Human fibroblast stromelysin catalytic domain: expression, purification, and characterization of a C-terminally truncated form.

Stromelysin-1 is a member of a tissue metalloproteinase family whose members are all capable of degrading extracellular matrix components. A truncated form of human fibroblast prostromelysin 1 lacking the C-terminal, hemopexin-like domain has been expressed in Escherichia coli and purified to homogeneity. Treatment of this short form of prostromelysin with (aminophenyl)mercuric acetate resulted in activation and loss of the propeptide in a manner identical with the wild-type, full-length protein. Kinetic comparisons using Nle11-substance P as a substrate showed that the wild-type stromelysin and the truncated form of the enzyme had similar kcat and Km values. Likewise, both enzymes displayed similar Ki values for a hydroxamate-containing peptide inhibitor. Taken together, these results indicate that the C-terminal portion of stromelysin is not required for proper folding of the catalytic domain, maintenance of the enzyme in a latent form, activation with an organomercurial, cleavage of a peptide substrate, or interaction with an inhibitor. Moreover, the active short form of stromelysin displayed a reduction in the C-terminal heterogeneity, a characteristic degradation of the full-length stromelysin, and thereby provides a more suitable protein for future structural studies.     

Bioactive conformation of a potent stromelysin inhibitor determined by X-nucleus filtered and multidimensional NMR spectroscopy

The biologically active conformation of a novel, very potent, nonpeptidic stromelysin inhibitor was determined by X-nucleus filtered and multidimensional NMR spectroscopy. This bound conformer was subsequently docked into the stromelysin catalytic domain (SCD) using intermolecular distance constraints derived from NOE data. The complex showed the S1′ pocket of stromelysin to be the major site of enzyme-inhibitor interaction with other portions of the inhibitor spanning the S2′ and S1 binding sites. Theoretical predictions of SCD-inhibitor binding from molecular modeling studies were consistent with the NMR data. Comparison of modeled enzyme-inhibitor complexes for stromelysin and collagenase revealed an alternate binding mode for the inhibitor in collagenase, suggesting a similar binding interaction might also be possible for stromelysin. The NMR results, however, revealed a single SCD-inhibitor binding mode and provided a structural template for the design of more potent stromelysin inhibitors.     

Stromelysin-1: three-dimensional structure of the inhibited catalytic domain and of the C-truncated proenzyme.

The proteolytic enzyme stromelysin-1 is a member of the family of matrix metalloproteinases and is believed to play a role in pathological conditions such as arthritis and tumor invasion. Stromelysin-1 is synthesized as a pro-enzyme that is activated by removal of an N-terminal prodomain. The active enzyme contains a catalytic domain and a C-terminal hemopexin domain believed to participate in macromolecular substrate recognition. We have determined the three-dimensional structures of both a C-truncated form of the proenzyme and an inhibited complex of the catalytic domain by X-ray diffraction analysis. The catalytic core is very similar in the two forms and is similar to the homologous domain in fibroblast and neutrophil collagenases, as well as to the stromelysin structure determined by NMR. The prodomain is a separate folding unit containing three alpha-helices and an extended peptide that lies in the active site of the enzyme. Surprisingly, the amino-to-carboxyl direction of this peptide chain is opposite to that adopted by the inhibitor and by previously reported inhibitors of collagenase. Comparison of the active site of stromelysin with that of thermolysin reveals that most of the residues proposed to play significant roles in the enzymatic mechanism of thermolysin have equivalents in stromelysin, but that three residues implicated in the catalytic mechanism of thermolysin are not represented in stromelysin.     

Structural characterizations of nonpeptidic thiadiazole inhibitors of matrix metalloproteinases reveal the basis for stromelysin selectivity.

The binding of two 5-substituted-1,3,4-thiadiazole-2-thione inhibitors to the matrix metalloproteinase stromelysin (MMP-3) have been characterized by protein crystallography. Both inhibitors coordinate to the catalytic zinc cation via an exocyclic sulfur and lay in an unusual position across the unprimed (P1-P3) side of the proteinase active site. Nitrogen atoms in the thiadiazole moiety make specific hydrogen bond interactions with enzyme structural elements that are conserved across all enzymes in the matrix metalloproteinase class. Strong hydrophobic interactions between the inhibitors and the side chain of tyrosine-155 appear to be responsible for the very high selectivity of these inhibitors for stromelysin. In these enzyme/inhibitor complexes, the S1'' enzyme subsite is unoccupied. A conformational rearrangement of the catalytic domain occurs that reveals an inherent flexibility of the substrate binding region leading to speculation about a possible mechanism for modulation of stromelysin activity and selectivity.     

