©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Structural Basis for the Elastolytic Activity of the 92-kDa and 72-kDa Gelatinases
ROLE OF THE FIBRONECTIN TYPE II-LIKE REPEATS (*)

(Received for publication, October 16, 1995; and in revised form, November 16, 1995)

J. Michael Shipley (1) Glenn A. R. Doyle (1) Catherine J. Fliszar (2) Qi-Zhuang Ye (4) Linda L. Johnson (4) Steven D. Shapiro (1) (3) Howard G. Welgus (2) Robert M. Senior (1)(§)

From the  (1)Respiratory and Critical Care Division and the (2)Division of Dermatology, Departments of Medicine and (3)Cell Biology and Physiology, Washington University School of Medicine at Jewish Hospital, St. Louis, Missouri 63110 and the (4)Department of Biochemistry, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48105

ABSTRACT
INTRODUCTION
MATERIALS and METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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, (^1)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 alpha2(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.


MATERIALS and METHODS

Reagents

Bovine ligament elastin, obtained from Elastin Products, Owensville, MO, was radiolabeled with [^3H]sodium borohydride (DuPont NEN) to a specific activity of 1000 cpm/µg (29) . Isopropyl-1-thio-beta-D-galactoside, heparin agarose, gelatin agarose, 5,5`-dithiobis-(2-nitrobenzoic acid) (DTNB), trypsin, soybean trypsin inhibitor, and Luria broth (LB) were from Sigma. Ac-Pro-Leu-Gly-S-Leu-Leu-Gly-OEt (thiopeptolide) was obtained from Bachem Bioscience, King of Prussia, PA. 72-kDa gelatinase, recombinantly expressed in vaccinia virus and free of TIMP-2(30) , was kindly provided by W. Stetler-Stevenson, NCI, National Institutes of Health, Bethesda, MD. Bovine dermal type I collagen (Vitrogen 100) was from CELLTRIX, Palo Alto, CA. The Pichia pastoris expression system was obtained from Invitrogen. Truncated stromelysin-1 was prepared by expression of the CD in Escherichia coli as described previously(20) . TIMP-1 was from Synergen (Boulder, CO). The BCA reagent for determining protein concentration was from Pierce.

Expression, Purification, and Activation of the Full-length 92-kDa Gelatinase

92-kDa gelatinase was expressed in E1A- and 92-kDa gelatinase-transfected fibroblasts and purified to homogeneity as described previously(31) . In this system, the 92-kDa enzyme is obtained free of TIMP-1. The 92-kDa progelatinase was activated with a truncated version of stromelysin-1 as previously reported(32) . Full conversion of the 92-kDa gelatinase to its active form was assessed by SDS-PAGE. Full catalytic activity was established by assays against gelatin and elastin as compared to previous batches of the enzyme.

Activation of the 72-kDa Gelatinase

The 72-kDa progelatinase was activated with 1 mM aminophenylmercuric acetate at 37 °C for 2 h(33) . Complete conversion of the 72-kDa proenzyme to its active form was assessed by SDS-PAGE.

Construction of the CDs of the 92-kDa Gelatinase, 72-kDa Gelatinase, and MME

92 CD

The fibronectin type II-like repeats of the 92-kDa gelatinase, which split the CD, were deleted by recombinant PCR(34) . The region of the CD 5` to the fibronectin type II-like repeats was amplified with the primers 5`-CTGTGCCATGGGATTCCAAACCTTTGAG-3` and 5`-CGAGGAACAAACTGTATCCGACGCCCTTGCCCAGGG-3`. The introduction of the NcoI site in the 5` primer created an additional Met-Gly dipeptide at the N terminus in order to start translation at this point within the cDNA. The region 3` to the fibronectin type II-like repeats was amplified with the primers 5`-CCCTGGGCAAGGGCGTCGGATACAGTTTGTTCCTCG-3` and 5`-CAACTCTCGAGTCAACCATAGAGGTGCCG-3`. A stop codon was placed in the 3` primer after residue 443, thereby eliminating the collagen type V and hemopexin-like domains. After gel purification of both PCR products, the products were annealed to each other by virtue of the complementary design of the internal PCR primers and amplified using the outside primers. The final PCR product was digested with NcoI and XhoI and subcloned into the pET-14b vector (Novagen, Madison, WI) for expression in E. coli. The resulting fragment coded for a protein lacking residues 217-390 of the parent molecule which encompass the three fibronectin type II-like repeats. Thus, the 92 CD contained residues 107-216 of the parent molecule fused to residues 391-443. Constructs were confirmed by DNA sequencing.

