(Received for publication, February 24, 1995; and in revised form, May 29, 1995)
From the
Soluble recombinant human fibroblast collagenase catalytic
domain was highly expressed and purified from Escherichia coli. The expression construct utilized the T7 gene 10 promoter for
transcription of a two-cistron messenger RNA which encoded the
ubiquitin-collagenase catalytic domain fusion protein as the second
cistron. The ubiquitin domain was attached to the collagenase catalytic
domain with the linker sequences Gly-Gly-Thr-Gly-Asp-Val-Ala-Gln (wild
type) or Gly-Gly-Thr-Gly-Asp-Val-Gly-His (mutant) which served as
cleavage sites for in vitro activation. The last four residues
of the linker were included based on the crystal structure of human
prostromelysin-1 catalytic domain. Soluble fusion proteins purified
from E. coli retained the proteolytic activity of the
collagenase catalytic domain. The collagenase catalytic domain was
released by either autoproteolytic or stromelysin-1-catalyzed cleavage,
purified to homogeneity, and separately possess Phe-81, Val-82, or
Leu-83 as the amino-terminal residue. Very similar k/K
values were
determined for the Phe-81 and Val-82 forms using continuous fluorogenic
and chromogenic peptide cleavage assays.
Connective tissue cells are embedded within an extracellular
matrix of glycoproteins. The integrity of this matrix is dependent upon
a balanced rate of cell division, matrix synthesis, and degradation.
Most connective tissue cells, including fibroblasts, chondrocytes,
osteoblasts, and endothelial cells, have been reported to secrete
matrix metalloproteinases (MMPs)()(1) . The MMP
family of enzymes, acting synergistically, can break down all the
components of the extracellular matrix at physiological pH.
Uncontrolled matrix metalloproteinase activity may lead to the loss of
connective tissue integrity and has been implicated in a variety of
connective tissue disorders such as rheumatoid arthritis, tumor
invasion, and metastasis. Thus, MMPs have become targets for
therapeutic intervention in these pathological processes.
Fibroblast
collagenase (MMP-1) belongs to the MMP family and has the unique
ability of cleaving the triple helical native type I, II, and III
collagens(2, 3) . MMP-1 is secreted as a proenzyme
which can be activated by the removal of an autoinhibitory prosequence
containing a conserved cysteine switch region present in all
MMPs(4) . Mature MMP-1 is a 41-kDa protein composed of an
NH-terminal catalytic domain containing the zinc-binding
region of the active site and a COOH-terminal hemopexin-like domain.
Activation in vitro can be facilitated by proteinases (e.g. trypsin, plasmin), oxidants, mercurials, or
stromelysin-1 (MMP-3). Activation of MMP-1 by proteinases, mercurials,
or oxidants produces a variety of active enzymes having either Met-72,
Phe-81, Val-82, or Leu-83 as the NH
-terminal
residue(5, 6, 7, 8, 9, 10) .
The Val-82 and Leu-83 forms accumulate only after extended incubation
with p-aminophenyl mercuric acetate or proteinases. Activation
with MMP-3, however, gives rise to a 41-kDa mature enzyme with Phe-81
as its NH
terminus. This 41-kDa Phe-81 form, with an intact
carboxyl-terminal hemopexin domain, has higher proteolytic activity
against native substrate type I collagen as compared with the partially
activated Val-82 and Leu-83
forms(10, 11, 12, 13) .
The MMP-1 catalytic domain (MMP-1cd) alone can cleave substrates such as casein, gelatin, and synthetic peptide substrates. However, unlike the full-length enzyme, MMP-1cd cannot cleave collagen(14) . And, the missing COOH-terminal hemopexin domain is probably responsible for this difference. With peptide substrates, the catalytic activities of both MMP-1cd and the full-length enzyme are very similar; they differ by only 21%(14) . This suggests that the active site structure of the catalytic domain is an appropriate template for the design of low molecular weight inhibitors. It is intriguing whether the structural differences between the Phe-81 and Val-82 or Leu-83 forms of MMP-1cd will be reflected by any changes in enzyme kinetics. Such understanding may provide additional insight in the design of inhibitors for therapeutic applications.
