mock-transfected insect cells (mock),
in the absence or presence of 0.6 m M APMA, as indicated Additional lanes also contained the following enzyme inhibitors: 6 m M EDTA (EDTA), 8 m M 1,10-phenanthroline (1,10), 2.3 lg TIMP-2 (TIMP-2), serine and cysteine protease inhibitors at the manufacturer’s recom-mended working strength (serine ⁄ cysteine) or the ethanol solvent used for 1,10-phenanthroline (EtOH) The efficacy of the trypsin was demonstrated by cleavage of denatured substrate (denat) The micr-ogram enzyme–substrate ratio of MMP-9–type I collagen is 1 : 54 The positions of the uncut collagen b and a chains (b 11 , b 12 , a 1 and a 2 ) are indicated Cleavage of type I collagen by proMMP-9 combined with APMA was investigated in five separate experi-ments, with similar results each time (B) The effect of 4 lg mono-clonal anti-(human MMP-9) serum (MAB911) on cleavage mediated
by 0.2 lg MMP-1 (MMP-1), 0.2 lg MMP-13 (MMP-13) or 0.1 lg recombinant human proMMP-9 (proMMP-9) combined with APMA (+ APMA) is shown The microgram enzyme–substrate ratio of MMP-9–type I collagen is 1 : 270 The positions of the uncut colla-gen b and a chains (b11, b12, a1and a2) are indicated.
Trang 4abolished by EDTA, 1,10-phenanthroline and tissue
inhibitor of metalloproteinases (TIMP)-2, but not by
serine and cysteine protease inhibitors, or the ethanol
vehicle for the 1,10-phenanthroline (Fig 2A), thereby
demonstrating metalloproteinase-mediated activity No
cleavage was observed in the presence of crude insect
cell culture medium (16.5-fold concentrate) conditioned
by mock-transfected cells (chloramphenicol acetyl
transferase vector) (Fig 2A, mock), therefore excluding
the possibility of a contaminating insect cell protease
Furthermore, collagenolytic digestion with a lower level
of MMP-9 was blocked by a monoclonal anti-MMP-9
antibody, whereas cleavage mediated by MMP-1 or
MMP-13 was unaffected or affected only slightly
(Fig 2B, –⁄ +MAB911) Taken together, these data
convincingly demonstrate that recombinant human
MMP-9 is capable of cleaving native, trypsin-resistant,
soluble type I collagen
The initial cleavage of type I collagen by MMP-9
is at the three-quarters⁄ one-quarter locus
A hallmark of MMP collagenolytic activity is the
abil-ity to perform the initial cleavage of native substrate
at the three-quarters⁄ one-quarter site To investigate
the initial cleavage site of MMP-9-mediated type I
col-lagen digestion, we examined its ability to digest
mutated type I collagen which is completely resistant
to collagenolytic cleavage, due to the mutation of
Gln774 (P2) and Ala777 (P¢2) of the a1(I) chain
three-quarters⁄ one-quarter site to proline The wild-type
a2(I) chain of each triple helix is also not cleaved, due
to the presence of two mutated a1(I) chains [25]
Mutated type I collagen was not cleaved by MMP-1
or MMP-13, as expected (Fig 3, mutated, MMP-1,
MMP-13), although telopeptidase activity was evident
in the presence of MMP-1 Under identical conditions,
wild-type collagen I was digested by both these
enzymes; the characteristic three-quarters fragments
are not seen, because at 36C, these cleavage products
spontaneously denature and are susceptible to further
gelatinolytic degradation Mutated type I collagen was
also resistant to cleavage mediated by human
recom-binant MMP-9 (Fig 3, mutated, proMMP-9, APMA)
Under identical conditions, wild-type collagen I was
digested by MMP-9 (Fig 3, wild-type, proMMP-9,
APMA) to give extensive degradation without the
appearance of partially digested fragments, as for
Fig 2 The resistance of the mutated type I collagen to
MMP-9-mediated digestion demonstrates that this
enzyme makes the initial cut at the three-quarters⁄
one-quarter locus, which is a characteristic of MMP
collagenolytic action Importantly, these data also
exclude gelatinolytic degradation of partially unfolded wild-type collagen I by MMP-9, as under the same conditions, this mechanism would also result in suscep-tibility of the mutated collagen
MMP-9 cleaves native, triple helical type III collagen to generate a 3/4 fragment The ability of recombinant human MMP-9 to cleave collagen type III was investigated in assays with sol-uble substrate and compared with the ability to cleave type I Type III collagen was cleaved at 25C by recombinant MMP-9, to produce a fragment with a similar mobility to that of the MMP-1-generated three-quarter piece (Fig 4, type III, compare MMP-1 with + proMMP-9, + APMA) [26] Digestion with proMMP-9 and APMA was abolished by EDTA, 1,10-phenanthroline and TIMP-2, but not by serine and cysteine protease inhibitors, or the ethanol vehicle for the 1, 10-phenanthroline, demonstrating metallo-proteinase-mediated cleavage In addition, no digestion was observed with concentrated crude insect cell culture medium from mock-transfected cells (Fig 4, type III, mock) A low level of cleavage was seen in the absence of APMA (Fig 4, type III, – APMA, + proMMP-9), indicating some spontaneous activation
