(Received for publication, October 16, 1995; and in revised form, November 16, 1995)
From the
Several matrix metalloproteinases, including the 92-kDa and 72-kDa gelatinases, macrophage metalloelastase (MME), and matrilysin degrade insoluble elastin. Because elastolytically active MME and matrilysin consist only of a catalytic domain (CD), we speculated that the homologous CDs of the 92-kDa and 72-kDa gelatinases would confer their elastolytic activities. In contrast to the MME CD, the 92 and 72 CDs expressed in Escherichia coli (lacking the internal fibronectin type II-like repeats) had no elastase activity, although both were gelatinolytic and cleaved a thiopeptolide substrate at rates comparable to the full-length gelatinases. To test the role of the fibronectin type II-like repeats in elastolytic activity, we expressed the 92-kDa gelatinase CD with its fibronectin type II-like repeats (92 CD/FN) in yeast. 92 CD/FN degraded insoluble elastin with activity comparable to full-length 92-kDa gelatinase. 92 and 72 CDs lacking the fibronectin type II-like repeats did not bind elastin, whereas the parent enzymes and 92 CD/FN did bind elastin. Furthermore, recombinant 92-kDa fibronectin type II-like repeats inhibited binding of the 92-kDa gelatinase to elastin. We conclude that the 92- and 72-kDa gelatinases require the fibronectin type II-like repeats for elastase activity.
Elastin is an extracellular matrix protein composed of highly
cross-linked, hydrophobic tropoelastin monomers which provides
resilience to elastic fibers. The hydrophobicity and extensive
cross-linking of tropoelastin monomers result in an insoluble elastic
fiber which is highly resistant to proteolysis(1) . Thus, under
normal physiologic conditions, elastin undergoes minimal
turnover(2) . However, certain pathologic situations, including
pulmonary emphysema (3) and abdominal aortic
aneurysm(4) , are characterized by proteolytic destruction of
elastic fibers. The involvement of serine proteases in such pathologies
has long been suspected. More recently, participation of cysteine
proteinases (5) and matrix metalloproteinases (MMPs, ()6-8) in these diseases has been proposed.
The
MMPs comprise a gene family that collectively is capable of degrading
all components of extracellular matrix in physiologic and pathologic
states(9, 10) . As presently recognized, this family
consists of fibroblast(11) , neutrophil(12) , and
breast carcinoma-derived (13) collagenases, three stromelysins,
92-kDa and 72-kDa gelatinases, macrophage metalloelastase (MME,
MMP-12), matrilysin, and a recently described 66-kDa membrane-type
metalloproteinase(14) . These enzymes are organized into
homologous structural domains, with some differences in domain
composition and number. All members share a zymogen domain, containing
a conserved PRCGXPD motif involved in enzyme latency, and a
zinc-binding CD. Most members also contain a hemopexin-like domain at
their C terminus, the exception being matrilysin, which lacks this
domain completely. Unique to the 72-kDa and 92-kDa gelatinases is an
additional domain composed of three fibronectin type II-like repeats
inserted in tandem within the zinc-binding CD. The 92-kDa gelatinase
also contains an 2(V) collagen-like domain not found in any of the
other family members.
The issue of substrate specificity has received considerable attention recently in MMP biology. The determinants which confer substrate specificity to these enzymes appear to be localized within discrete structural domains. For example, the ability of the collagenases to degrade triple-helical collagen requires the presence of the C-terminal hemopexin-like domain(15, 16, 17) . In contrast, the stromelysins degrade a variety of substrates in a manner which is independent of the C-terminal hemopexin-like domain(16, 18, 19, 20) . Likewise, the C-terminal domain of the 92-kDa and 72-kDa gelatinases is not required for these enzymes to degrade gelatin(21, 22) . However, the fibronectin type II-like repeats within the CDs of the gelatinases confer high affinity binding of these enzymes to gelatin(23, 24, 25, 26) .
