THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 267, No. 19, Issue of July 5. PP. 13763-13767.1992 Printed in U. S. A.

Collagen Binding Site in Collagenase Can Be Determined Using the Concept of Sense-Antisense Peptide Interactions*

(Received for publication, October 10, 1991)

Sandro Jose de SouzaS and Ricardo BrentaniQ From the Ludwig Institute for Cancer Research, Rua Professor Antonio Prudente, 109, 4, 01509 Sao Paulo, Brazil

Tissue degradation and invasion are hallmarks of the metastatic phenotype. While several extracellular matrix components can be digested by proteases, deg- radation of interstitial collagen is selectively initiated by collagenase. It is obvious that inhibitors of collagen- ase activity would be extremely useful in preventing tissue destruction and tumor cell invasion and thus prove invaluable therapeutic agents. We describe here the possible development of such inhibitors through the use of the principle of complementary hydropathy. A peptide was deduced from the nucleotide sequence com- plementary to that coding for the region in interstitial collagen surrounding the bond between Gly776 and Ile778 which is cleaved by the enzyme. Labeled collagen binds specifically and quantitatively to this peptide. A polyclonal mouse serum raised against this peptide recognized purified human collagenase, was able to immunoprecipitate collagenase from cultured human keratinocyte supernatants and was effective in inhib- iting collagenolytic activity with a Kiapp = 0.3 FM.

Enzymatic dissolution of the ECM’ and cell locomotion are key steps in many physiological phenomena such as tropho- blast implantation, morphogenesis, tissue remodeling and in- flammation. They are likewise important in pathological states such as rheumatoid, parasitic or bacterial diseases, as well as in tumor invasion and metastasis. Dissemination of tumor cells throughout the body is ultimately the cause of death of most cancer patients. Degradation of the ECM is mainly effected by a large family of Zn2+-dependent metallo- proteinases. This degradation can be produced by tumor cells either through the induction of such enzymes from surround- ing stromal cells (Basset et al., 1990) or by enzyme synthesis by the tumor cells themselves (Templeton et al., 1990). Trans- fection of cells with several oncogenes as well as treatment with cytokines has led to an increase both in metalloprotei- nase production and metastatic propensity (Liotta et al., 1991). The use of tissue inhibitors of metalloproteinases (TIMP) abrogated the metastatic phenotype in many cell types (Schultz et al., 1988; Alvarez et al., 1990), whereas transfection of cells with an antisense TIMP RNA, blocking TIMP-1 expression, led to an increased metastatic capacity (Khokha et al., 1989). Clearly, inhibition of metalloproteinase

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

do Estado de Sao Paulo. $ Supported by a fellowship from Fundagao de Amparo a Pesquisa

5 To whom correspondence should be addressed. The abbreviations used are: ECM, extracellular matrix; ELISA,

enzyme-linked immunosorbent assay; SDS-PAGE, sodium dodecyl- sulfate polyacrylamide gel electrophoresis.

activity would be of great clinical significance for the man- agement of advanced cancer.

Mammalian collagenases cleave interstitial collagens, the most abundant ECM component, between positions 775, which is always a glycine, and 776, which is either leucine or isoleucine. Residues at vicinal positions, both upstream and downstream of the cleavage site are likewise conserved. Work with peptide analogues (Fields et al., 1987) and with collagen produced by cells transfected with collagen al(I) mutants obtained by site-directed mutagenesis (Wu et al., 1990) has indeed indicated that conservation of the vicinal positions is necessary for the maintenance of collagenase susceptibility. Collagenase, like all other metalloproteinases, has a zinc- binding site, which is believed to be within the catalytic site (Birkedal-Hansen et al., 1988). An understanding of the struc- tural and functional basis of the very specific interaction between collagen and collagenase has been hindered by the lack of knowledge of the collagen-binding site in collagenase. This information is critical to the development of specific inhibitors. Since information about the nucleotide sequence coding for that particular collagen domain, which is attacked by collagenase, is available, we decided to employ the hydro- pathic complementarity (sense-antisense peptides) approach (Blalock and Smith, 1984; Brentani, 1988) in order to try to define a domain in the collagenase molecule responsible for binding to collagen.

