©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Rat Uterine Matrilysin and Its cDNA
RELATIONSHIP TO HUMAN PUMP-1 AND ACTIVATION OF PROCOLLAGENASES (*)

Susan R. Abramson (4) (1)(§), Gregory E. Conner (2), Hideaki Nagase (5), Isaac Neuhaus (1), J. Frederick Woessner , Jr. (1) (3)

From the (1)Departments of Biochemistry and Molecular Biology, (2)Cell Biology and Anatomy, and (3)Medicine, University of Miami School of Medicine, Miami, Florida 33136, the (4)Division of Research, Cleveland Clinic Florida, Ft. Lauderdale, Florida 33309, and the (5)Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A small uterine metalloproteinase of the rat has been shown by amino acid and cDNA sequencing to be orthologous to human pump-1. Both proteinases are now designated as matrilysin or matrix metalloproteinase 7. The properties of purified uterine metalloproteinase and recombinant pump-1 were compared. Their specificities on substrates (gelatins, fibronectin, transferrin, elastin, Azocoll, and (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3,[2, 4-dinitrophenyl]-L-2, 3-diaminopropionyl)-Ala-Arg-NH) are similar and distinct from those of the stromelysins and gelatinases. The two matrilysins have similar sensitivity to hydroxamate and pseudopeptide inhibitors. Rat matrilysin selectively cleaves the 2(I) chain of rat gelatin, producing major cuts at Gly--Ile, Gly--Leu, and Gly--Ile. Rat matrilysin produces maximum activation of latent human interstitial collagenase 1 (pro-matrix metalloproteinase 1) when added in the presence of 4-aminophenylmercuric acetate (APMA) by cleaving the Gln--Phe bond. Rat and human matrilysin do not directly activate latent rat collagenase 3 (matrix metalloproteinase 13) and do not enhance its activation when added together with APMA. Autoactivation of collagenase 3 in the presence of APMA results in cleavage at Val--Tyr corresponding to the Gln--Phe cleavage in collagenase 1. Thus collagenase 3 is capable of maximal autoactivation, whereas collagenase 1 is dependent upon another matrix metalloproteinase in order to be activated to its full potential.


INTRODUCTION

The connective tissue matrix metalloproteinases constitute a family of proteinases, the matrixin family, which is often divided into three groups: collagenases, gelatinases, and stromelysins(1, 2, 3) . In exploring the post-partum involuting rat uterus, a tissue in which activities of many of these proteinases are elevated, we found that stromelysin was not present(4) . In its stead, we found a small metalloproteinase (4) that was subsequently purified and characterized as uterine metalloproteinase(5) . cDNA encoding a similar small proteinase was cloned from a human tumor cell line; this enzyme was given the name pump-1 or putative (or punctuated) metalloproteinase 1 (6). Subsequent expression of this cDNA yielded a proteinase with properties comparable with those of the uterine enzyme(7) , but sequence comparison between the two enzymes has been lacking. The enzymes have been designated matrix metalloproteinase 7, and the Enzyme Commission has recommended the name matrilysin, EC 3.4.24.23(8) .

Matrilysin is the smallest known member of the matrixin family, having M 28,000 for the zymogen and M 19,000 for the active form. It is capable of digesting gelatins of type I, III, IV, and V collagens, elastin, proteoglycans, fibronectin, casein, transferrin, insulin B-chain and DNP-peptide()(5, 9) . Human matrilysin is capable of enhanced activation of procollagenase 1(7) , but it is not known if the cleavage site corresponds to that produced by stromelysin 1. We first emphasized the importance of matrilysin in the degradation of matrix that accompanies post-partum involution of the rat uterus (4); its activity was low during pregnancy and peaked at 1 day postpartum when tissue breakdown reached its maximum rate. Matrilysin is expressed in the human endometrium in the proliferative, late secretory, and menstrual phases but not in early or midsecretory phases (10). The human enzyme is elevated in various tumors and cancers of the stomach, colon, lung, head, and prostate(11, 12, 13, 14, 15) . It is found in promonocytic cells (16) and in mesangial cells of the kidney(17) , where it may cleave pro-urokinase to a smaller size(18) .