of the proMMP-9 during the assay Minor cleavage was also seen with trypsin, in agreement with a previ-ous report demonstrating specific cleavage of native type III collagen with this enzyme (Fig 4, type III, T) [27] The more extensive digestion of denatured type III collagen by trypsin (Fig 4, type III, T, denat)
mutated wild-type
buff er
M
P-1
T T
dena t
M
P-1 3
proM
MP -9
buff M
P-1
t M
P-1 3
proM
MP -9
β 11
β 12
α 1
α 2
AP
MA
AP
MA
β 11
β 12
α1
α2
Fig 3 Recombinant human MMP-9 cleaves native type I collagen
at the three-quarters ⁄ one-quarter locus Soluble type I collagen (27 lgÆlane)1) from bovine skin (wild-type) or mouse skin (mutated) was digested for 98 h at 36 C with buffer alone, 0.6 lg MMP-1, 0.2 lg trypsin (T), 0.5 lg MMP-13 or 0.5 lg recombinant human proMMP-9 in the additional presence of 0.6 m M APMA The effic-acy of the trypsin was demonstrated by cleavage of denatured sub-strate (denat) The positions of the uncut collagen b and a chains (b11, b12, a1and a2) are shown Cleavage of wild-type and mutated type I collagen by proMMP-9 combined with APMA was compared
in two separate experiments, with similar results on each occasion.
Trang 5confirms that all other conditions represent cleavage
of native rather than denatured substrate Taken
together, these data clearly demonstrate the ability of
recombinant human MMP-9 to cleave native type III
collagen Gel-scanning densitometry of the data in
Fig 4 indicates cleavage of 42% of the type III
sub-strate by MMP-9
Under the same conditions as the type III assay,
MMP-1 cleaved type I collagen to give characteristic
three-quarter fragments, but no digestion was observed
with proMMP-9 and APMA (Fig 4, type I) This
indi-cates that recombinant MMP-9 cleaves type III collagen
more effectively than type I, as digestion of type III was
seen at 25C whereas digestion of type I occurred only
at the higher temperatures of 36C or 37 C (Figs 2 and
3) Recombinant MMP-9 was also able to digest
colla-gen III at 35 and 36C, as well as at 25 C; however, at
36C, extensive substrate digestion was also seen in the
presence of trypsin, making it difficult to ascertain that
the collagen retained its native conformation at this tem-perature (data not shown)
Discussion
A number of previous reports have investigated the ability of MMP-9 to degrade native collagen types I and III [5,6,18–23] with disparate results In this study, recombinant human MMP-9 was expressed in insect cells and the ability of enzyme purified from this source
to digest native collagens I and III was evaluated Importantly, the possibility of contaminating, endog-enous collagenolytic activity was excluded, as shown by the lack of substrate cleavage seen with concentrated, crude insect cell culture medium from mock-transfected cells The data in this report therefore conclusively demonstrate that MMP-9 is able to digest soluble, native collagen types I and III at 37 and 25C, respect-ively Furthermore, the location of substrate cleavage sites was also investigated, demonstrating for the first time that MMP-9 attacks native collagens I and III initially at the three-quarters⁄ one-quarter site
Several previous studies report that MMP-9 is unable
to digest native collagen I [5,6,18,19,23] In two of these [19,23], the precise assay conditions are not described and it is therefore difficult to compare these findings with the data reported here Aimes and Quigley [5], Konttinen et al [6] and Murphy et al [18] performed assays at either 22 or 25C and reported no digestion
at these temperatures, in agreement with the findings of this study The latter study [18] also reported no degra-dation of native collagen I at 37C, but these data are described in the text only and therefore cannot readily
be compared with the data reported here In agreement with our study, a further report [20] describes digestion
at both 30 and 37C, but in this case, the collagen I substrate was pepsin-treated and therefore possibly sus-ceptible to a gelatinolytic attack; furthermore, resist-ance to trypsin was not demonstrated
Three previous studies [6,21,22] have shown digestion
of native collagen III by MMP-9 at 22, 25 and 27 C, respectively, in agreement with the data reported here However, another study [18] reported no degradation
at either 25 or 37C The discrepancy at 25 C may be due to differences in the quantity of enzyme and assay period; although the amounts of substrate were similar,
we used more enzyme (4.5·) in a longer assay (5·)
A shorter assay time (5· less) with less enzyme (0.6·) may also explain the reported lack of digestion
at 30 C [20]; the same study indicated that collagen III
is degraded under these conditions at 37C
Of the existing MMPs, MMP-9 is most closely rela-ted to MMP-2 at the structural level The C-terminal,
Type III
β 11
α 1
buff
er
M
P-1
T T
nat
APMA
-+ + + + + +
E T
EtO H
1,10 TIM
P-2
proMMP-9
moc k
+ + + + + + +
seri
ne/
cyst ne
mock
- +
-proMM
P-9
Type I
β 12
β 11
α 1
α 2
buff er MM
P-1
T Tde
t
AP MA
Fig 4 Recombinant human MMP-9 cleaves soluble, triple helical
type III collagen to generate a three-quarters ⁄ one-quarter fragment.