MATERIALS AND METHODS

Peptides and Antiserum-TKKTLRT heptapeptide coupled to keyhole limpet hemocyanin as well as TKKTLRT, SQNPVQP, and LTTTKKR were purchased from Neosystem, Strasbourg, France. Mice were injected intraperitoneally with 200 pg of total protein at 2-week intervals. After the fourth injection, animals were bled, and titers against the uncoupled peptide were checked by ELISA as described (Brentani et al., 1988). For IgG purification, serum from mice immunized with TKKTLRT was diluted in PBS and chromat- ographed on protein A-Sepharose CL4B (Pharmacia, Upsala, Swe- den). Material bound to the column was eluted with 0.05 M glycine, pH 3.0, neutralized with 1 M Tris, pH 8.0, collected, and dialyzed against PBS overnight. The same procedure was applied to normal mouse serum.

Collagen-Type I collagen used in all experiments was purified from rat tail tendon, as described (Shor, 1982) and labeled to a specific activity of 0.9 pCi/wg using the chloramine-T procedure. After labeling, collagen was extensively dialyzed against water, which improves the rate of triple-helix formation. Its molecular integrity after labeling was checked by SDS-PAGE and only pepsin-resistant collagen (collagen treated with 0.1% pepsin in 0.1 M acetic acid at 25 “C for 1 h) was used in all assays.

Binding Assays-TKKTLRT was adsorbed (50 pg/ml) on plastic microtiter plates (Costar) for 24 h at 4 “C. After discarding the unbound peptide solution, a 3% bovine serum albumin solution (Inlab, Sao Paulo, Brazil) was added to each well for 2 h at room temperature. Plates were then incubated with a defined concentration of labeled collagen for 8 h at room temperature. After extensive washing with TBS containing 0.05% Tween 20, radioactive material bound to the plate was determined using a 127s Minigamma gamma

13763

13764 Determination of a Collagen Binding Site in Collagenase counter (LKB-Wallag, Finland). For the inhibition assays, labeled collagen was preincubated with increasing concentrations of TKKTLRT, SQNPVQP, and LTTTKKR before its addition to the plates.

ELISA-Direct binding assays as well as inhibition in liquid phase were previously described (Brentani et al., 1988; Brentani and Pas- qualini, 1990). In the direct binding assay, microtiter plates (Costar) were coated overnight at 4 "C with 50 pl of the different antigens at 50 pg/ml in PBS. After discarding the coating solution, plates were blocked with PBS containing 3% bovine serum albumin (Inlab), and serial dilutions of antibodies were added for 2 h at room temperature. After extensive washing with PBS containing 1% Tween 20 (Riede- De Haen AG, Srelze, Hannover), a goat anti-mouse IgG peroxidase conjugate (Sigma) was added. After further extensive washing, the developing substrate was added. For liquid-phase competition assays, a mixture of antibodies (at a constant concentration) and competitor (serial dilutions) which had been preincubated together for 2 h at room temperature was added to the plates. After 2 h at room temper- ature, peroxidase-labeled goat anti-mouse IgG was added, followed by the specific substrate. The washing steps were the same as used for direct binding assays.

Cells and Supernatants-Human keratinocytes obtained from neo- natal foreskins were grown to early confluency at 37 "C in tissue cultures dishes in serum-free KGM (Clonetics' Corp., San Diego, CA). The medium was collected and treated with ammonium sulfate to 20% saturation. After centrifugation at low speed the supernatant was dialyzed against 1 liter of 50 mM Tris-HC1, pH 7.5, containing 10 mM CaCL2 for 8 h with two buffer changes. After this step, the supernatant was 10 X concentrated in a Speed-Vac concentrator (Savant). 100 g1 of this sample were activated with 10 p1 of trypsin, 1 mg/ml (Boehringer, Mannheim, Germany) at 37 'C for 10 min. The reaction was terminated by addition of 10 p1 of soybean trypsin inhibitor, 10 mg/ml (Sigma).

SDS-PAGE and Western Blots-Proteins were separated on SDS- PAGE as described (Laemmli, 1970) using 4% stacking and 7.5% resolving gels. After this, the proteins were transferred to nitrocellu- lose filters (0.45-pm pore size). Filters were stained with 0.5% Pon- ceau S (Sigma) to verify the extent of transfer. For Western blots, filters were blocked with PBS containing 5% fat-free dry milk and incubated 2 h at room temperature with the respective antibodies. After extensive washing with PBS containing 0.5% Tween 20 and 0.5% of fat-free dry milk, phosphatase-conjugated secondary antibody was added and incubated for 1 h at room temperature. After extensive washing with the same solution previously used, a revelation buffer containing 5-bromo-4-chloro-3-indolyl phosphate (p-toluidine salt) and nitro blue tetrazolium chloride (Bethesda Research Laboratories) was added.