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-beta-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(2), 30 mM NaCl and sonicated 5 times 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 times 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(2) and 0.05% Brij and finally against urea-free buffer (50 mM Tris, pH 7.5, 10 mM CaCl(2), 30 mM NaCl, 0.05% Brij) in which ZnCl(2) 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.

72 CD

The 72 CD was expressed in E. coli as described recently(25) . Briefly, a synthetic gene coding for the 72 CD (lacking the fibronectin type II-like repeats) was generated by PCR, inserted into the vector pGEMEX-1, and used to transform the E. coli strain BL21(DE3)pLysS (Novagen, Madison, WI). This construct contained an additional Met-Ala-Ser tripeptide at the amino terminus to initiate translation of the synthetic gene, and the methionine was removed during E. coli expression. The resulting protein contained residues 110-213 of the parent molecule fused to residues 387-444.

MME CD

The MME CD was amplified with the primers 5`-CTTGCCATGGGTCTGCGTGCAGTGCCCCAGAGGTCA-3` and 5`-TACGGGATCCTTATGTCAAGGATGGGGGTTT-3` containing NcoI and BglII sites, respectively, for subcloning into the pET-14b expression vector. The introduction of the NcoI site in the 5` primer creates an additional Met-Gly dipeptide at the amino terminus in order to start translation at this point within the cDNA. The 3` primer replaces Lys with a stop codon, eliminating the carboxyl-terminal hemopexin-like domain. The resulting protein contains residues 93-265 of the parent enzyme. This protein was expressed and purified as described for the 92 CD except that the zinc chelate chromatography step was replaced by chromatography over a 1-ml heparin-agarose column. The MME CD was eluted in a buffer containing 50 mM Tris, pH 7.5, 10 mM CaCl(2), 1 M NaCl, 0.05% Brij and dialyzed against the same buffer containing 30 mM NaCl.

Expression of the CD of the 92-kDa Gelatinase Containing the Fibronectin Type II-like Repeats (92 CD/FN) and the Fibronectin Type II-like Repeats Alone in Yeast

PCR was used to generate the CD of the 92-kDa gelatinase containing the fibronectin type II-like repeats as well as the repeats themselves. The 92 CD/FN (amino acids 18-444) was amplified using the forward primer 5`-CTACGGAATTCGCTGCCCCCAGAC-3` and reverse primer 5`-CTACGGAATTCAACCATAGAGGTGCCG-3`. Both primers contained EcoRI sites for subcloning. The reverse primer incorporated a conversion of Pro to a stop codon. The fibronectin type II-like repeats alone (amino acids 217-391) were amplified with the forward primer 5`-CTTCGCTCGAGGATTCCAAACCTTTGAG-3` and the reverse primer 5`-CAACTCTCGAGTCATTGGTCCGGGCAGAA-3`. Both primers incorporate XhoI sites for subcloning, and the reverse primer incorporates the conversion of Gly to a stop codon. The PCR products were subcloned into the pHIL-S1 yeast expression vector. The 92 CD/FN contains an additional Arg-Glu dipeptide at its N terminus after processing of the yeast acid phosphatase signal sequence, and the fibronectin type II-like repeats contain an additional amino-terminal Arg residue.

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(2), 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(2), 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(2), 150 mM NaCl, 0.02% Brij) containing 20 µM ZnCl(2). The 92 CD/FN was activated using truncated stromelysin as described previously(32) .