The probable role of matrilysin in vivo is to degrade a broad spectrum of connective tissue components and to activate other extracellular proteinases. Therefore, we have characterized matrilysin with respect to its substrate and inhibitor profiles and its ability to activate two distinct collagenases, human collagenase 1 (matrix metalloproteinase 1) and rat collagenase 3 (matrix metalloproteinase 13), which until recently were believed to be orthologous enzymes(19) . Further questions to be addressed are whether uterine metalloproteinase is truly the rat equivalent of human pump-1 and, if so, whether these two matrilysins have a specificity similar to the stromelysins or form a distinct metalloproteinase subgroup. These questions are approached by determination of the cDNA sequence of the rat enzyme, by comparison of characteristics of the rat and human enzymes, and by comparison to stromelysin.


EXPERIMENTAL PROCEDURES

Cloning

A gt10 rat uterus cDNA library (CLONTECH, Palo Alto, CA) was screened by PCR and plaque hybridization. Initially, the library was used as a template for PCR with degenerate primers. The resulting PCR product was sequenced to confirm that it was a portion of the matrilysin gene, and it was then used as a probe for screening the library by plaque hybridization(20) . The screening was carried through three rounds, and the cDNA inserts from DNA from 3 isolated plaques were subcloned into pGEM3Zf(+) (Promega, Madison, WI). One of these clones was sequenced completely (both strands), and the other two were partially sequenced. Sequencing was performed with the Sequenase Version 2.0 kit (U.S. Biochemical Corp.).

Enzyme Preparations

Uterine rat matrilysin was purified as described previously(5) . Human matrilysin was a kind gift from Andrew J. P. Docherty, Celltech Ltd., Slough, United Kingdom. Gelatinase A (matrix metalloproteinase 2, EC 3.4.24.24) and stromelysin 1 (matrix metalloproteinase 3, EC 3.4.24.17) were purified from human synovial fibroblast culture media (21, 22). Digestions with stromelysin 1 employed the high M active form(45, 0) . Rat procollagenase 3 was a generous gift from John J. Jeffrey, Albany Medical College. Human procollagenase 1 was purified from the culture medium of rheumatoid synovial cells as described previously(23) .

Assay of Matrilysin

Routine assay of the enzyme was performed by digestion of Azocoll (Calbiochem) as described(5) . The assay buffer (used in all assays, unless otherwise noted) was 50 mM Tris, 200 mM NaCl, 10 mM CaCl, 0.02% NaN, pH 7.5. The rat matrilysin preparation was titrated with human tissue inhibitor of metalloproteinases 1 to 50% inhibition, permitting exact calculation of the protein content, which was too dilute for standard protein determination methods. One unit of enzyme digests 1 µg of Azocoll/min at 37 °C; the specific activity of rat matrilysin is 17,000 units/mg proenzyme.

Amino Acid Sequencing of Matrilysin and Collagenases

The amino-terminal sequences of rat promatrilysin and of two CNBr fragments were determined by Edman degradation using an Applied Biosystems model 120A analyzer and model 900A data analysis system. Cyanogen bromide fragments were separated by high performance liquid chromatography (Hewlett Packard model 1090, Palo Alto, CA), using a C-18 narrow bore column. The amino-terminal sequence of active matrilysin was determined following activation of proenzyme with 1 mM APMA. Human collagenase 1 was activated in the presence of 1 mM APMA with and without added rat matrilysin (4.6 µg of collagenase 1, 3 µg of matrilysin in 1.3 ml) in assay buffer for 2 h at 37 °C. Purified rat procollagenase 3 was activated by 1 mM APMA. After activation, samples were subjected to SDS-PAGE (24) and transferred to Immobilon P polyvinylidene difluoride (Millipore Corp., Bedford, MA). Protein was visualized with Coomassie Blue, and the appropriate band was excised for sequencing at the University of Florida Core Protein Facility (Gainesville, FL).