Immunoprecipitation-Cultured keratinocyte supernatant was la- beled with by the chloramine-T method. The labeled medium was clarified and preabsorbed with rabbit serum as previously described (Pasqualini et al., 1989). All immunoprecipitation steps were per- formed at 4 "C. 100 pl of labeled clarified supernatant were incubated for 3 h with 5 p1 of anti-TKKTLRT antiserum or normal serum. 30 p1 of protein A-Sepharose (Pharmacia) were added to each sample and incubated for 1 h. Samples were washed 10 times with a buffer containing 0.05% Tween 20, 0.5 M NaCl, 0.05 M Tris-HC1, pH 7.4. Sample buffer was added to each sample in order to perform SDS- PAGE.

Zymogram-The material immunoprecipitated by anti-TKKTLRT and normal serum was subjected to gel eletrophoresis in 7.5% SDS- PAGE co-polymerized with 1 pg of 1251-labeled collagen. The method is a modification of that reported by Irvine et al. (1990). Samples were mixed with Laemmli sample buffer (without 8-mercaptoethanol and containing 5% SDS), and eletrophoresis was performed at 4 "C. After the run, the gel was incubated in 2% Triton X-100 for 30 min at 37 'C followed by 16 h at 37 "C in 0.05 M Tris-HC1, pH 7.4, containing 5 mM CaC12. The gel was then incubated in the same buffer containing 100 rg/ml of trypsin for 4 h and dried. Collagenol- ytic activity was visualized by autoradiography.

Collagenolytic Assay-The method of Yoshioka et al. (1987) was used in the inhibition assays using the anti-TKKTLRT as competi- tor. Briefly, collagen (100 pg/ml) was placed on plastic plates wells for more than 7 days at 4 "C. After washing with TBS containing 0.05% Tween 20 (pH 7.4), 100 p1 of the cultured keratinocyte super- natant were added and then incubated for 6 h at room temperature. After washing with the same washing solution containing 50 mM EDTA, anti-rat collagen rabbit antiserum (a kind gift from Dr. Wilson Savino, Fundacao Oswaldo Cruz, Rio de Janeiro) diluted

1:200 in TBS containing 0.05% Tween 20 was added to the microplate and incubated for 2 h at room temperature. The plate was washed and incubated with goat anti-rabbit IgG peroxidase conjugate (Sigma) diluted in TBS containing 0.05% Tween 20 and 0.5% bovine serum albumin. After extensive washing, a substrate solution was placed in each well and incubated for 15 min at room temperature. Reaction was stopped by adding 8 N sulfuric acid, and the color developed was measured with a TITERTEK spectrophotometer (Bio-Rad, Japan). Inhibition studies were performed by preincubating keratinocyte su- pernatants with the appropriated IgG solutions for 2 h at room temperature. One unit of collagenase is defined as the amount of enzyme that degrades 1 pg of interstitial collagen at room tempera- ture. For inhibition assays using the peptides as competitors, 10 pg of 1125-labeled collagen was adsorbed in plastic wells for 12 h at room temperature. Keratinocyte supernatant was added for 6 h at room temperature. For inhibitions, labeled collagen was preincubated with different concentrations of the peptides before the addition of super- natant. The radioactive material released from the plates was counted using a 127s Minigamma counter (LKB).

RESULTS AND DISCUSSION

The principle of complementary hydropathy stipulates that because codons for hydrophilic amino acids are complemented by hydrophobic amino acid coding ones, peptides thus encoded would bind one another (Blalock and Smith, 1984; Brentani, 1990). The chemical plausibility of such hydrophobic-hydro- philic interactions has been already discussed (Brentani, 1988; 1990). In Fig. 1 peptides TKKTLRT and SSNTLRS were found to be coded for by the nucleotide sequences comple- mentary to those which code for the domain spanning posi- tions 772 through 778 from human ( ~ ~ ( 1 ) and mouse ( ~ ~ ( 1 ) collagen chains, respectively (de Wett et al., 1987; Monson et al., 1982). As shown in Fig. 2, both in human fibroblast and polymorphonuclear collagenases (Goldberg et al., 1986; Hasty