This paper demonstrates
high level Escherichia coli expression of soluble 19-kDa
rMMP-1cd covering the region between Phe-81 and Gln-249 using the
ubiquitin fusion-protein method. We also report in vitro activation methods used to generate homogeneous Phe-81, Val-82, or
Leu-83 at the NH terminus as well as the purification and
comparative kinetic analysis.
The
expression plasmid pUMColl.1 was constructed by standard methods (18) . The first step involved PCR amplification and cloning of
ubiquitin into the final expression plasmid from an existing ubiquitin
plasmid Pnmhub-poly. The 5`-ubiquitin PCR primer contained a unique NdeI site that also encoded the ATG for translational
initiation. The 3`-ubiquitin PCR primer contained DNA sequence for a
unique KpnI site and a downstream EcoRI cloning site.
PCR mutagenesis was performed for the collagenase portion of the fusion
protein. The 5` primer introduced a silent mutation to create a BstEII site at Gly-86 codon in the MMP-1 coding region. The 3`
primer contained a unique EcoRI cloning site downstream of
Gln-249 codon and the stop codon. This amplified fragment was digested
and ligated with a synthetic KpnI-BstEII linker into
a cloning vector. The final expression construct was made by excising
the KpnI-EcoRI DNA fragment and ligating to the
ubiquitin expression plasmid digested with the same two restriction
enzymes (Fig. 1). The first Gly residue in the linker region was
provided by the ubiquitin coding region at the KpnI site.
Plasmid constructs were confirmed by restriction enzyme mapping and
dideoxynucleotide sequencing(19) . Fusion proteins were
expressed in E. coli strain BL21(DE3) (Novagen, (20) )
which were grown at 37 °C to mid-log phase and induced with 0.2
mM isopropyl-1-thio--D-galactopyranoside for
protein production. Cells were harvested 3 h post-induction.
Figure 1: Map of the expression plasmid pUMColl.1. The open bar represents the first cistron, and the filled bar between NdeI and EcoRI sites denotes the ubiquitin fusion as the second cistron. The ubiquitin region ends with a Gly residue at the KpnI site. Included is sequence of the wild type synthetic linker between ubiquitin and the MMP-1 catalytic domain starting with the second Gly residue.
k/K
was calculated
from the relationship k
/K
= k
/[rMMP-1cd], where
is the active enzyme concentration determined by
the active site titration described below.
Production of human proMMP-1 full-length enzyme in E.
coli has been reported to be problematic as it could only be
accumulated in E. coli either at relatively low levels or as
an insoluble protein sequestered within inclusion
bodies(4, 24) . Expression levels and solubility of
proMMP-1 were improved when the heat shock chaperone protein GroESL or
DnaK was co-expressed in E. coli(24) . Lowry et
al.(25) expressed a recombinant 19 kDa MMP-1cd without
the prosequence in E. coli. Again, it existed only as
inclusion bodies and required refolding to gain solubility and
proteolytic activity(25) . When the collagenase catalytic
domain was initially expressed as a proenzyme (proMMP-1cd) in E.
coli in this laboratory, it was also observed that only low
amounts of insoluble protein could be produced. Clearly a better system
was needed to improve the expression and solubility of proMMP-1cd.