Assays

Protein

Protein concentrations were determined using the BCA method.

Elastase Activity

Elastase activity was determined by quantifying the solubilization of insoluble ^3H-labeled elastin(29) . Reactions were carried out at 37 °C in 50 mM Tris HCl, pH 7.5, 10 mM CaCl(2), 150 mM NaCl, 0.02% Brij, in a final volume of 100 µl with an excess of elastin (70 µg). Following centrifugation, radioactivity released into the supernatant was collected and counted. Buffer blanks were subtracted to determine net values. Initial velocities were calculated in ranges which were linear over time and enzyme concentration. When assaying 92-kDa gelatinase and 92 CD/FN, blanks also contained stromelysin-1 which was used to activate the gelatinase. Stromelysin had no activity in these assays. For assays of 72 CD, the buffer included 1 µM ZnCl(2)(25) . The addition of 1 µM ZnCl(2) was necessary to retard autocatalytic degradation of the enzyme.

Hydrolysis of Ac-Pro-Leu-Gly-S-Leu-Leu-Gly-OEt (Thiopeptolide)

Hydrolysis of the thiopeptolide substrate was determined as described previously(35) . Accordingly, 7 mg of the thiopeptolide substrate was dissolved in 50 µl of methanol and brought to 1 ml by the addition of 950 µl of assay buffer (0.05 M HEPES, pH 7.0, 0.01 M CaCl (2)). DTNB, 40 mg, was dissolved in 2 ml of ethanol and brought up to 10 ml with assay buffer. Reaction mixtures contained 100 µl each of enzyme solution (5 times 10M) and DTNB and 10 µl of the thiopeptolide substrate in a total volume of 1 ml. Reactions were run at room temperature and monitored at A (Gilford RESPONSE) for the first 5 min after the addition of enzyme.

Hydrolysis of Gelatin

Type I collagen was dialyzed against 400 mM NaCl and then denatured by heating at 60 °C for 15 min. To determine the gelatinolytic activity of the 92 CD and 72 CD, enzymes (6.5 times 10M) were incubated at 37 °C for periods up to 60 min with 10 µg of gelatin in a final volume of 60 µl containing 50 mM Tris HCl, pH 7.5, 10 mM CaCl(2), 400 mM NaCl, 0.001 mM ZnCl(2). For comparisons of the full-length 92-kDa gelatinase with the 92 CD and 92 CD/FN, enzymes (2 times 10M, 0.4 times 10M, and 0.08 times 10M) were incubated with 10 µg of gelatin for 20 min in the same buffer. Aliquots of the reaction mixtures were mixed with sample buffer and dithiothreitol and subjected to SDS-PAGE, after which gels were stained with Coomassie Blue. In some experiments, the enzymes were preincubated (37 °C, 15 min) with TIMP-1 at 5 times the molar concentration of the enzyme.

Binding of Full-length MMPs and CD Mutants to Insoluble Elastin

To determine binding to elastin, full-length enzymes or CD mutants (5 times 10M) were mixed with 63 µg of insoluble elastin for 10 min at room temperature in 50 mM Tris, pH 7.5, 10 mM CaCl(2), 150 mM NaCl, 0.02% Brij in a volume of 120 µl, after which the mixture was centrifuged for 30 s, and the supernatant was removed and analyzed for activity against the thiopeptolide substrate as described above. The thiopeptolide activity of the supernatant was compared to the thiopeptolide activity in control reaction mixtures in which the enzyme was not exposed to insoluble elastin. Binding to elastin was expressed as the percent reduction in thiopeptolide activity produced by incubation with elastin. The fibronectin type II-like repeats of the 92-kDa gelatinase (6.6 times 10M, 13-fold excess over enzyme) were included in some experiments. In these experiments, the repeats were preincubated with elastin for 10 min at room temperature prior to the addition of enzyme.


