From the
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
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
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.
A
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
Routine assay of the enzyme was performed by digestion of
Azocoll
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).
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 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.
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.
There are
two potential translation start sites: Met
Several highly conserved regions in
the matrixin family are also conserved in the rat matrilysin: the
``cysteine switch'' sequence PRC
The
IntelliGenetics search programs identified sequences in
GenBank
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
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
In contrast, collagenase
3 is capable of autolysis directly at the NH
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
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
Peptide sequences are compared with deduced amino acid sequences
from mouse
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) 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.
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) .
Cloning
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
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
(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
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 M
versus distance, taking M
of intact
2(I) chain as 100,000, its
TC
fragment as 75,000, and TC
as 25,000.
The
1(I) and
2(I) Collagen Digests
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
Collagenase Assay
Sequence Analysis
The NH-terminal
amino acid sequences obtained from Edman degradation of rat uterine
metalloproteinase and isolated peptides are as follows.
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.
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) .
GVPD; the
structural Zn
ligands His
,
Asp
, His
, and His
; the
structural Ca
ligands Asp
,
Asp
, and Glu
; and the catalytic
Zn
binding site H
ELGHSLGLGH, 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.
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.
, 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.
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) .
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.
-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.
` 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) .
,
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
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
; 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.
, accession number
L24374.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.