Four members of the MMP family have the capacity to degrade insoluble elastin. These are the 92-kDa and 72-kDa gelatinases(27, 28) , macrophage metalloelastase(7, 8) , and matrilysin(28) . In their fully processed active forms, matrilysin and MME consist only of a zinc-binding CD. Because the zinc-binding CDs of other MMPs such as collagenase and stromelysin have been implicated in conferring substrate cleavage specificity, we speculated that the zinc-binding CDs of the 92-kDa and 72-kDa gelatinases might contain all of the necessary elements for elastolytic activity, as do those of matrilysin and MME. In this report, we tested various mutants of the 92-kDa and 72-kDa gelatinases for elastolytic activity. We found that gelatinase mutants consisting only of their respective zinc-binding CDs, lacking both the fibronectin type II-like and C-terminal domains, neither bound to, nor degraded, insoluble elastin. Notably, these mutants degraded other substrates cleaved by the parent enzymes. Inclusion of the fibronectin type II-like repeats within the CD of the 92-kDa gelatinase fully restored its elastolytic activity. We conclude that the structural determinants required for elastin cleavage by the gelatinases are distinct from those of matrilysin or MME.
The resulting 92 CD construct in pET-14b was transformed into the E. coli BL21(DE3) strain (Novagen) for expression. Colonies
were grown in 10 ml of LB media containing 50 µg/ml ampicillin to
log phase and induced with O.4 mM isopropyl-1-thio--D-galactoside for 4 h. After
centrifugation, the pellet was resuspended in 2.5 ml of 50 mM Tris, pH 7.5, 10 mM CaCl
, 30 mM NaCl
and sonicated 5
15 s on ice. 8 M deionized urea in the
same buffer was added to a final concentration of 6 M, and the
extract was rocked gently at 4 °C overnight, prior to
centrifugation at 12,000
g for 10 min in a Sorvall
SS34 rotor. The sample was dialyzed successively against 4 M,
2 M, and 1 M urea in the same buffer containing 20
µM ZnCl
and 0.05% Brij and finally against
urea-free buffer (50 mM Tris, pH 7.5, 10 mM CaCl
, 30 mM NaCl, 0.05% Brij) in which
ZnCl
was omitted. The enzyme was purified over a 1-ml zinc
chelate chromatography column, and bound material was eluted in
equilibration buffer containing 0.1 M imidazole. Imidazole was
removed by further dialysis against equilibration buffer.
Expression constructs were transformed into P.
pastoris strain GS115 by the spheroplasting method as described by
the manufacturer. Colonies were screened for high level protein
expression and secretion by Western analysis upon induction with
methanol. Briefly, yeast clones were grown to a high density in minimal
glycerol medium for 2 days, then shifted to culture in 1/5 volume of
inducing minimal methanol complex media, containing 0.5-5%
methanol, and allowed to grow for an additional 2-4 days.
Conditioned yeast media were collected, equilibrated to gelatin column
buffer (10 mM Tris, pH 7.5, 5 mM CaCl,
150 mM NaCl), and 92-kDa gelatinase proteins were purified by
affinity chromatography over a gelatin-agarose column (Sigma).
Gelatin-agarose columns were loaded with sample, washed with 10 volumes
of column buffer followed by 20 volumes of high salt column buffer (10
mM Tris, pH 7.5, 5 mM CaCl
, 1 M NaCl). Bound protein was eluted with high salt column buffer
containing 10% dimethyl sulfoxide (v/v). Eluted fractions were analyzed
for protein by Western blot. Fractions containing the appropriate
proteins were pooled and dialyzed to completion against elastin assay
buffer (50 mM Tris, pH 7.5, 10 mM CaCl
,
150 mM NaCl, 0.02% Brij) containing 20 µM ZnCl
. The 92 CD/FN was activated using truncated
stromelysin as described previously(32) .
Figure 1: Alignment of the peptide sequences of the 92-kDa gelatinase, the 72-kDa gelatinase, MME, and interstitial collagenase CDs. The peptide sequences of the 92-kDa gelatinase, 72-kDa gelatinase, MME, and interstitial collagenase CDs were aligned using the Geneworks program (Intelligenetics). Residues of identity among the four enzymes are boxed. Gaps are indicated by dashes. The Met-Gly dipeptide (MME CD and 92 CD) and the Met-Ala-Ser tripeptide (72 CD) that were introduced to initiate translation of the CDs are not shown. The fibronectin type II-like repeats located within the 92 CD and 72 CD have been deleted, and their normal position within the gelatinase CDs is indicated. The percent identity between CDs are as follows: overall (92 CD versus 72 CD versus MME CD versus collagenase CD), 37%; 92 CD versus 72 CD, 60%; 92 CD versus MME CD, 49%; 92 CD versus collagenase CD, 54%; 72 CD versus MME CD, 56%; 72 CD versus collagenase CD, 53%; MME CD versus collagenase CD, 55%.