RESULTS

Comparison of the CDs of Elastolytic and Nonelastolytic MMPs

An alignment of the amino acid residues of the MME CD, the gelatinase CDs (lacking the fibronectin type II-like repeats), and the CD of collagenase is shown in Fig. 1. The CDs of the elastolytic MMPs (MME and the gelatinases) share no more homology with each other (49-60%) than they do with fibroblast collagenase (53-55%), a nonelastolytic MMP. Consequently, regions within the CDs that might be involved in elastin-degrading functions are not inherently obvious by inspection of these primary sequences.


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%.



Expression and Purification of Recombinant Proteins

The purified 92 CD, 72 CD, and MME CD proteins expressed in E. coli are shown in Fig. 2. Purification as described under ``Materials and Methods'' yielded a protein band of M(r) 20,000 for each construct.


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.



Enzymatic Activity of CD Mutants

To determine whether the CD mutants purified and reconstituted from E. coli were enzymatically competent and elastolytic, three substrates were used. First, we employed a previously described thiopeptolide substrate assay (35) . Fig. 3demonstrates that recombinantly expressed 92 CD, 72 CD, and MME CD are at least as active against the thiopeptolide substrate as the full-length enzymes.


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 times 10M as described under ``Materials and Methods.'' The DeltaA 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 times 10M) 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 times 10M (lanes 1 and 4), 0.4 times 10M (lanes 2 and 5), and 0.08 times 10M (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 ^3H-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 times 10M (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).

Binding of the Gelatinases to Elastin Occurs through the Fibronectin Type II-like Repeats

To determine whether the lack of elastase activity of the 92 CD and 72 CD proteins related to elastin binding, we compared the elastin binding potential of these constructs with that of the full-length parent proteins (Fig. 6). As shown in Fig. 6A, the full-length 92-kDa and 72-kDa gelatinases, as well as the MME CD, demonstrated substantial binding to insoluble elastin. In contrast, the 92 CD and 72 CD showed virtually no binding to insoluble elastin. Interstitial collagenase, a MMP devoid of elastase activity, also did not bind to elastin in this assay. This suggested that binding of the gelatinases, but not of MME, to elastin might require the fibronectin type II-like or carboxyl-terminal domains. Steffensen et al.(36) recently demonstrated that the 72-kDa gelatinase fibronectin type II-like domains bind to elastin. Indeed, inclusion of the fibronectin type II-like repeats in the 92 CD (92 CD/FN) restores binding to elastin. To further investigate the role of the fibronectin type II-like repeats in elastin binding, we expressed the tandem repeats of the 92-kDa gelatinase in a yeast system. Fig. 6B shows that exogenously added recombinant fibronectin type II-like repeats inhibited binding of the full-length 92-kDa gelatinase to elastin, while having little effect on the binding of the MME CD. We conclude that the fibronectin type II-like repeats of the 92-kDa gelatinase participate in the binding of this enzyme to insoluble elastin, and that MME and the gelatinases represent two distinct classes of elastolytic MMPs.


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 times 10M 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 times 10M, 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.




DISCUSSION

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 Me(2)SO 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. (^2)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.


FOOTNOTES

*
This work was supported by Grants RO-1 HL47328 and PO-1 HL29594 from NHLBI, National Institutes of Health, the Montsanto-Searle/Washington University Biomedical Agreement, a Research Fellowship (to J. M. S.) and a Career Investigator Award (to S. D. S.) from the American Lung Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Medicine, Jewish Hospital, 216 South Kingshighway, St. Louis, MO 63110. Tel.: 314-454-7113; Fax: 314-454-8605.

(^1)
The abbreviations used are: MMP, matrix metalloproteinase; MME CD, murine macrophage elastase catalytic domain; DTNB, 5,5`-dithiobis-(2-nitrobenzoic acid); FN, fibronectin; 92 CD, 92-kDa gelatinase catalytic domain; 92 CD/FN, 92-kDa gelatinase catalytic domain containing the three fibronectin type II-like repeats; PCR, polymerase chain reaction; 72 CD, 72-kDa gelatinase catalytic domain; TIMP, tissue inhibitor of metalloproteinases; PAGE, polyacrylamide gel electrophoresis.