Protein kinase C-independent expression of stromelysin by platelet-derived growth factor, ras oncogene, and phosphatidylcholine-hydrolyzing phospholipase C.

Changes in the expression of several genes play critical roles in cell growth and tumor transformation. A number of proteases are increased in some tumors, and the level of these enzymes correlates with the metastatic potential of several cancer cell lines. Stromelysin, with the widest substrate specificity, can degrade the extracellular matrix conferring metastatic potential to tumor cells. The mechanisms whereby growth factors and oncogenes control the expression of stromelysin are beginning to be characterized. In the study shown here we also identify a region in the stromelysin promoter which is involved in the induction of stromelysin in response to platelet-derived growth factor, phosphatidylcholine-hydrolyzing phospholipase C, and ras oncogene. Our results are consistent with the notion that platelet-derived growth factor/phosphatidylcholine-hydrolyzing phospholipase C induces stromelysin gene expression through a phorbol myristate acetate/protein kinase C-independent mechanism by acting through elements in the stromelysin promoter distinct from the 12-O-tetradecanoylphorbol-13-acetate-responsive element.     

Cleavage of cartilage proteoglycan between G1 and G2 domains by stromelysins

Normal and pathological turnover of proteoglycans in articular cartilage involves its cleavage close to the N-terminal G1 domain responsible for aggregation. A fragment containing G1 and G2 N-terminal domains of pig cartilage proteoglycans was therefore used as a substrate to investigate its degradation by the metalloproteinase stromelysin and related recombinant stromelysin enzymes. The stromelysins produced an apparent single cleavage yielding a G1 fragment of 56 kDa and a G2 fragment of 110 kDa. Rabbit bone stromelysin was much more active against the G1-G2 fragment and against proteoglycan aggregates than recombinant human stromelysin-1 and stromelysin-2. All metalloproteinase preparations were active against proteoglycan and the G1-G2 fragment at acid (pH 5.5) and neutral pH (7.4). N-terminal sequencing of the G2 fragment derived from the action of recombinant human stromelysin-1 revealed that cleavage between G1 and G2 occurred at the N-terminal end of the interglobular domain, close to the last cysteine in G1. The specific cleavage site was between an asparagine and a pair of phenylalanine residues, where the asparagine corresponds to residue 341 in human and rat mature core protein sequence.     

Interaction of human rheumatoid synovial collagenase (matrix metalloproteinase 1) and stromelysin (matrix metalloproteinase 3) with human alpha 2-macroglobulin and chicken ovostatin. Binding kinetics and identification of matrix metalloproteinase cleavage sites

The homologous proteinase inhibitors, human alpha 2-macroglobulin (alpha 2M) and chicken ovostatin, have been compared with respect to their "bait" region sequences and interactions with two human matrix metalloproteinases, collagenase and stromelysin. A stretch of 34 amino acid residues of the ovostatin bait region sequence was determined and the matrix metalloproteinase cleavage sites identified. Collagenase cleaved a X-Leu bond where X was unidentified, whereas the major cleavage site by stromelysin was at the Gly-Phe bond, 4 residues on the COOH-terminal side of the collagenase cleavage site. Collagenase cleaved the alpha 2M bait region at the Gly679-Leu680 bond, and stromelysin at Gly679-Leu680 and Phe684-Tyr685 bonds. Sequence similarity in the bait region of members of the alpha-macroglobulin family is strikingly low. The kinetic studies indicate that alpha 2M is a 150-fold better substrate for collagenase than type I collagen. Structural predictions based on the bait region sequences suggest that a collagen-like triple helical structure is not a prerequisite for the efficient binding of tissue collagenase to a substrate. The binding of stromelysin to alpha 2M is slower than that of collagenase. Stromelysin reacts with ovostatin even more slowly. Despite the preference of chicken ovostatin for metalloproteinases, human alpha 2M, a far less selective inhibitor, reacts more rapidly with collagenase and stromelysin. These results suggest that alpha 2M may play an important role in regulating the activities of matrix metalloproteinases in the extracellular space.     