Soluble type III or type I collagen (27 lgÆlane)1), as indicated, was
digested for 98 h at 25 C with buffer alone, 0.6 lg MMP-1, 0.2 lg
trypsin (T), 0.5 lg recombinant human proMMP-9 or 24 lg of
pro-tein from the concentrated crude culture medium of
mock-trans-fected insect cells (mock) in the absence or presence of 0.6 m M
APMA, as indicated Additional lanes also contained the following
enzyme inhibitors: 6 m M EDTA, 8 m M 1,10-phenanthroline (1,10),
2.3 lg TIMP-2, serine and cysteine protease inhibitors at the
manu-facturer’s recommended working strength or the ethanol solvent
used for 1,10-phenanthroline (EtOH) The efficacy of the trypsin
was demonstrated by cleavage of denatured substrate (denat) The
microgram enzyme–substrate ratio of MMP-9–collagen is 1 : 54.
The positions of the uncut collagen b and a chains (b11, b12, a1and
a 2 ) are indicated Cleavage of type III collagen by proMMP-9
com-bined with APMA was investigated in four separate experiments,
with similar results each time.
Trang 6hemopexin-like domain of MMP-2 is essential for its
collagenolytic activity [7], as is also the case for the
‘classical’ collagenases [28–33] In contrast, the three
fibronectin type II repeats, which are shared with
MMP-9 but not other MMPs, are not absolutely
required for collagenolysis, although their presence
does enhance activity [7] MMP-9, unlike MMP-2,
contains a unique, 54 amino acid, proline-rich
inser-tion, immediately before the hinge region, which is
homologous to the a2 chain of type V collagen [10]
This extra domain, the function of which is currently
unknown, may play a role in the ability of MMP-9 to
cleave native collagens MMP-2 cleaves all three of the
interstitial collagen types I, II and III [5–7] with a
sub-strate preference of type III > type II > type I [6]
The data shown here indicate that MMP-9 also has a
clear substrate preference for type III above type I;
digestion of type II was not investigated, but in
con-trast to MMP-2, all previous studies report a lack of
cleavage by MMP-9 [6,18,21,22]
The data presented in this report demonstrate for
the first time that MMP-9 performs the initial cleavage
of native collagens I and III at the three-quarters⁄
one-quarter site Type I collagen, which is completely
resistant to collagenolysis due to mutation of residues
close to the three-quarters⁄ one-quarter site in the a1(I)
chain, was not digested by MMP-9 In addition,
com-parison of the fragments generated by MMP-1 and
MMP-9-mediated cleavage of type III collagen
indica-ted digestion at the three-quarters⁄ one-quarter locus
by MMP-9 In this respect, MMP-9-mediated collagen
cleavage resembles that of the MMP collagenases,
since cleavage at this site is a hallmark of these
enzymes However, MMP-9 does not completely fit the
description of a stereotypical MMP collagenase, as it
does not appear to cleave type II collagen [6,18,21,22];
all the existing mammalian collagenases are able to
cleave all three interstitial collagen types [2–4,6,8,34]
MMP-9 was also unable to cleave soluble type I
colla-gen at 25C, but this finding does not preclude a
phy-siological role in the degradation of native soluble
substrate at 37C and the ability of MMP-9 to digest
the latter in vitro is clearly demonstrated in this study
A clear association of MMP-9 with rheumatoid and
inflammatory arthritis and a correlation between
syn-ovial fluid levels of proMMP-9 and the collagen
degra-dation product, hydroxyproline, in rheumatoid
arthritis [35] indicate a role for MMP-9 in these
dis-eases in the destruction of type I collagen in bone and
type III collagen in synovium MMP-9 levels in
rheu-matoid and inflammatory synovial fluids are higher
than those from noninflammatory or osteoarthritic
patients [12–14]; furthermore, net gelatinolytic activity