Figure 2: SDS-PAGE of the MME CD, 92 CD, and 72 CD expressed in E. coli. The CDs of the 92-kDa gelatinase, the 72-kDa gelatinase, and MME were expressed in E. coli and purified as described under ``Materials and Methods.'' The predicted molecular masses are 20194, 18428, and 19154 daltons for the MME CD (lane 13), 92 CD (lane 2), and 72 CD (lane 3), respectively. The minor band in lane 3 represents autolytic degradation of the 72 CD.
Figure 3:
Cleavage of a synthetic thiopeptolide
substrate by the 92 CD, 72 CD, MME CD, and the full-length gelatinases.
Thiopeptolide assays were carried out at an enzyme concentration of 5
10
M as described under
``Materials and Methods.'' The
A
was measured every minute up to 5 min, and the assay was linear
up to 20 min. Results shown represent the mean and standard deviations
of triplicate determinations in a single
experiment.
A second substrate tested was gelatin prepared from type I collagen. Although the fibronectin type II-like repeats of the gelatinases have been implicated in gelatin binding, it has been shown that removal of these repeats from the 72-kDa gelatinase results in an enzyme with reduced, but detectable, gelatinolytic activity(24) . Similarly, the 92-kDa gelatinase actively degrades gelatin in the presence of 10% dimethyl sulfoxide, which disrupts the binding of the fibronectin type II-like domain to gelatin(23) . However, in each of these studies, the gelatinase constructs contained intact carboxyl-terminal domains. Therefore, we assessed whether the 92 CD and 72 CD, which lack both the fibronectin type II-like domains and the carboxyl-terminal domains, were capable of degrading gelatin. Fig. 4A demonstrates that both CDs were readily capable of digesting gelatin, although the activity of the 92 CD appeared greater than that of the 72 CD. The gelatinolytic activity was specific to the gelatinase CDs, as it was completely inhibited by TIMP-1 (data not shown). As reported for the 72 CD relative to the full-length 72-kDa gelatinase(25) , the 92 CD exhibits a reduced capacity to degrade gelatin relative to the full-length activated 92-kDa gelatinase (Fig. 4B). The 92 CD appears to be 20-30% as active as the full-length 92-kDa gelatinase. However, inclusion of the fibronectin type II-like repeats into the 92 CD restored full gelatinolytic activity (data not shown). In fact, this construct is more active against gelatin than the full-length 92-kDa gelatinase.
Figure 4:
Digestion of type I gelatin by the 92 CD,
72 CD, and the 92-kDa gelatinase. A, digestion of type I
gelatin by the 92 CD and 72 CD. The 92 CD and 72 CD (6.5
10
M) were incubated with 10 µg of
gelatin for 20 min (lanes 1 and 4), 40 min (lanes
2 and 5), and 60 min (lanes 3 and 6) as
described under ``Materials and Methods.'' Reaction aliquots
were boiled in sample buffer and subjected to SDS-PAGE on a 10% gel.
Bands were visualized by Coomassie staining. Digestion of gelatin was
fully inhibited by preincubation with TIMP-1 (not shown). B,
relative gelatinase activities of the 92 CD and the 92-kDa gelatinase.
10 µg of gelatin was digested for 20 min using 2
10
M (lanes 1 and 4), 0.4
10
M (lanes 2 and 5), and 0.08
10
M (lanes 3 and 6) of either the 92 CD or the
activated full-length 92-kDa gelatinase.
Finally, to determine whether the CDs of the gelatinases were elastolytic, the CDs were tested for their ability to degrade insoluble elastin. Although the native gelatinases and the MME CD were elastolytic, both the 92 CD and 72 CD were completely inactive against insoluble elastin (Fig. 5). Even very high concentrations of the gelatinase CDs (in excess of 100 µg/ml) produced only barely detectable activity against elastin (data not shown).
Figure 5:
Degradation of H-insoluble
elastin by full-length MMPs and MMP CDs. Elastase assays were for
1-5 h at 37 °C as described under ``Materials and
Methods.'' The 92-kDa gelatinase and 92 CD/FN were activated with
truncated stromelysin (1:100, mol/mol) as described(32) . The
truncated stromelysin had no activity against elastin. In some
experiments (not shown), the 92 CD and 72 CD were assayed at
concentrations as high as 5
10
M (
100 µg/ml) to confirm the absence of elastase activity.