Pump-1 cDNA codes for a protein with characteristics similar to those of classical collagenase family members

Pump-1 cDNA has recently been isolated by screening a human tumor cDNA library with a transin (rat stromelysin) probe under low-stringency hybridization conditions. The cDNA codes for a potential protein with significant sequence similarity to the metalloproteinases collagenase and stromelysin, but which lacks the hemopexin-like domain characteristic of these enzymes. Expression of pump-1 cDNA in cos cells using an expression vector leads to secretion of a protein of Mr 28,000 with latent, organomercurial-activatable proteinase activity. Cos cells transfected with a partial pump-1 cDNA in the vector pPROTA secrete a fusion protein between the IgG-binding domains of staphylococcal protein A and pump-1. The fusion protein binds to IgG-Sepharose, and the bound fusion protein undergoes apparent autocleavage in the presence of 4-aminophenylmercuric acetate with elution of active pump-1 species of Mr 21,000 and 19,000. Active pump-1 degrades casein, gelatins of types I, III, IV, and V, and fibronectin and can activate collagenase. Active pump-1 is inhibited by EDTA, 1,10-phenanthroline, and the tissue inhibitor of metalloproteinases. These results show that, despite the absence of a hemopexin-like domain, pump-1 is a latent secreted metalloproteinase. Postpartum rat uteri contain elevated levels of rat pump-1 mRNA. On the basis of this observation, its size, and its substrate specificity, we suggest that pump-1 might correspond to a previously described uterine metalloproteinase, matrix metalloproteinase 7.     

A fluorescence resonance energy transfer method for measuring the binding of inhibitors to stromelysin.

A sensitive fluorescence resonance energy transfer method was developed for the direct measurement of the dissociation constants of stromelysin inhibitors. The method is applied to the thiadiazole class of stromelysin inhibitors and it takes advantage of the fact that, upon binding to the active site of enzyme, the thiadiazole ring, with its absorbance centered at 320 nm, is able to quench the fluorescence of the tryptophan residues surrounding the catalytic site. The changes in fluorescence are proportional to the occupancy of the active site: Analysis of the fluorescence versus inhibitor concentration data yields dissociation constants that are in agreement with the corresponding competitive inhibitory constants measured by a catalytic rate assay. The affinity of nonthiadiazole inhibitors of stromelysin-such as hydroxamic acids and others-can be determined from the concentration-dependent displacement of a thiadiazole of known affinity. Using this displacement method, we determined the affinities of a number of structurally diverse inhibitors toward stromelysin. Since the three tryptophan residues located in the vicinity of the active site of stromelysin are conserved in gelatinase and collagenase, the method should also be applicable to inhibitors of other matrix metalloproteinases.     

The role of the C-terminal domain in collagenase and stromelysin specificity.

Recombinant human interstitial collagenase, an N-terminal truncated form, delta 243-450 collagenase, recombinant human stromelysin-1, and an N-terminal truncated form, delta 248-460 stromelysin, have been stably expressed in myeloma cells and purified. The truncated enzymes were similar in properties to their wild-type counterparts with respect to activation requirements and the ability to degrade casein, gelatin, and a peptide substrate, but truncated collagenase failed to cleave native collagen. Removal of the C-terminal domain from collagenase also modified its interaction with tissue inhibitor of metalloproteinases-1. Hybrid enzymes consisting of N-terminal (1-242) collagenase.C-terminal (248-460) stromelysin and N-terminal (1-233) stromelysin.C-terminal (229-450) collagenase, representing an exchange of the complete catalytic and C-terminal domains of the two enzymes, were expressed in a transient system using Chinese hamster ovary cells and purified. Both proteins showed similar activity to their N-terminal parent and neither was able to degrade collagen. Analysis of the ability of the different forms of recombinant enzyme to bind to collagen by ELISA showed that both pro and active stromelysin and N-terminal collagenase.C-terminal stromelysin bound to collagen equally well. In contrast, only the active forms of collagenase and N-terminal stromelysin.C-terminal collagenase bound well to collagen, as compared with their pro forms.     

Identification of the active site of gelatinase B as the structural element sufficient for converting a protein to a metalloprotease

Gelatinase B is a member of the matrix metalloproteinase family that efficiently cleaves gelatin, elastin, and types V and X collagen. To understand the contribution of the active site of the enzyme (amino acid residues 373-456) in these activities, we studied catalytic properties of a fusion protein consisting of maltose binding protein and the active site region of gelatinase B. We found that addition of the active site of gelatinase B, which corresponds to 12% of the total protein molecule, to maltose binding protein is sufficient to endow the protein with the ability to cleave the peptide substrates Mca-PLGL(Dpa)AR-NH(2) and DNP-PLGLWA-(D)-R-NH(2). The fusion protein hydrolyzed the Mca-PLGL(Dpa)AR-NH(2) peptide with the same efficiency as that of the stromelysin, k(cat)/K(m) approximately 1.07 x 10(6) M(-)(1) h(-)(1). The fusion protein, however, was not able to degrade the large substrate, gelatin. Inhibition of the activity of the protein by EDTA suggested that its activity was metal dependent. ESR analyses indicated that the fusion protein bound one molecule of Zn(2+). In addition, Z-Pro-Leu-Gly-hydroxamate and TIMP-1 inhibited the activity of the protein, suggesting that the structure of the active site of the fusion protein is similar to that of the other metalloproteinases. These data provide fundamental information about the structural elements required for transforming a protein to a metalloprotease.     