Substrate Digestions

Synthetic Substrate

The Mca-peptide (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3,[2, 4-dinitrophenyl]-L-2, 3-diaminopropionyl)-Ala-Arg-NH(25) was kindly supplied by Dr. Alan Jacobson, OsteoArthritis Sciences, Inc., Cambridge, MA. This internally quenched fluorescent peptide is cleaved by various matrix metalloproteinases at the Gly-Leu bond. Peptide was made up as 53 µM stock solution in 15% dimethyl sulfoxide and stored at -80 °C; the peptide is light-sensitive. The assay mixture contained enzyme and 2 µM substrate in 630 µl of assay buffer. During incubation at 37 °C, aliquots of 70 µl were removed at 5-min intervals and diluted into 0.75 ml of 0.1 N sodium acetate, pH 4.0, to stop the reaction. Fluorescence was determined at an excitation wavelength of 328 nm and emission at 393 nm. Standards were prepared with 7-methoxycoumarin made up in 2 µM Mca-peptide solution. Blanks contained 1 mM 1,10-phenanthroline. Digestion was first standardized with stromelysin 1 at levels of 10-30 ng/assay. Matrilysin was then examined at levels of 0.5-1.24 ng of human and 2-3 ng of rat enzyme/assay.

Transferrin

Bovine transferrin (Sigma) was reduced, alkylated, and used for the protease assay as described(26) . Briefly, 50 µg of tritiated transferrin were digested in an assay volume of 60 µl with 0.06-37 nM or 14-21 nM of rat or human matrilysin, respectively, for 2 h at 37 °C. The undigested transferrin was precipitated with 3% (w/v) trichloroacetic acid, and the supernatant was counted for radioactivity.

Elastin

Insoluble, tritiated elastin was prepared as described previously(5) . Digestion of 100 µg of the elastin by 500 ng of enzyme (rat or human matrilysin) was carried out in a volume of 200 µl in assay buffer at pH 8.0 at 37 °C for 18 h. After incubation, undigested elastin was centrifuged off, and the supernatant was counted for radioactivity. Porcine pancreatic elastase (Sigma) was used as a positive control.

Gelatin

Type I collagen was purified from rat skin and digested with pepsin to remove the telopeptides(27) . Collagen was digested with rat uterine collagenase to produce the tropocollagen fragments TC and TC, followed by heating to 60 °C in 20 mM EDTA to convert these to gelatin and to destroy collagenase. After removal of the EDTA by dialysis, 45 µg of the collagenase-digested gelatin was digested further with 36 ng of matrilysin in a total volume of 250 µl for 4 h at 37 °C in assay buffer. Alternatively, 75 µg of intact gelatin was digested with 100 ng of either rat or human matrilysin in a total volume of 500 µl at 37 °C in assay buffer. The resultant products of both reactions were subjected to SDS-PAGE. The relative sizes of the fragments were estimated from a graph of log Mversus distance, taking M of intact 2(I) chain as 100,000, its TC fragment as 75,000, and TC as 25,000.

1(I) and 2(I) Collagen Digests

The 1 and 2 chains of rat telopeptide-free collagen type I were heat-denatured and separated on a CM52 cellulose column(28) . Digestions were carried out in assay buffer in a total volume of 50 µl, with 5.7 ng of matrilysin, 3 µg of substrate, and 2 mM 4-(2-aminoethyl)-benzenesulfonylfluoride, a serine protease inhibitor, at 30 °C for varying times. Blanks had added 40 mM EDTA. Reactions were terminated by the addition of 17 µl of 4 SDS-PAGE sample buffer containing EDTA. For sequencing of digestion fragments, the 2(I) chain was digested with rat matrilysin for 15 or 30 min to make the initial cuts. The samples were separated on a 7.5% SDS-PAGE gel (24) and transblotted onto Immobilon-P polyvinylidene difluoride membrane. Protein was stained with Coomassie Blue. Bands were excised for sequencing at the University of Florida Core Protein Facility.

Synthetic Inhibitors of Matrilysin

BB94 was a kind donation from Dr. Alan Galloway at British Bio-Technology, Ltd, Oxford, U.K. This metalloproteinase inhibitor contains a hydroxamic acid group that binds the catalytic zinc and is attached to a peptide backbone. A stock solution was prepared by first dissolving BB94 in dimethyl sulfoxide to a concentration of 21 µM and then diluting it down to 21 nM in assay buffer. Two other inhibitors of metalloproteinases, SC 40827 (a pseudopeptide) and SC 44463 (a hydroxamate compound) were kindly provided by R. A. Mueller of G. D. Searle & Co., Skokie, IL. These compounds were added to the assay from 1 mg/ml stocks in dimethyl sulfoxide. Inhibition studies were performed using each of the three inhibitors with both the Azocoll assay system (containing 10 ng of rat matrilysin with overnight incubation) and the Mca-peptide assay system (using 0.3 ng of rat matrilysin or 0.2 ng of human matrilysin for 1-h incubations).