G,,, P a G L L G,,,

5" ACC AAG AAG ACC CTG AGG ACC -3 ' c 5" GGT CCT CAG GGT CTT CTT GGT -3 ' b

5" TCC AGC AAT ACC CTG AGO TCC -3' f S S N T L R S s

5" GGA CCT CAG GGT ATT GCT GGA - 3 ' p

G ~ W P Q G I A G 7 ~ e h

T K K T L R T d

FIG. 1. Sequences of the domains in interstitial collagen which are attacked by mammalian collagenases and the pu- tative collagen-binding domains of collagenase. a, amino acid sequence of human a2 chain of type I collagen; b, corresponding nucleotide sequence from cloned a2(I) collagen chain; c, nucleotide sequence of the DNA strand complementary to the 4 1 ) collagen chain depicted in b; d, corresponding amino acid sequence; e, amino acid sequence coded for the DNA strand, shown in f , complementary to that coding for the collagenases susceptible domain in al(I) collagen chain; f , nucleotide sequence coding for the amino acid sequence shown in e; g, nucleotide sequence from ~ ~ ( 1 ) collagen gene which codes for the collagenase-susceptible domain; h, corresponding amino acid sequence.

FIG. 2. Hydropathy plot of predicted peptides and peptide

0, SSNTLRS; A, SQNPVQP, a sequence found in fibroblast colla- domains present in mammalian collagenases. 0, TTKKTLRT;

genase; 0, SSNPIQP, a sequence present in neutrophil collagenase.

Determination of a Collagen Binding Site in Collagenase 13765

et al., 1990: Clarke et al., 1990), peptides exist, at exactly the same alignment position in the molecule, which show striking similarity with respect to their hydropathic profiles to those predicted from cloned information, concerning the cleavage site, both of human az(I) and mouse al(I) chains. Remarkably however, there is divergence as far as primary structure is concerned. We have discussed earlier the conservation of shape or of hydropathy despite the evolutionary drift of primary structure (Brentani, 1988,1990).

Labeled collagen can bind in a saturable fashion to the theoretically predicted peptide TKKTLRT (Fig. 3a). Fig. 3b shows that this binding can be inhibited both by TKKTLRT itself and by SQNPVQP, a peptide present in fibroblast collagenase, but not by the "scrambled" peptide LTTTKKR which contains the same amino acid residues as TKKTLRT but displays a completely different hydropathic profile (Fig. 36, insert). This result suggests that SQNPVQP mimics the predicted peptide TKKTLRT in its collagen binding activity.

To gain further insight into the problem, TKKTLRT was used to raise polyclonal mouse antibodies. As seen in Fig. 4, these antibodies can bind to purified human collagenase ( A ) and this binding can be inhibited, in a dose-dependent fash- ion, by TKKTLRT and SQNPVQP but not by the scrambled peptide LTTTKKR ( B ) . This inhibition pattern suggests therefore that antibodies directed toward TKKTLRT recog- nize the sequence SQNPVQP in collagenase. It is important to note that additional control peptides (WTVPTA and GAV- STA) with different hydropathic profiles were used in the assays shown in Figs. 3b and 4B and they did not show any

M M --la

Inhlbltlan

80 -

60 -

40 -

20 -

' ' L 8 8 ' 8 8

0.6 6 60 600 Peptides (uM)

FIG. 3. a, binding of labeled collagen to TKKTLRT (0). Incubat- ing increasing concentrations of labeled collagen in TKKTLRT- coated wells, a saturation curve was obtained. Specific binding was defined as total hound minus nonspecific binding (on bovine serum albumin) For the inhibition assays shown in b, labeled collagen was preincubated for 2 h with increasing concentration of TKKTLRT (O), SQNPVQP (A), and LTTTKKR (0). Every data point is the average of triplicates.