X-ray structure of a novel matrix metalloproteinase inhibitor complexed to stromelysin

A new class of matrix metalloproteinase (MMP) inhibitors has been identified by screening a collection of compounds against stromelysin. The inhibitors, 2,4,6-pyrimidine triones, have proven to be potent inhibitors of gelatinases A and B. An X-ray crystal structure of one representative compound bound to the catalytic domain of stromelysin shows that the compounds bind at the active site and ligand the active-site zinc. The pyrimidine triones mimic substrates in forming hydrogen bonds to key residues in the active site, and provide opportunities for placing appropriately chosen groups into the S1' specificity pocket of MMPS: A number of compounds have been synthesized and assayed against stromelysin, and the variations in potency are explained in terms of the binding mode revealed in the X-ray crystal structure.     

Structure-activity relationships by mass spectrometry: identification of novel MMP-3 inhibitors

A novel class of nonpeptide inhibitors of stromelysin (MMP-3) has been discovered with the use of mass spectrometry. The method relies on the development of structure-activity relationships by mass spectrometry (SAR by MS) and utilizes information derived from the binding of known inhibitors to identify novel inhibitors of a target protein with a minimum of synthetic effort. Noncovalent complexes of known inhibitors with a target protein are analyzed; these inhibitors are deconstructed into sets of fragments which compete for common or overlapping binding sites on the target protein. The binding of each fragment set can be studied independently. With the use of competition studies, novel members of each fragment set are identified from compound libraries that bind to the same site on the target protein. A novel inhibitor of the target protein was then constructed by chemically linking a combination of members of each fragment set in a manner guided by the proximity and orientation of the fragments derived from the known inhibitors. In the case of stromelysin, a novel inhibitor composed of favorably linked fragments was observed to form a 1:1 complex with stromelysin. Compounds that were not linked appropriately formed higher order complexes with stoichiometries of 2:1 or greater. These linked molecules were subsequently assessed for their ability to block stromelysin function in a chromogenic substrate assay.     

The expression of a xylanase targeted to ER-protein bodies provides a simple strategy to produce active insoluble enzyme polymers in tobacco plants

Background

Xylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs).

Methodology/Principal Findings

Tobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase.

Conclusion/Significance

In the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for industrial purposes.     

Ultrastructure of the cell surface cellulosome of Clostridium thermocellum and its interaction with cellulose.

The ultrastructural distribution of the cellulosome (a cellulose-binding, multicellulase-containing protein complex) on the cell surface of Clostridium thermocellum YS was examined by cytochemical techniques and immunoelectron microscopy. When cells of the bacterium were grown on cellobiose, cellulosome complexes were compacted into quiescent exocellular protuberant structures. However, when the same cells were grown on cellulose, these polycellulosomal organelles underwent extensive structural transformation; after attachment to the insoluble substrate, the protuberances protracted rapidly to form fibrous "contact corridors." The contact zones mediated physically between the cellulosome (which was intimately attached to the cellulose matrix) and the bacterial cell surface (which was otherwise detached from its substrate). In addition, cell-free cellulosome clusters coated the surface of the cellulose substrate. The cellulose-bound cellulosome clusters appear to be the site of active cellulolysis, the products of which are conveyed subsequently to the cell surface via the exocellular contact zones.     

Secretion of stromelysin by cultured dermal papilla cells: differential regulation by growth factors and functional role in mitogen-induced cell proliferation.

To understand better the molecular nature of the epithelial-mesenchymal interactions that govern folliculogenesis and hair growth, we have studied the behavior of cultured rat dermal papilla cells (rDP), the mesenchymal component of the hair follicle. Basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) both potentiated the growth of rDP in culture, and transforming growth factor-beta (TGF-beta) inhibited rDP proliferation. Biosynthetic labeling studies demonstrated that both PDGF and bFGF induced synthesis of a major secreted protein(s) with Mr = 55-60 kD. It was noted that PDGF and bFGF differentially regulated synthesis of this major secreted protein; PDGF-mediated induction was found to be transient, while bFGF allowed prolonged synthesis of the protein. Sodium dodecyl sulfate (SDS)-substrate gel analysis of rDP-conditioned media revealed that this protein is a metalloproteinase with casienolytic activity and Mr approximately 51 kD (unreduced). We have identified the growth factor-regulated rDP protein as the matrix metalloproteinase stromelysin by immunoprecipitation. Northern analysis established that increased secretion of stromelysin was accompanied by an increased expression of stromelysin-specific mRNA. Remarkably, stromelysin antisera interfere with stimulation of dermal papilla cell growth, demonstrating that stromelysin production serves a functional role in mitogen-induced proliferation in these cells. These findings provide insight into the mechanism by which the connective tissue remodeling required for formation of hair embryonically and the postembryonic hair cycle may be regulated.     