Therefore, expression constructs such as pUMColl.1 (Fig. 1) were
made. pUMColl.1 contains the T7 gene 10 promoter for efficient
transcription. To ensure efficient translation, we applied a
two-cistron approach. The ribosome binding site and the first 20 codons
of the dihydrofolate reductase gene from a trimethoprim-resistant E. coli strain were used as the first cistron (26) followed by the ubiquitin-MMP-1cd fusion as the second
cistron. Yeast ubiquitin was chosen since it had been used as the
NH-terminal leader to produce various soluble fusion
proteins at high levels(16) . The Gly-Gly-Thr-Gly sequence was
chosen as the first half of the linker sequence between the ubiquitin
and rMMP-1cd domains to introduce both flexibility and distance between
the two protein domains and to create a unique KpnI
restriction enzyme site for cloning purposes. Based on the solved x-ray
structure of proMMP-3cd, (
)it was seen that the last four
amino acid residues from the prosequence were exposed to solvent and
susceptible to proteolytic attack. Assuming three-dimensional
structures around this region of MMP-1 and MMP-3 are similar, we chose
to include only the last four residues from the prosequence as the
second half of the cleavage site linker sequence. It was anticipated
that the linker region between two protein domains would be flexible
and susceptible to either autoprocessing or proteolytic attack by
MMP-3cd at the His-Phe bond. As shown in Fig. 2, the ubiquitin
fusion protein was indeed expressed at a higher level than proMMP-1cd
in E. coli. Furthermore, the vast majority of fusion protein
was found in the soluble fraction of crude E. coli lysates.
Figure 2: SDS-polyacrylamide gel electrophoresis analysis of recombinant collagenase expressed in E. coli BL21(DE3). Lane 1, molecular mass markers; lanes 2 and 3 and 4 and 5 are pairs of total E. coli lysates prepared from uninduced and induced cultures bearing the procollagenase and fusion-collagenase expression constructs, respectively. After electrophoresis(22) , the gel was stained with Coomassie Brilliant Blue. Arrowheads indicate the positions of the two recombinant proteins. Molecular masses of markers are indicated in kilodaltons.
Initial purification of the fusion protein containing the wild type
linker sequence showed that in the absence of 1,10-phenanthroline,
fusion protein that eluted off the Q-Sepharose column was able to
undergo uncontrolled autoproteolytic conversion to mature rMMP-1cd.
This autoprocessing event occurred since both the cysteine switch
region and the upstream prosequence were omitted from the fusion
proteins. The fusion protein was also shown to be fully active prior to
autoactivation or proteolytic processing since it cleaved the
Mca-peptide substrate (data not shown). Nevertheless, mature rMMP-1cd
enzymes with different but homogeneous NH-terminal residues
could be generated from the fusion protein containing the last four
wild type residues of the proMMP-1 sequence. Based on
NH
-terminal sequencing, the Phe-81 form was generated by
MMP-3cd-catalyzed activation ( Fig. 3and Table 1).
Approximately 6 mg of purified Phe-81 rMMP-1cd was obtained per liter
of induced E. coli culture. MMP-1cd activated by spontaneous
autoproteolysis generated rMMP-1cd with Val-82 as the predominate
NH
-terminal residue. However, autoactivation at higher
concentrations of fusion protein resulted in accumulation of both
Val-82 and Leu-83 in roughly equal proportions. Thus, enzyme
concentration seems to shift the proteolytic specificity of this
autoprocessing reaction. It is not known whether the Leu-83 NH
terminus is generated by a primary cleavage or from subsequent
cleavage of the Val-82 form. It is noteworthy that low concentrations
of 1,10-phenanthroline were necessary for purifying the Phe-81 rMMP-1cd
to high homogeneity, since it inhibited autoproteolysis and the
accumulation of the Val-82 and Leu-83 forms. The purification scheme
was therefore optimized to obtain the Phe-81 and Val-82 forms to high
homogeneity (Fig. 3). SDS-polyacrylamide gel electrophoresis
analysis of the Phe-81 form purification is shown in Fig. 4.
Figure 3: Purification scheme. Purification schematic yielding rMMP-1cd with Phe-81, Val-82, or Leu-83 as the amino terminus.