Collagenase Assay

Collagenase was assayed by use of telopeptide-free collagen (27); one unit of enzyme digests 1 µg of collagen/min at 30 °C. Procollagenase was preincubated with APMA with and without matrilysin at 37 °C. Aliquots were taken out at varying times for assay and analysis by SDS-PAGE (24) followed by staining with silver(29) . Assay samples were diluted so that 10-15 fmol of collagenase were in each assay.


RESULTS

Sequence Analysis

The NH-terminal amino acid sequences obtained from Edman degradation of rat uterine metalloproteinase and isolated peptides are as follows.

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

Uncertain residues are lowercase, and residues that could not be determined are indicated by X. Degenerate oligonucleotide primers were based on the sequences underlined above as well as on comparison with human matrilysin sequence data(6) . Several of the residues shown above proved to be inconsistent with the protein sequence deduced from cDNA (see Fig. 1), but overall it is clear that the deduced sequence corresponds to that found by Edman degradation.


Figure 1: cDNA sequence of rat matrilysin with corresponding amino acid residues. The third line shows the amino acid sequence of human pump-1 with residues shown only when different from those of the rat (*, no corresponding residues in pump-1). Underlined sequences were those determined by amino acid sequencing. Verticalarrows show the intron-exon borders. Nucleotides in the shadedbox (289-294) are the site of EcoRI cleavage. Nucleotides in boldface (958-963; 982-987) are transcription stop sites. Amino acids in boldfacecapitals are as follows: Met, translation start; Leu, first residue of proenzyme form; Cys, cysteine switch; Phe, first residue of active enzyme; His, first His in the putative zinc binding sequence.



The nucleotide sequence()obtained from the isolated gt10 clones is shown in Fig. 1. This sequence was determined from both strands of a single clone, partial sequences of two separate clones, and sequence from the initial PCR product; all matched identically. The cDNA is 1057 base pairs in length, with an open reading frame of 804 base pairs and 5`- and 3`-untranslated regions of 33 and 220 base pairs, respectively (Fig. 1). The cDNA was subcloned into pGEM3Zf(+) in two fragments due to an EcoRI site (bases 289-294) within the cDNA (the library was cloned into by use of EcoRI). This EcoRI site was verified by directly sequencing the product from the original PCR on the gt10 rat uterus cDNA library. The open reading frame encodes for a protein of 277 residues. The deduced amino acid sequence is shown in Fig. 1, and the residues corresponding to the actual amino acid sequences obtained from Edman degradation of the isolated protein and the two CNBr fragments are indicated by underlining. The deduced human matrilysin amino acid sequence (6) is indicated in this figure only where the two sequences differ. The amino acid sequences are 70% identical, while the nucleotide sequences are 74% identical. The similarity in the primary structure, together with substrate specificity (see below), establishes that uterine metalloproteinase is orthologous to human pump-1, i.e. the same protein, but produced in different species.

There are two potential translation start sites: Met and Met in Fig. 1. According to Kozak's scanning model for translation initiation(30) , it is likely that the first AUG codon is used. In either case, the signal peptide is removed, and the protein is secreted as a zymogen, which has NH-terminal Leu, as shown by amino acid sequencing. This matches the NH terminus of human matrilysin(7) . The resulting zymogen comprises 257 residues, having M of 27,772. The calculated M for the active form of the enzyme is 18,934, based on the new NH terminus at Phe found by amino acid sequencing after APMA activation; the Phe residue in the rat enzyme corresponds to the NH-terminal Tyr of the active form of human matrilysin(31) .