(^2)
R. Mecham and R. Senior, manuscript in preparation.


REFERENCES

  1. Mecham, R. P., and Heuser, J. E. (1991) in Cell Biology of Extracellular Matrix (Hay, E. D., ed) pp. 79-109, Plenum Press, New York
  2. Shapiro, S. D., Endicott, S. K., Province, M. A., Pierce, J. A., and Campbell, E. J. (1991) J. Clin. Invest. 87, 1828-1834 [Medline] [Order article via Infotrieve]
  3. Janoff, A. (1985) Am. Rev. Respir. Dis. 132, 417-433 [Medline] [Order article via Infotrieve]
  4. Thompson, R. W., Holmes, D. R., Mertens, R. A., Liao, S., Botney, M. D., Mecham, R. P., Welgus, H. G., and Parks, W. C. (1995) J. Clin. Invest. 96, 318-326 [Medline] [Order article via Infotrieve]
  5. Chapman, H. A., Jr., Munger, J. S., and Shi, G.-P. (1994) Am. J. Respir. Crit. Care Med. 150, S155-S159
  6. Senior, R. M., Connolly, N. L., Cury, J. D., Welgus, H. G., and Campbell, E. J. (1989) Am. Rev. Respir. Dis. 139, 1251-1256 [Medline] [Order article via Infotrieve]
  7. Shapiro, S. D., Griffin, G. L., Gilbert, D. J., Jenkins, N. A., Copeland, N. G., Welgus, H. G., Senior, R. M., and Ley, T. J. (1992) J. Biol. Chem. 267, 4664-4671 [Abstract/Free Full Text]
  8. Shapiro, S. D., Kobayashi, D. K., and Ley, T. J. (1993) J. Biol. Chem. 268, 23824-23829 [Abstract/Free Full Text]
  9. Birkedal-Hansen, H., Moore, W. G., Bodden, M. K., Windsor, L. J., Birkedal-Hansen, B., DeCarlo, A., and Engler, J. A. (1993) Crit. Rev. Oral Biol. Med. 4, 197-250 [Abstract]
  10. Murphy, G., and Docherty, A. J. P. (1992) Am. J. Respir. Cell Mol. Biol. 7, 120-125 [Medline] [Order article via Infotrieve]
  11. Goldberg, G. I., Wilhelm, S. M., Kronberger, A., Bauer, E. A., Grant, G. A., and Eisen, A. Z. (1986) J. Biol. Chem. 261, 6600-6605 [Abstract/Free Full Text]
  12. Hasty, K. A., Pourmotabbed, T. F., Goldberg, G. I., Thompson, J. P., Spinella, D. G., Stevens, R. M., and Mainardi, C. L. (1990) J. Biol. Chem. 265, 11421-11424 [Abstract/Free Full Text]
  13. Freije, J. M. P., Diez-Itza, I., Balbin, M., Sanchez, L. M., Blasco, R., Tolivia, J., and Lopez-Otin, C. (1994) J. Biol. Chem. 269, 16766-16773 [Abstract/Free Full Text]
  14. Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994) Nature 370, 61-65 [CrossRef][Medline] [Order article via Infotrieve]
  15. Schnierer, S., Kleine, T., Gote, T., Hillemann, A., Knauper, V., and Tschesche, H. (1993) Biochem. Biophys. Res. Commun. 191, 319-326 [CrossRef][Medline] [Order article via Infotrieve]
  16. Sanchez-Lopez, R., Alexander, C. M., Behrendtsen, O., Breathnach, R., and Werb, Z. (1993) J. Biol. Chem. 268, 7238-7247 [Abstract/Free Full Text]
  17. Hirose, T., Patterson, C., Pourmotabbed, T., Mainardi, C. L., and Hasty, K. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2569-2573 [Abstract]
  18. Sanchez-Lopez, R., Nicholson, R., Gesnel, M.-C., Matrisian, L. M., and Breathnach, R. (1988) J. Biol. Chem. 263, 11892-11899 [Abstract/Free Full Text]