Standard deviations from three to six different experiments are shown.
The data for the 92 CD/FN represents the average of duplicate
measurements of two separate preparations.
The inability of the gelatinase CDs to degrade insoluble elastin distinguished them from the CDs of MME and matrilysin and raised the question of what domains of the parent molecules, other than the zinc-binding CD, are required for elastolytic activity. To investigate this question, we restored the fibronectin type II-like repeats to the CD of the 92-kDa gelatinase (92 CD/FN). When expressed in E. coli, this construct was inactive against all substrates tested. We speculate that the lack of activity was due to improper protein folding in E. coli because the three fibronectin type II-like repeats contain a total of 12 disulfide-bonded cysteine residues. To circumvent this problem, a similar construct containing the propeptide, catalytic, and fibronectin type II-like domains was expressed as a soluble secreted protein in a yeast system. The secreted enzyme was activated by removal of the propeptide with a small amount of a truncated form of stromelysin which activates the full-length 92-kDa gelatinase(32) , but has no elastolytic activity of its own (data not shown). The activity of the 92 CD/FN protein against insoluble elastin is shown in Fig. 5. Notably, the 92 CD/FN mutant has elastolytic activity comparable to that of full-length 92-kDa gelatinase. The elastase activity was specific to the 92 CD/FN because conditioned medium from nonexpressing yeast clones purified in parallel with the 92 CD/FN had no activity against elastin, and the observed elastolytic activity from expressing clones was inhibitable by TIMP-1 (data not shown).
Figure 6:
Binding of the gelatinases to elastin
occurs through the fibronectin type II-like repeats. A,
binding of elastolytic versus nonelastolytic MMPs to insoluble
elastin. Binding was carried out as described under ``Materials
and Methods'' using 5 10
M enzyme. The thiopeptolide activity of unbound enzyme in the
supernatant relative to that of the total amount of enzyme added was
used to determine the percent binding. Results and standard deviations
of 3-4 experiments are shown. B, inhibition of
gelatinase binding by the fibronectin type II-like repeats. Binding
assays with the 92-kDa gelatinase as well as the MME CD were carried
out in the presence or absence of exogenous recombinant fibronectin
type II-like repeats of the 92-kDa gelatinase. The fibronectin type
II-like repeats (6.6
10
M, 13-fold
excess over enzyme) were preincubated with elastin prior to the
addition of enzyme as described under ``Materials and
Methods.''
, -fibronectin type II-like repeats;
&cjs2113;, +fibronectin type II-like
repeats.
The property of degrading insoluble elastin is restricted to select members of the MMP family. Previous studies have demonstrated that interstitial collagenases and stromelysins have virtually no elastolytic activity, whereas the 92-kDa and 72-kDa gelatinases, MME, and matrilysin are elastolytic(7, 8, 27, 28) . However, the overall amino acid homology among the CDs of the elastolytic MMPs does not distinguish them from the nonelastolytic members of the family (Fig. 1). In fact, the elastolytic MMPs are as similar to fibroblast collagenase, a nonelastolytic enzyme, as they are to each other. Consequently, regions of these enzymes which may be involved in elastin binding and degradation are not readily apparent by inspection.
Because MME and matrilysin in their activated forms are functional elastases consisting only of the typical catalytic zinc-binding domain, we speculated that the CDs of the 92-kDa and 72-kDa gelatinases would be functional against elastin. Accordingly, we expressed constructs in E. coli encoding the CDs of the 92-kDa gelatinase, the 72-kDa gelatinase, and MME. The gelatinase CDs lacking the fibronectin type II-like repeats which split the CD in the native enzymes were devoid of elastase activity although they did display catalytic activity against both a synthetic thiopeptolide substrate and gelatin, indicating that they were enzymatically active. The MME CD expressed in the same system was elastolytically active, showing that this activity can be reconstituted in an E. coli expression system. Restoration of the fibronectin type II-like repeats into the CD of the 92-kDa gelatinase restored elastin-binding and elastin-degrading activity. This finding indicated that the fibronectin type II-like repeats in the 92-kDa gelatinase are necessary for the elastolytic activity of this enzyme. It also revealed that the carboxyl-terminal type V collagen-like and hemopexin-like domains of the native enzyme are not required for elastase activity. These carboxyl-terminal domains are also not required for gelatin degradation by either of the gelatinases(21, 22) .