Several highly conserved regions in the matrixin family are also conserved in the rat matrilysin: the ``cysteine switch'' sequence PRCGVPD; the structural Zn ligands His, Asp, His, and His; the structural Ca ligands Asp, Asp, and Glu; and the catalytic Zn binding site HELGHSLGLGH, based on comparison to the sequence and x-ray structure of human collagenase 1 (32). Examination of the sequences immediately before the catalytic zinc-binding His suggests a close relationship between the rat and human enzymes; both have a deletion of Leu (six residues before His) and the insertion of a Thr (one residue before His) not found in any of the other 16 sequences of the matrixin family. This deviation is somewhat surprising in view of its close juxtaposition to the most highly conserved sequence of these enzymes. Interestingly, there is an RGD sequence at residues 143-145 in rat, but not human, matrilysin that could possibly serve as a recognition site for integrins. A corresponding RGD sequence is also found in human collagenase 1(33) .

Gelatin Digestion

Gelatinase A readily degrades both 1 and 2 chains of type I gelatin into small fragments (not shown). Stromelysin 1 also produces extensive digestion; however, an enzyme level 200 times higher than that of gelatinase A is required (not shown). Rat matrilysin, on the other hand, has substantial action on the 2(I) chain and only a weak action on the 1(I) chain (Fig. 2, lanes2 and 3). The 2(I) chain appears to be digested into two main fragments of 70 and 78 kDa. Furthermore, digestion of collagen sequentially with rat collagenase followed by matrilysin produced a 70-kDa band and a reduction of the TC band from 25 to 22 kDa (Fig. 2, lanes6 and 7). It might be argued that a low level of digestion of the 1(I) chain could produce products that confound the picture in Fig. 2. Therefore, we purified 1(I) and 2(I) chains, digested them separately with matrilysin, and found that all the digestion product bands come from the 2(I) chain (not shown). Three short peptides of 22, 25, and 30 kDa resulting from limited digestion of the purified 2(I) chain with rat matrilysin were subjected to amino acid sequencing. The N-terminal sequences of the three peptides are shown in . As can be seen, one of the matrilysin cleavage sites is the same as the collagenase site. The two sites on either side of the collagenase site are approximately 5722 and 3085 Da from the collagenase cutting site (assuming hydroxylation of appropriate Pro residues). At higher enzyme levels the fragments of 2(I) are cleaved to a final single band of 40 kDa (not shown). Human matrilysin produces an almost identical pattern of gelatin digestion but with the addition of a further fragment at about 65 kDa (not shown).


Figure 2: Digestion of type I gelatin by rat matrilysin. The digestions and SDS-PAGE are described under ``Experimental Procedures.'' The rightside of the figure shows the positions of known collagen chains and their collagenase fragments. Lane1, molecular mass markers; lanes2, 3, and 4, digestion of gelatin by matrilysin for 4, 2 and 0 h, respectively; lanes5-9, matrilysin digestion of denatured fragments of collagen produced by the action of rat collagenase 3; lane5, 4-h EDTA blank; lanes6, 7, and 8, 4-, 2-, and 0-h digests; lane9, substrate alone.



Digestion of Other Proteins

Digestion of fibronectin (Fig. 3) by gelatinase A, stromelysin 1, and matrilysin yields patterns with all 3 enzymes that show a number of corresponding bands, but it is evident that the three enzymes have distinct specificities. Human matrilysin produces patterns similar to those of rat (not shown). Fig. 3is representative of results we obtained with various substrates, including casein and transferrin. summarizes the specific activities of rat and human matrilysin acting on various substrates.


Figure 3: Digestion of fibronectin by gelatinase A, stromelysin 1, and matrilysin. Human fibronectin (10 µg, Collaborative Research Inst., Waltham, MA) was incubated with enzyme in assay buffer in a total volume of 50 µl; 11 ng of gelatinase A (MMP-2), 650 ng of stromelysin 1 (MMP-3), or 28 ng of matrilysin (UMP) were added, and digestion was for 18 h at 37 °C. Reactions were stopped with EDTA (final concentration, 30 mM), and aliquots were boiled with sample buffer with or without -mercaptoethanol and applied to SDS-PAGE (24). Proteins were stained with silver (29). Molecular mass standards are shown along the left; FN, fibronectin.