  19. Marcy, A. I., Eiberger, L. L., Harrison, R., Chan, H. K., Hutchinson, N. I., Hagmann, W. K., Cameron, P. M., Boulton, D. A., and Hermes, J. D. (1991) Biochemistry 30, 6476-6483 [Medline] [Order article via Infotrieve]
  20. Ye, Q.-Z., Johnson, L. L., Hupe, D. J., and Baragi, V. (1992) Biochemistry 31, 11231-11235 [Medline] [Order article via Infotrieve]
  21. Murphy, G., Willenbrock, F., Ward, R. V., Cockett, M. I., Eaton, D., and Docherty, A. J. P. (1992) Biochem. J. 283, 637-641 [Medline] [Order article via Infotrieve]
  22. O'Connell, J. P., Willenbrock, F., Docherty, A. J. P., Eaton, D., and Murphy, G. (1994) J. Biol. Chem. 269, 14967-14973 [Abstract/Free Full Text]
  23. Collier, I. E., Krasnov, P. A., Strongin, A. Y., Birkedal-Hansen, H., and Goldberg, G. I. (1992) J. Biol. Chem. 267, 6776-6781 [Abstract/Free Full Text]
  24. Murphy, G., Nguyen, Q., Cockett, M. I., Atkinson, S. J., Allan, J. A., Knight, C. G., Willenbrock, F., and Docherty, A. J. P. (1994) J. Biol. Chem. 269, 6632-6636 [Abstract/Free Full Text]
  25. Ye, Q.-Z., Johnson, L. L., Yu, A. E., and Hupe, D. (1995) Biochemistry 34, 4702-4708 [Medline] [Order article via Infotrieve]
  26. Banyai, L., Tordai, H., and Patthy, L. (1994) Biochem. J. 298, 403-407 [Medline] [Order article via Infotrieve]
  27. Senior, R. M., Griffin, G. L., Fliszar, C. J., Shapiro, S. D., Goldberg, G. I., and Welgus, H. G. (1991) J. Biol. Chem. 266, 7870-7875 [Abstract/Free Full Text]
  28. Murphy, G., Cockett, M. I., Ward, R. V., and Docherty, A. J. P. (1991) Biochem. J. 277, 277-279 [Medline] [Order article via Infotrieve]
  29. Banda, M. J., Dovey, H. F., and Werb, Z. (1981) in Methods for Studying Mononuclear Phagocytes (Adams, D. O., Edelson, P. J., and Koren, H., eds) pp. 603-618, Academic Press, New York
  30. Fridman, R., Fuerst, T. R., Bird, R. E., Hoyhtya, M., Oelkuct, M., Kraus, S., Komarek, D., Liotta, L. A., Berman, M. L., and Stetler-Stevenson, W. G. (1992) J. Biol. Chem. 267, 15398-15405 [Abstract/Free Full Text]
  31. Frisch, S. M., Reich, R., Collier, I. E., Genrich, L. T., Martin, G., and Goldberg, G. I. (1990) Oncogene 5, 75-83 [Medline] [Order article via Infotrieve]
  32. Shapiro, S. D., Fliszar, C. J., Broekelmann, T. J., Mecham, R. P., Senior, R. M., and Welgus, H. G. (1995) J. Biol. Chem. 270, 6351-6356 [Abstract/Free Full Text]
  33. Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A., Seltzer, J. L., Kronberger, A., He, C., Bauer, E. A., and Goldberg, G. I. (1988) J. Biol. Chem. 263, 6579-6587 [Abstract/Free Full Text]
  34. Higuchi, R., Krummel, B., and Saiki, R. K. (1988) Nucleic Acids Res. 16, 7351-7367 [Abstract]
  35. Weingarten, H., and Feder, J. (1985) Anal. Biochem. 147, 437-440 [Medline] [Order article via Infotrieve]
  36. Steffensen, B., Wallon, U. M., and Overall, C. M. (1995) J. Biol. Chem. 270, 11555-11566 [Abstract/Free Full Text]

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