Figure 4: SDS-polyacrylamide gel electrophoresis analysis of protein samples collected throughout purification of rMMP-1cd Phe-81 form from E. coli. Lane 1, molecular mass markers; lane 2, soluble fraction of the E. coli lysate; lane 3, pooled Q-Sepharose fractions; lane 4, pooled S-100 HR fractions; lane 5, after activation by rMMP-3cd cleavage; lane 6, pooled zinc chelate column fractions. The single and double arrowheads denote rMMP-1cd and ubiquitin released from the fusion protein after activation, respectively.
The second fusion protein, with the last four amino acid residues
from the prosequence of MMP-3, was made to determine if the chimeric
cleavage site became resistant to autoproteolytic cleavage and thus
could result in higher yields of the Phe-81 form after the MMP-3
activation. During purification, more intact fusion protein was indeed
observed in pooled Q-column fractions indicating a reduced level of
autoproteolysis (data not shown). However, only 80% homogeneity of the
Phe-81 form was attained after MMP-3 activation. On the other hand,
NH-terminal sequencing showed that the purified rMMP-1cd
possessed Leu-83 homogeneously when allowed to autoprocess at 37
°C. The yield of the Leu-83 form was slightly higher than 6
mg/liter of E. coli culture, since its purification did not
require the S-100 column chromatographic step needed for the Phe-81
form. Therefore, with these two fusion proteins, we have generated
three forms of rMMP-1cd: Phe-81, Val-82, and Leu-83. The degree of
homogeneity for each form was determined by NH
-terminal
sequence analysis. The data are summarized in Table 1.
The
carboxyl-terminal integrity of the Phe-81 form of rMMP-1cd was
confirmed by mass spectroscopy. The main component of this sample had a
molecular mass of 18,896.2 Da, which is essentially identical to the
calculated rMMP-1cd (Phe-81 to Gln-249) molecular mass of 18,895.7 Da.
However, two minor components comprising less than 5% of the total mass
had molecular masses of 18,750 and 18,653 Da. These two minor
components correspond to the Val-82 and Leu-83 forms of rMMP-1cd,
respectively. Both the mass spectroscopy and NH terminal
sequence analysis data indicated that purified rMMP-1cd protein
possessed a high degree of homogeneity. This purified rMMP-1cd protein
is stable and can be stored at 12 mg/ml and 4 °C for at least 6
weeks.
Accurate determination of k/K
requires an accurate
measurement of the active enzyme concentration. To address this, enzyme
samples were titrated, under conditions which promote stoichiometric
binding, with the strong competitive inhibitor BB-94. For
stoichiometric binding, it is essential that the enzyme concentration
be kept high and the K
of the titrating ligand
low, since low enzyme concentrations and/or weaker ligands results in
falsely high estimates of the active site concentration. The activity
titration of rMMP-1cd was linear as shown in Fig. 5. The
abscissal intercept of a linear fit gave a cuvette concentration for
the Val-82 form of rMMP-1cd of 1009 nM. The stock
concentration of rMMP-1cd was then calculated to be 778
µM. After three determinations, the stock concentration of
the Val-82 form was 764 ± 25 µM and that of the
Phe-81 form was 448 ± 30 µM.
Figure 5: Active site titration of the Val-82 form of rMMP-1cd. Each data point corresponds to the residual enzymatic activity at each concentration of BB-94. The data were fitted with a straight line, and the concentration of enzyme was calculated by the abscissal intercept. The activity beyond this concentration of BB-94 is indistinguishable from that of the rMMP-1cd-independent degradation of the thioester-peptilide.
k/K
values for both the
Phe-81 and the Val-82 forms of rMMP-1cd were determined by first order
progress curve analysis with both the Mca-peptide and the
thioester-peptilide. rMMP-1cd was fully stable during the collection of
the progress curves. Table 2shows the k
/K
values obtained for the
Mca-peptide and the thioester-peptilide substrates with the two forms
of rMMP-1cd. The k
/K
for
the Mca-peptide shows that Val-82 rMMP-1cd is roughly 19% more active
than the Phe-81 form. With the thioester-peptilide, Val-82 is only 7%
more active than Phe-81. These data show that the activity of the
Phe-81 and Leu-82 forms of rMMP-1cd are very similar. Initially, an
attempt was made to determine the individual values of k
and K
for the Mca-peptide
by saturation analysis. Independent determination of such may have
shown more significant but counteracting effects on these parameters.