It may be argued that the CDs of the gelatinases contain the elements necessary for elastin binding and degradation, and that removal of the fibronectin type II-like domain results in a steric alteration which prohibits these elements from acting in concert. We believe this scenario is unlikely for the following reasons. First, the CDs retain activity on other substrates. More importantly, exogenous fibronectin type II-like domains strongly inhibit binding of the full-length enzyme to elastin. These data, coupled with the observation of Steffensen et al.(36) that the fibronectin type II-like repeats of the 72-kDa gelatinase bind directly to elastin, strongly suggest that the gelatinases bind elastin through the fibronectin type II-like domain.
The requirement of the fibronectin type II-like domains for elastase
activity was unexpected, but there is precedence for the participation
of these repeats in matrix binding. Several investigators have shown
that these repeats confer high affinity binding to type IV collagen and
type I gelatin(23, 24, 25, 26) .
However, there is some question as to whether gelatin binding through
the fibronectin type II-like domain is rate-limiting for catalysis. A
72-kDa gelatinase mutant lacking the fibronectin type II-like repeats
had only 10% the gelatinolytic activity of the native enzyme,
suggesting that this was a rate-limiting event(24) . Likewise,
the 72 CD also has a reduced ability to degrade gelatin relative to the
native enzyme(25) . This result is in contrast to the data of
Collier et al.(23) regarding the 92-kDa
gelatinase(23) . These investigators demonstrated that
MeSO concentrations which inhibit >90% of the binding of
the recombinant fibronectin type II-like domain to gelatin inhibit only
20% of its gelatinolytic activity, suggesting that binding of the
92-kDa gelatinase to gelatin through the fibronectin type II-like
repeats is not rate-limiting for catalysis. We found that the 92 CD has
only 20-30% the gelatinolytic activity of the full-length
gelatinase (Fig. 4B). However, restoration of the
fibronectin type II-like repeats resulted in gelatinase specific
activity which was greater than that of the full-length 92-kDa
gelatinase (data not shown). These data suggest that gelatin binding
through the fibronectin type II-like domain is rate-limiting for
catalysis.
With respect to elastin, the ability of the fibronectin type II-like repeats to play a role in elastase activity received support from a recent study showing that the fibronectin type II-like repeats of the 72-kDa gelatinase themselves bind elastin with high affinity(36) . In this report, we present evidence indicating that the fibronectin type II-like repeats of the 92-kDa gelatinase bind elastin. First, the CD of this enzyme lacking these repeats does not bind to elastin. Second, the CD containing the repeats both binds to and degrades elastin. Third, exogenous recombinant 92-kDa gelatinase fibronectin type II-like repeats inhibit the binding of the 92-kDa gelatinase to elastin. Collier et al.(23) demonstrated that the second fibronectin type II-like repeat of the 92-kDa gelatinase is responsible for most of the gelatin binding and speculated that the other repeats may be involved in binding to other matrix substrates. We are currently investigating the role of the individual repeats of the 92-kDa gelatinase in elastin binding.
The necessity of the fibronectin type II-like repeats of
the gelatinases for elastolytic activity indicates that the gelatinases
differ from MME and matrilysin in their mechanism of elastolysis.
Moreover, the inability of exogenous fibronectin type II-like repeats
to inhibit binding of the MME CD to elastin while they do inhibit
binding of the 92-kDa gelatinase suggests that the gelatinases and MME
bind to different sites on the elastin molecule. Interestingly, the
92-kDa gelatinase and MME have different cleavage site preferences
within insoluble elastin. ()The binding of the gelatinases
to elastin through the fibronectin type II-like repeats is an
attractive model since the repeats interrupt the CD immediately
adjacent to the active site, thereby potentially bringing the active
site into close proximity with the substrate.
In conclusion, these studies reveal unexpected complexity in the domain requirements of MMPs having elastolytic activity. There appear to be two classes of elastolytic MMPs: 1) macrophage metalloelastase and matrilysin that require only the CD for elastin binding and degradation, and 2) the gelatinases which require the fibronectin type II-like repeats for elastin binding. Thus, structure/function relationships that apply to one elastolytic MMP cannot be assumed to apply to other elastolytic MMPs.