Synthetic Inhibitors of Matrilysin

Inhibition studies with BB94, SC 40827, and SC 44463 were performed using Mca-peptide as substrate for both rat and human matrilysin; the IC values for the rat and human enzymes were 0.8 nM and 1.8 nM; 8 nM and 16 nM; and 3 µM and 20 µM, respectively. These values were verified for the rat enzyme using Azocoll as a substrate. In each case, rat matrilysin was more readily inhibited than the human enzyme. BB94 is the most potent inhibitor of matrilysin among the three tested, while the other hydroxamate, SC 44463 is about 10 times less powerful, and the pseudopeptide is considerably weaker.

Activation of Collagenases by Matrilysin

Activation of human procollagenase 1 by equimolar rat matrilysin and 1 mM APMA resulted in a 2-kDa reduction in molecular mass compared with activation by APMA alone (Fig. 4). In addition, we found that the specific activity of the collagenase 1 activated by matrilysin plus APMA was at least 4-fold higher than when activated by APMA alone; its NH terminus was determined to be Phe, the same site cleaved upon activation by stromelysin 1. A 10-kDa decrease in molecular weight was seen by SDS-PAGE after APMA activation of collagenase 3 (not shown), and amino acid sequencing confirmed that the resulting NH terminus is Tyr. In contrast to collagenase 1 activation, neither of the matrilysins could activate rat collagenase 3 either directly or in the presence of APMA. However, partially purified preparations of rat uterine procollagenase were readily activated (as determined by collagenase assay) by matrilysin near its physiological concentration (1.5 µg/ml) without the addition of APMA. This collagenase is assumed to be the same as the uterine collagenase cloned by Quinn et al.(35) and now thought to be collagenase 3 (19).


Figure 4: Comparison of activation of human procollagenase 1 by APMA with or without rat matrilysin at 37 °C. Reducing 10% SDS-PAGE is shown. Lane1, procollagenase 1, 0 h; lane2, procollagenase 1 + 1 mM APMA, 3 h; lane3, procollagenase 1 + 1 mM APMA + rat promatrilysin, 3 h. Molecular mass markers are indicated on the left. proMMP-1, procollagenase 1; MMP-7, active matrilysin.




DISCUSSION

Is Uterine Metalloproteinase the Same as Pump-1?

Analysis of the cDNA and protein sequences of the two enzymes indicates that they are indeed orthologous and distinct from other known enzymes in the matrixin family. The cloning of rat matrilysin cDNA establishes that the enzyme contains the minimum number of domains reported for matrixins; it has not lost a hemopexin domain by post-translational processing as in the case of macrophage elastase(36) . Although it has been suspected that uterine metalloproteinase and pump-1 are the same, the sequence data give definitive proof that has been lacking until now.

The IntelliGenetics search programs identified sequences in GenBank, listed under transin-3, that proved to be four unpublished exon sequences from genomic clones of rat matrilysin (accession numbers X07821, X07822, X07823, and X07824, submitted by R. Breathnach, 1988). The exon junctions between exons 2, 3, 4, and 5 are marked by arrows in Fig. 1based on comparisons between our sequence, the transin-3 sequences, and consensus sequences for intron/exon junctions(37) . These junctions are in exact agreement with the junctions for the human matrilysin gene(38) . It is likely that the rat gene has two additional exons, to match the human gene. Additionally, exon 2 of transin-3 contains two extra bases compared with our sequence; these insertions create a frameshift that disrupts the open reading frame and are likely to be errors in that sequence.

Comparative Substrate Digestions

All three matrix metalloproteinases (gelatinase A, stromelysin 1, and matrilysin) compared in this study have certain similarities in their substrate specificities: they digest the collagenase octapeptide substrate DNP-peptide (5, 39, 40) at the Gly--Ile bond, the Mca-peptide at the Gly--Leu bond (25) and the oxidized B-chain of insulin at the Ala--Leu and Tyr--Leu bonds(5, 41) . However, they each cleave these bonds at different rates. With various protein substrates, there are many points where the three enzymes also appear to produce the same products (but at different rates) plus a number of additional bands where each enzyme shows its own particular specificity. Matrilysin has a more limited action on gelatin than does gelatinase A and stromelysin 1 and has no action on type IV collagen compared with the rapid digestion by the other two matrixins. A recent study by Mayer et al.(42) confirms that matrilysin has quite distinct specificity from gelatinase A and stromelysin 1 in the cleavage of nidogen. However, it is shown here that matrilysin has the same ability as stromelysin 1 to activate procollagenase 1 maximally. We conclude that the matrilysins should be considered a distinct subclass within the matrixin family but are most similar to the stromelysins.