is not found in osteoarthritic synovial fluids but does occur in approximately one-quarter of rheumatoid samples [15] Rheumatoid synovial tissue also contains significantly more MMP-9 than osteoarthritic syno-vium, although increased levels are observed in osteo-arthritic tissue with a heightened inflammatory response [12] In addition, immunostaining of MMP-9
in experimental models of inflammatory arthritis shows
a correlation with disease progression [36]; further-more, antibody-induced arthritis, which is one of the murine models of rheumatoid disease, is less severe in MMP-9 knockout mice [37]
Immunolocalization studies also show intense stain-ing for MMP-9 in the osteoclasts of both normal and rheumatoid knee joints [38], suggesting a role in both normal and pathological bone resorption Osteoclasts also produce very low levels of TIMP-1 compared with MMP-9 [39]; the majority of MMP-9 produced should therefore be free from complex formation with this inhibitor The gelatinolytic activity of MMP-9 is opti-mal at pH 7.5, but 50–80% of the full activity is retained at pH 5.5–6.0 [38] MMP-9 is also activated
by acidic conditions [38,40] These observations, com-bined with its ability to solubilize collagen from demin-eralized bone particles [41] and to produce visible degradation of the collagen fibrils, as shown by elec-tron microscopy [38], selec-trongly suggest a role in colla-gen degradation during bone resorption Digestion of the a2N-terminal telopeptides of native type I collagen
by MMP-9 [21] is also likely to be important in bone collagen resorption, as highly cross-linked type I colla-gen, such as that found in bone collagen fibrils, is reported to be resistant to enzymes such as MMP-1, without prior removal of the cross-link-containing ter-minal peptides by telopeptidases Surprisingly, the bone histology of MMP-9 knockout mice does not reveal a lack of osteoclastic resorption of mineralized matrix [42], but other enzymes may be compensating for its absence Indeed, another study [43], using a spe-cific neoepitope antibody, detected collagen fragments resulting from a three-quarters⁄ one-quarter cut along the spicules of trabecular bone in the developing tibiae
of wild-type, but not MMP-9 knockout mice, provi-ding in vivo evidence for the involvement of MMP-9 in bone collagen turnover
Experimental procedures
Antibodies
A rabbit polyclonal anti-MMP-9 serum was purchased from Sigma-Aldrich (Gillingham, UK), and a mouse mono-clonal antibody against human MMP-9 (MAB911) was
Trang 7from R & D Systems Europe Ltd (Abingdon, UK) A
sheep polyclonal antibody against porcine MMP-9
(A560⁄ 8) was a generous gift from R Hembry (University
of East Anglia, Norwich, UK)
MMP and TIMP proteins
Human proMMP-1 was expressed in Escherichia coli,
refold-ed and purifirefold-ed as previously describrefold-ed [44,45] and
quantitat-ed by enzyme-linkquantitat-ed immunosorbent assay [46] Refolding
caused activation of proMMP-1 to the fully active form or
conversion to an intermediate lacking the polyhistidine tag
and the first four amino acids of the proenzyme Activation
with APMA resulted in conversion of the intermediate form
to the fully active enzyme Enzyme assays were performed
without APMA activation and therefore measured only the
fully active form present in the refolded sample
The cDNA for human MMP-13 was generously provided
by V Kna¨uper (University of York, York, UK)
Oligonu-cleotides to amplify the mature MMP-13 sequence were
designed, incorporating an initiating ‘ATG’ in the forward
primer and a ‘TAG’ stop codon in the reverse primer
Polymerase chain reaction was performed with Pfu DNA
polymerase (Promega, Southampton, UK) and the
frag-ment was ligated into pRSETA (Invitrogen Ltd, Paisley,
UK), followed by expression in E coli cells, inclusion body
extraction and refolding as for proMMP-1 [44,45] Active
MMP-13 was quantitated by BCA assay (Pierce
Biotechno-logy, Inc., Rockford, IL, USA) and stored in the presence
of 10)5m CI-C, to prevent autocatalytic fragmentation
CI-C, a broad-spectrum peptide hydroxamic acid MMP