However, this was not possible, since the K
significantly exceeded its solubility of 250 µM. For
the same reason, K
could not be determined through
progress curve analysis using the integrated Michaelis-Menten equation.
In the chromogenic assay, the thioester-peptilide K
could not be determined at pH 7.5, since this molecule was quite
unstable and underwent significant spontaneous hydrolysis at this pH.
At greater than 500 µM thioester-peptilide, the rate of
enzyme independent hydrolysis was large, and therefore, partial
saturation could not be reached. It is clear that the K
for both these substrates with rMMP-1cd is large. Hence, the
condition [substrate] K
is satisfied
and progress curves are pseudo-first order.
This report demonstrates that rMMP-1cd could be produced abundantly as part of a soluble ubiquitin fusion protein. The increased solubility precludes the need to refold out of urea or other denaturants and therefore results in a more stable protein. The fusion protein retains proteolytic activity as shown by its ability to catalyze its own release from fusion protein as well as to cleave peptide substrates. This is not surprising since both the cysteine switch region and the upstream prosequence were omitted from the fusion proteins. The presence of these two regions is crucial for maintaining the latency of MMPs in general(4) . High homogeneity of the purified Phe-81 and Val-82 forms of the rMMP-1cd allowed us to perform comparative kinetic studies.
It is important to note that the k/K
values obtained with
Phe-81 and Val-82 MMP-1cd are very similar. But, they differ
significantly from the values previously reported. Knight et
al.(21) , using a full-length MMP-1 purified from NSO
myeloma cells, reported a k
/K
for the Mca-peptide of 14,800 M
s
. This is roughly half the value we
determined for the Phe-81 and Val-82 forms. However, it has been shown
that the MMP-1 catalytic domain is 21% less active than full-length
MMP-1 against the peptide substrate
2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-Arg(14) . Although
the MMP-1 preparations were titrated with tissue inhibitor of
metalloproteases for effective in-study comparative purposes, tissue
inhibitor of metalloprotease concentration was not standardized.
Comparison of resulting data with independent active site titrations
must be made with caution. For the thioester-peptilide, the Weingarten
group used full-length MMP-1 and reported a k
/K
of 26,353 M
s
at pH
6.5(22) , whereas our value is 508,917 M
s
for the Phe-81 form at pH 7.5. The higher pH
in our assays may be partially responsible for the increased k
/K
, since the
thioester-peptide scissile bond is significantly more labile at pH 7.5.
Other factors contributing to the disparity were that the enzyme used
by Weingarten and Feder (22) was full-length and partially
activated by trypsin digestion. Other authors have shown this
activation method to result in partially activatedMMP-1 with an
NH
-terminal extension that is detrimental to activity (9, 10, 11) . It is commonly observed that
full-length MMP-1, as well as other MMPs, are relatively
unstable(27) . An estimate of active enzyme concentration based
on protein content can introduce significant errors in k
/K
calculations.
Concentrations of the active enzymes in the present report were more
accurately determined by active site titration with BB-94, a small
molecular weight synthetic tight binding metalloproteinase inhibitor.
These points together could explain the 20-fold difference in the k
/K
values determined by
Weingarten and us. It is quite possible that the abundant expression
and solubility of our rMMP-1cd has allowed higher purity, stability,
and proteolytic activity when measured with peptide substrates.
Previous reports have shown that MMP-1 with Phe-81 as the
NH-terminal residue has higher collagenolytic activity than
the other
forms(9, 10, 11, 12, 13) .