indicates that the rat and human matrilysins have specific activities that differ by a maximum of a factor of 3 with various protein substrates. Our value of 273,000 for human matrilysin digestion of the Mca-peptide is higher than the value of 169,000 reported by Knight et al.(25) ; this is attributed to minor differences in the assay method and the use of a lower level of enzyme over a longer period. The digestion of elastin by either enzyme was very slight; the human matrilysin value is about one-seventh that reported by Murphy et al.(9) . This discrepancy may be due to differences in the insoluble elastin preparations.

Matrilysin has no action on type I collagen and only limited action on gelatin, with a preference for the 2(I) chain(7) ; it cleaves 2(I) at the collagenase site and at two positions to either side of this. The resultant six fragments are still large enough to be precipitated by trichloroacetic acid in the usual gelatinase assays (43), causing the gelatinase activity of the enzyme to appear much lower than it actually is. Since matrilysin and collagenase have some similarity in specificity, cleaving the Gly--Ile bond of DNP-peptide and the Tyr--Leu bond of the B-chain of insulin(44) , it is not surprising that matrilysin can cleave the 2(I) chain at the same site as collagenase when it acts on the intact triple-helical collagen. However, based on sequence comparison of the three matrilysin sites in the 2(I) chain with the collagenase site in the 1(I) chain, it is surprising that matrilysin does not readily cleave at the 1(I) collagenase site. The other two sites are quite different between the 1(I) and 2(I) chains. Perhaps these differences can help to elucidate the subtleties of matrilysin's specificity.

Activation of Procollagenases by Matrilysin

Quantin et al.(7) have shown that human matrilysin is able to fully activate human procollagenase 1 in the presence of APMA, increasing the collagenase 1 activity 5-fold above that resulting from APMA alone. We find similar results using rat matrilysin to activate human procollagenase 1. Indeed, it is reported here that the site at which procollagenase 1 is cleaved by matrilysin is the same site at which stromelysin 1 activates collagenase 1(23) .

It is interesting to note the difference in susceptibility of partially- and completely-purified procollagenase 3 to activation by matrilysin. In addition, maximal activation of the partially purified collagenase 3 by APMA is achieved at only 5 µM, whereas activation of purified collagenase 3 occurs at 1 mM. We speculate that a second factor facilitates activation of partially purified collagenase 3, perhaps by changing its conformation so that the activation site is more readily accessible. These results are consistent with the findings of Tyree et al.(45) , who reported a stoichiometric protein activator in human skin and rat uterus. Such a factor could be important in an in vivo activation pathway.

The differences in activation of procollagenases 1 and 3 may be related to structural differences. When Phe and Tyr of collagenases 1 and 3, respectively, are the NH termini of their activated enzymes, their ammonium group probably interacts with the carboxylate group of Asp (Asp in collagenase 3). This interaction is predicted from the crystal structure of neutrophil collagenase (matrix metalloproteinase 8) (46) and would be expected to stabilize the enzyme in an optimal conformation for catalytic activity. If the N terminus is to either side of the Phe or Tyr residue, this interaction is weakened or nonexistent. Thus, collagenase 1 in the presence of APMA can undergo autolysis at the Val--Met bond, the Phe--Val bond, and the Val--Leu bond; in each case the specific activity is greatly reduced(23) . However, cleavage of the Gln--Phe bond is only achieved by stromelysin(23) , matrilysin, or gelatinase A(47) , and not by collagenase 1 itself(23) . Autocleavage at the Val--Met bond in the presence of APMA occurs rapidly, while the Phe--Val and Val--Leu bonds are cleaved only upon extended incubation(23) . Thus, the APMA-activated collagenase seen in Fig. 4is the Met species, and a 2-kDa drop is seen upon matrilysin activation due to the loss of the Met-Gln peptide.