inhibitor [47] was donated by SmithKline Beecham
Phar-maceuticals (Harlow, UK)
Human proMMP-9 cDNA, including the stop codon,
was subcloned from pCOC-MMP-9 (a kind gift from
G Murphy, University of Cambridge, Cambridge, UK)
into the pIB⁄ V5-His-TOPO expression vector (Invitrogen)
Following sequence confirmation, the pIB-proMMP-9
con-struct was transfected into Hi-5 insect cells (Invitrogen)
grown in serum-free HyClone HyQ SFX-Insect culture
medium (Perbio Science UK Ltd, Cramlington, UK)
Highly expressing clones were identified by gelatine
zymo-graphy [48] and dot-blot, using the anti-MMP-9 rabbit
polyclonal serum from Sigma-Aldrich One litre of culture
supernatant from these cells was adjusted to pH 7.6 with
Tris⁄ HCl Brij-35 and NaN3 were added to 0.025% and
0.02% (w⁄ v), respectively, and proMMP-9 was then
puri-fied by affinity chromatography on gelatine-agarose
(Sig-ma-Aldrich) according to [16] The purified enzyme was
quantitated using the absorbance at 280 nm combined with
the predicted molar extinction coefficient, which was
deter-mined using lasergene, Protean software (DNAStar, Inc,
Madison, WI, USA)
Recombinant TIMP-2 was purified by Ultrogel AcA44
gel filtration and heparin-Sepharose (GE Healthcare UK
Limited, Chalfont St Giles, UK) chromatography from COS cell conditioned medium (supplied by British Biotech, Oxford, UK)
Electrophoretic techniques Reduced or nonreduced (± 0.17 m 2-mercaptoethanol) sam-ples were heat-denatured (105C, 5 min) and analysed by SDS⁄ PAGE on 10% gels, which were silver-stained using a Plus OneTM kit (GE Healthcare UK Limited) Samples analysed by zymography were applied nonreduced and without heat denaturation to 10% gels copolymerized with
1 mgÆmL)1 gelatine Gels were renatured by two 10-min washes in 5% (v⁄ v) Triton X-100 and digested for 15 h at
37C, followed by staining with Coomassie brilliant blue G-250 For western blotting, heat-denatured samples were run-reduced or nonreduced on 10% gels followed by transfer
to nitrocellulose membranes (PROTRAN; Whatman plc, Brentford, UK) Blots were probed with primary antibodies
as appropriate and horseradish peroxidase-conjugated secon-dary antibodies (Dako UK Ltd, Ely, Cambridgeshire, UK), followed by detection of immunoreactive bands by enhanced chemiluminescence (GE Healthcare UK Limited)
Soluble collagen assay for collagenase activity Bovine skin type I collagen was prepared as previously described [49,50]; bovine type III collagen was a generous gift from V Duance (Cardiff University, Cardiff, UK) Mutated type I collagen from murine skin was generously provided by S Krane (Harvard Medical School and Massa-chusetts General Hospital, Boston, MA, USA) This type I collagen is completely resistant to collagenolytic cleavage, due to the mutation of Gln774 (P2) and Ala777 (P¢2) of the
a1(I) chain to proline The wild-type a2(I) chain of each tri-ple helix is also resistant to cleavage, due to the presence of two mutated a1(I) chains [25]
Activity was measured using 27 lgÆsample)1 of collagen
in 50 mm Tris⁄ HCl, pH 7.6, 1 m glucose, 200 mm NaCl,
1 mm CaCl2, 0.02% (w⁄ v) NaN3 In some reactions, the collagen was denatured by incubation at 56C for 30 min prior to the assay Samples were digested for 72 or 98 h at
25, 36 or 37C, followed by electrophoresis on 6.5% SDS⁄ PAGE gels Reactions were visualized by staining with Coomassie brilliant blue G-250 Protease inhibitor cocktail tablets containing serine and cysteine protease inhibitors (Roche Products Ltd, Welwyn Garden City, UK) were used at the manufacturer’s recommended working strength
Acknowledgements
We thank Dr N McKie for critically reviewing the manuscript We are also grateful to Professor S Krane
Trang 8for generously providing the mutated type I collagen.
This work was funded by the Arthritis Research
Cam-paign, UK; The Health Foundation, UK; and the
Dunhill Medical Trust, UK
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