In some cases, NH
-terminal sequencing has identified the
NH
terminus to be Val-82 or
Leu-83(10, 27) . In others, there was a 15-amino acid
extension on the NH
terminus(10) . All these MMP-1
proteins were full-length 41- or 43-kDa species containing the
hemopexin domain and collagenolytic activity. Murphy et al.(14) showed that the hemopexin domain is responsible for
binding collagen and that the short form is unable to do so. Not only
is the hemopexin domain important for binding but also must be
responsible for local denaturation or unwinding of the collagen triple
helix. The x-ray crystal structure clearly shows that only one strand
of the collagen can fit into the active site of MMP-1(28) . The
short forms of MMP-1 generated in this report do not retain
collagenolytic activity. The loss of collagenolytic activity is not due
to a change in the active site but to a loss of its distant binding
site within the hemopexin domain.
Higher activity was observed with
the Phe-81 form of full-length MMP-1 and primarily with collagen as
substrate. An explanation of the cause of superactivation of MMP-1 has
been proposed based on an observed salt bridge of Phe-79 and Asp-232
which stabilizes the NH terminus in neutrophil
collagenase(29) . Similar interaction between the
NH
-terminal Phe-81 residue and the conserved aspartic acid
has been observed in solved structures of MMP-1cd, MMP-3cd,
and MMP-7(30) . The loss of this salt bridge results in
the disorder of the NH
terminus, which, in turn, may lead
to less efficient catalysis. We considered that if Phe-81 were missing,
the Val-82 residue might contact the catalytic zinc and decrease the k
value. However, the disordered
NH
-terminal section is too short to do so. Besides,
positively charged amine can not form a stable interaction with the
catalytic zinc. The mobility of the disordered NH
termini
may perturb the architecture of the S3 and S4 pockets, thus causing
less favorable contacts with extended substrates which will be
reflected by the elevated K
value.
Knäuper et al.(9) did show 2-fold
higher activity of the Phe-81 form of full-length MMP-1 with a
2,4-dinitrophenyl octapeptide substrate, which has a binding site
presumably reaching the S5 pocket. Clark and Cawston (13) have
shown that the MMP-1 catalytic domain does cleave casein and gelatin.
However, they are not in vivo substrates for
MMP-1(13) . We expect the Phe-81 form of MMP-1cd may have a
slightly higher cleavage rate for these extended substrates than the
Val-82 form. However, the experimental determination of the Michaelis
constant and the K
and k
values for
these substrates will be very difficult and so will further
interpretation of the data.
Willenbrock et al.(31) have recently reported that full-length MMP-3, and
MMP-9 contains only one catalytic zinc atom and not the additional
structural zinc atom detected in the catalytic domain of MMP-1, MMP-3,
MMP-7, and MMP-8(28, 30, 32, 33) .
This indicates that the hemopexin domain may stabilize the catalytic
domain in a different manner, thus eliminating the need of the second
zinc. Although the reason for this difference in metal requirements is
unclear, the catalytic activities of the full-length and truncated
MMP-1 with small peptide substrates are similar as are the inhibition
constants of small molecular weight inhibitors. ()Collagenolytic activity data and the difference in zinc
content indicate that there can be structural differences between the
full-length enzyme and its truncated form. However, with currently
available kinetic and active-site structural information we feel that
the catalytic domain does provide us an adequate model for the design
of low molecular weight inhibitors. Ultimately, the structural study of
full-length MMP-1 will be needed for a full comprehension of this
enzyme. Instability of full-length MMPs has so far hampered such
studies.
Information from kinetic, small molecule inhibition, and structural observations indicate that the active site of rMMP-1cd is not significantly different from that of the full-length form. High level expression of soluble rMMP-1cd as a cleavable ubiquitin fusion protein has allowed for detailed kinetic and crystallographic studies, and a similar expression approach may be used for other proteins that are either poorly expressed or insoluble in E. coli.