In contrast, collagenase 3 is capable of autolysis directly at the NH-terminal side of the optimal Tyr residue following trypsin activation (35) or APMA addition (this paper). Collagenase 3 is also likely to be susceptible to matrilysin cleavage at Val--Tyr, but because collagenase 3 is capable of cutting at this site, no effect is seen upon the addition of equimolar amounts of matrilysin.

The difference in autolytic specificity between the two collagenases suggests that they may have different subsite requirements. Indeed, modeling of collagenase 3 structure based on the crystal structure of collagenase 1 and stromelysin 1 indicates that its S` pocket is more similar to that of stromelysin 1 than to that of collagenase 1.()Both collagenase 3 and stromelysin 1 have pockets that can accommodate an aromatic residue at the P` site, but an Arg side chain lies in the corresponding pocket of collagenase 1, effectively excluding the Phe aromatic side chain at P`(32) .

In conclusion, the sequence and enzyme specificity data presented here support the hypothesis that human pump-1 and rat uterine metalloproteinase are two species of the same enzyme, now called matrilysin or matrix metalloproteinase 7. These matrilysins do not fit well into the matrixin groups designated collagenases, gelatinases, and stromelysins and should be assigned to a fourth group, with macrophage elastase possibly forming a fifth group(36) . Matrilysin's role in vivo is probably to produce extensive degradation of a wide variety of matrix constituents. Its broad specificity may be facilitated by its lack of the additional large hemopexin/vitronectin-like domain found in other matrix metalloproteinases and believed to participate in substrate binding (48). Another important role suggested in this study may be to activate procollagenase 1 at its optimal site, Phe, although it is not established that this occurs in vivo. Finally, it is important to note the subtle differences in specificity and activation mechanisms, shown throughout the paper, since a better understanding of these differences will undoubtedly improve our ability to determine the natural and pathological roles of these matrix metalloproteinases.

  
Table: NH-terminal sequences of rat 2(I) collagen peptides produced by rat matrilysin digestion

Peptide sequences are compared with deduced amino acid sequences from mouse 2(I) collagen (lower row of each pair) (34). P` indicates hydroxyproline residues, and indicates the cleavage site. The second residue of peptide 1 is either Thr or Ser; uncertain residues are lowercase.


  
Table: Comparison of substrate digestions by rat and human matrilysins



FOOTNOTES

*
This investigation was supported by National Institutes of Health Grants HD-06773 (to J. F. W.), AR-39189 (to H. N.), and GM-35812 (to G. E. C.). 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.

§
Supported by National Institutes of Health Training Grant HD-07129 and a University of Miami Graduate Fellowship. To whom correspondence should be addressed: Cleveland Clinic Florida, Div. of Research, 3000 W. Cypress Creek Rd., Ft. Lauderdale, FL 33309. 305-978-5898; Fax: 305-978-5006.

The abbreviations used are: DNP-peptide, DNP-Pro-Leu-Gly-Ile-Ala-Gly-Glu-D-Arg; APMA, 4-aminophenylmercuric acetate; Mca-peptide, (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3,[2, 4-dinitrophenyl]-L-2, 3-diaminopropionyl)-Ala-Arg-NH; PAGE, polyacrylamide electrophoresis; BB94, [4-(N-hydroxyamino)-2R-isobutyl-3S-(thiopen-2-ylthiomethyl)-succinyl]-L-phenylalanine-N-methylamide; SC 40827, N-[3-N-(benzyloxycarbonyl)amino-1-(R)carboxypropyl]-L-leucyl-O-methyl-L-tyrosine-N-methylamide; SC 44463, N-hydroxy-N-{1S-[(4-methoxphenyl)methyl]-2-(methylamino)-2-oxoethyl}-2R-(2-methylpro-pyl)butane-diamide; PCR, polymerase chain reaction.

The cDNA sequence has been deposited in GenBank, accession number L24374.

K. Appelt, personal communication.


ACKNOWLEDGEMENTS

We thank Carolyn Taplin and Sonia Vittoria for their exceptional technical support. We would also like to thank Benne Parten and Ruth Davenport at the University of Florida Protein Chemistry Core Laboratory (which has been supported by the National Institutes of Health, the National Science Foundation, and the University of Florida) for protein sequencing.


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