5000

Absorband492 nm)

0.26 -

0.2 -

0.16 -

0.1 -

0 ,T. ,. m - -

0 100 200 300 400 600 600 700 800 Dilutions (1:~)

Inhibition 4i " - 1

0 100 200 300 400 500 600 PEPTIDE (uM)

FIG. 4. Characteristics of the binding between anti- TKKTLRT antibodies and purified collagenase (a kind gift from Dr. Arthur Eisen, Washington University, St. Louis, MO). A: A, binding of anti-TKKTLRT anti-serum to fibroblast collagenase as measured by an ELISA assay; 0, normal serum reac- tion with the same enzyme. B: to determine inhibition of the binding shown in A by peptides, anti-TKKTLRT serum was preincubated with TKKTLRT (O), SQNPVQP (A), and LTTTKKR (0) for 60 min before adding it to collagenase-coated wells.

inhibitory activity (data not shown). Furthermore, this mouse serum can recognize in Western blots of trypsin-activated supernatants from cultured human keratinocytes, a band displaying the same electrophoretical mobility as collagenase (about 55-57 kDa) (Fig. 5, lane 1 ); this serum can also immunoprecipitate collagenase from the same supernatant as demonstrated by zymographic analysis (Fig. 5, lane 3 ) and by Western blotting using a specific anti-collagenase anti-serum (Fig. 5, lane 5 ) . Petersen et al. (1987) have shown that kera- tinocyte-derivated collagenase is similar to that produced by human dermal fibroblasts both in a latent form as well as in an active one. Curiously, our active collagenase (Fig. 5 lane 3 ) shows a molecular weight corresponding to the latent form. An increase in collagenase activity occurred after the activa- tion step (data not shown). We can not find any explanation to this. However, as shown in Fig. 5, lane 5, a specific anti- collagenase antiserum recognizes the material brought down by anti-TKKTLRT antiserum. In addition, Birkedal-Hansen et al. (1988) have reported that 57-kDa collagenase may exist in an active as well as a latent form. In lane 5, the serum against fibroblast collagenase also recognizes a band running between 67 and 92 kDa. It is possible that this band corre- sponds to a complex of both collagenase and TIMP.

All the results thus far indicate that TKKTLRT and SQNPVQP share some characteristics and support collagen binding. In addition, antibodies raised against TKKTLRT seem to recognize epitopes present in the collagenase sequence

13766 Determination of a Collagen Binding Site in Collagenase

205 -

116 - 9 7 -

6 7 -

4 5 -

1 2 3 4 5 6 100, Inhibition %

. . i

I ,

!

I I

FIG. 5. Analysis of the protein recognized by anti- TKKTLRT antibody in cultured keratinocyte supernatant. The ahility of anti-TKKTLRT to recognize collagenase from cultured keratinocyte supernatants was assessed by different approaches. Lane I , Western blot analysis of cultured keratinocyte supernat.ant with anti-TKKTLRT antibodies; lane 2, normal serum reaction; lane 3, zymographic analysis of the material immunoprecipitated by anti- TKKTLRT antiserum from cultured keratinocyte supernatant; lane 4 , zymographic analysis of the material immunoprecipitated by nor- mal mouse serum from cultured keratinocyte supernatant; lane 5, Western blot of the material immunoprecipitated by anti-TKKTLRT antiserum from cultured keratinocyte supernatant using a specific anti-collagenase antiserum; lane 6, normal rabbit serum reaction.

SQNPVQP. If the assumption that the complement of the collagen cleavage site is contained within a functionally im- portant domain of the collagenase molecule is correct, anti- TKKTLRT antibodies should inhibit collagenolytic activity. This was tested by examining the effect of the TKKTLRT anti-serum IgG fraction on the collagenolytic activity from cultured keratinocyte supernatants. We decided to use the IgG fraction because human serum contains some metallopro- teinase inhibitors such as TIMP and oc2-macroglobulin. In- deed, anti-TKKTLRT antibodies were able to inhibit colla- genase (Fig. 6). From this graph it is possible to determine the apparent inhibition constant (Kin,,,,) of the competitor which is the order of 0.3 PM. One may ask if the inhibition of collagenolytic activity by a protein with 150 kDa is specific. Recently, Birkedal-Hansen et al. (1988) have produced many monoclonal antibodies against fibroblast collagenase, and most of them didn't inhibit its enzymatic activity. Moreover, because SQNPVQP can support collagen binding it is unlikely that this inhibition is due to steric hindrance. In addition, TKKTLRT and SQNPVQP themselves inhibited the colla-

60 I 1 ,A'!

FIG. 6. Effect of anti-TKKTLRT (0) and normal IgG (0) on the collagenolytic activity of cultured keratinocyte superna- tant as measured by the method of Yoshioka et al. (1987). From the curve we deduce a K,,, = 0.3 p ~ .