Basic calcium phosphate crystals cause coordinate induction and secretion of collagenase and stromelysin.

Synovial fluid basic calcium phosphate crystals (BCP) are often found in severely degenerated joints. Crystalline BCP is a growth factor stimulating fibroblast mitogenesis and acting as a competence factor similar to platelet-derived growth factor. In human fibroblasts (HF), the synthesis of collagenase and stromelysin is coordinately induced after stimulation with a variety of cytokines and growth factors. We sought to determine whether BCP, like other growth factors, might induce proteases that would damage articular tissue. Northern blot analysis of mRNA for collagenase and stromelysin in HF stimulated with BCP was performed. Secreted enzymes were analyzed by immunoblot using a monoclonal antibody to collagenase and by immunoprecipitation using a polyclonal antibody to stromelysin. Stromelysin activity was confirmed using casein substrate gels. A significant, dose-dependent accumulation of collagenase and stromelysin message was evident after 4 h and continued for at least 24 h in BCP-stimulated cultures. Forty-nine and 54 kD proteins immunoreacting with collagenase antibody were identified in the conditioned media (CM) from BCP-stimulated cultures while 50 and 55 kD proteins were identified by immunoprecipitation with stromelysin antibody. Collagenase activity was increased significantly in the CM from BCP treated cells; casein substrate gels showed casein degrading bands at molecular weights consistent with stromelysin. BCP stimulates coordinate induction of collagenase and stromelysin which may mediate the joint destruction associated with these crystals.     

N40, a novel nonacidic matrix protein from pearl oyster nacre, facilitates nucleation of aragonite in vitro

A novel nonacidic matrix protein from pearl oyster nacre has been purified by cation-exchange chromatography. It was designated N40 for the nacreous protein of approximately 40 kDa. On the basis of the extraction method (with Tris-buffered Milli-Q water) and amino acid compositions (Gly- and Ala-rich), N40 was inferred to be a conventional "insoluble matrix protein". Crystallization experiments showed that N40 could facilitate the nucleation of aragonite drastically. So far, among the macromolecules that have been purified from the shell, N40 is an exclusive protein that can nucleate aragonite by itself, without the need for adsorption to a substrate. Thus, the present study has proposed the possibility that the nonacidic shell protein (maybe a conventional "insoluble framework protein") can also directly participate in aragonite nucleation and even act as a nucleation site. It is a valuable supplement to the classic biomineralization theory, in which the soluble acidic proteins of the shell are generally believed to function as a nucleation site.     

Stromelysin generates a fibronectin fragment that inhibits Schwann cell proliferation

Our previous report (Muir, D., S. Varon, and M. Manthorpe. 1990. J. Cell Biol. 109:2663-2672) described the isolation and partial characterization of a 55-kD antiproliferative protein found in Schwann cell (SC) and schwannoma cell line-conditioned media and we concluded that SC proliferation is under negative autocrine control. In the present study the 55-kD protein was found to possess metalloprotease activity and stromelysin immunoreactivity. The SC-derived metalloprotease shares many properties with stromelysin isolated from other sources including the ability to cleave fibronectin (FN). Furthermore, limited proteolysis of FN by the SC-derived protease generated a FN fragment which itself expresses a potent antiproliferative activity for SCs. The active FN fragment corresponds to the 29-kD amino-terminal region of the FN molecule which was also identified as an active component in SC CM. Additional evidence that a proteolytic fragment of FN can possess antiproliferative activity for SCs was provided by the finding that plasmin can generate an amino-terminal FN fragment which mimicked the activity of the SC metalloprotease-generated antiproliferative FN fragment. Both the 55-kD SC metalloprotease and the 29-kD FN fragment could completely and reversibly inhibit proliferation of SCs treated with various mitogens and both were largely ineffective at inhibiting proliferation by immortalized or transformed SC lines. Normal and transformed SC types do secrete the proform of stromelysin, however, transformed cultures do not produce activated stromelysin and thus cannot generate the antiproliferative fragment of FN. These results suggest that, once activated, a SC-derived protease similar to stromelysin cleaves FN and generates an antiproliferative activity which can maintain normal SC quiescence in vitro.