80 1

A 8 C D E F

FIG. 7. Effect of TKKTLRT, SQNPVQP, and LTTTKKR on collagenolytic activity of keratinocyte supernatant. A , TKKTLRT, 2 mM; B, TKKTLRT, 5 mM; C, SQNPVQP; 2 mM; D, SQNPVQP, 5 mM; E, LTTTKKR, 2 mM; F, LTTTKKR, 5 mM.

genolytic activity of keratinocyte supernatant measured by the liberation of labeled collagen fragments (Fig. 7). These results strongly suggest that the sequence SQNPVQP may be involved in collagenase binding to collagen.

I t is well known that the hydropathy coefficient of amino acids is based on their chemical characteristics. Our results favor the idea already reported by us and others that peptides with similar hydropathic profile show the same binding activ- ity. This phenomenon could be due to the chemical charac- teristics of the peptides which impose restricted conforma- tions to them. This idea is still controversial despite a large body of evidence in its favor. Our group has addressed this issue by using anti-peptide monoclonal antibodies. It was shown that they recognize the conformation of the peptides used for immunization rather than their primary structures (Brigido et al., 1990). Since elucidation of structure-function relationships of proteins is one of the most attractive aims for those who are working in the field of protein chemistry, we believe that sense-antisense peptide interaction can be a useful tool in order to understand some critical points of this area.

Collagenase are members of a larger family of zinc-metal- loproteinases and their zinc binding as well as their cysteine switch domains are conserved in all family members (Gold-

Determination of a Collagen Binding Site in Collagenase 13767

berg et al., 1986; Hasty et al., 1990; Clarke et al., 1990; Wilhelm et al., 1987; Saus et al., 1988; Enghildet et al., 1989; Collier et al., 1988). Despite this, in no other well-characterized metal- loproteinases such as stromelysins 1-111 and type IV collagen- ase, peptides were found similar to those described in the present study, both with regard to their amino acid sequence or their hydropathic profile. It is important to note, however, that in native collagen-degrading members, the hydropathic profile of SQNPVQP is conserved (Fig. 2). In rat collagenase, however, the hydrophobic amino acid is replaced by a partially hydrophilic one (Quin et al., 1990). It is further interesting to observe that SQNPVQP localizes, in the enzyme molecule, close to the recognized zinc binding site (VAAHELGHSLG).

The principle of complementary hydropathy has been val- idated in a variety of peptide interactions (Fassina et al., 1989; Shai et al., 1989; Bost et al., 1985; Knutson, 1988; Fassina et al., 1989; Carr et al., 1989) and thus far, in only few models of full protein interactions: self-binding antibodies (autobodies) (Kang et al., 1988); binding of fibronectin to its PI and P3 integrin receptors (Brentani et al., 1988; Pasqualini et al., 1989); and binding of fibrinogen to integrin GPIIb/IIIa (Cal- vete et al., 1991). We have now shown that it is possible to predict, from the nucleotide sequence complementary to that coding for the cleavage site of type I collagen, a peptide which mimics very accurately the hydropathic profile of peptides which are present in cloned mammalian collagenases. This peptide binds collagen type I and such binding is inhibitable by the peptide itself and by that present in cloned fibroblast collagenase (SQNPVQP) but not by a “scrambled” peptide which contains the same amino acid residues but displays a different hydropathic profile. Furthermore, antibodies raised against it recognize collagenase in ELISAs and Western blots and can immunoprecipitate it from cultured keratinocyte supernatants. Additionally, they inhibit collagenolytic activ- ity. The extension of the principle of complementary hydrop- athy to a proteolytic enzyme-substrate interaction strengbh- ens the idea that sequences coding for interacting exons were once displayed in complementary DNA strands at the same locus as a means of speeding up evolution (Brentani, 1988).

Acknowledgments-We thank L. L. Villa for critical review of the manuscript, Dr. Arthur Eisen for purified human collagenase and anti-collagenase antiserum, K. B. Vieira for cultured keratinocyte supernatant, M. M. Brentani for type I collagen, and Wilson Savino for anti-type I collagen rabbit antiserum.

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