©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Degradation of the COL1 Domain of Type XIV Collagen by 92-kDa Gelatinase (*)

(Received for publication, August 1, 1994; and in revised form, October 31, 1994)

Ulrike I. Sires (1) (2)(§) Bernard Dublet (3) Elisabeth Aubert-Foucher (3) Michel van der Rest (3) Howard G. Welgus (1)

From the  (1)Division of Dermatology, Department of Medicine, Washington University School of Medicine at The Jewish Hospital, St. Louis, Missouri 63110, the (2)Department of Pediatrics, St. Louis Children's Hospital, St. Louis, Missouri 63110, and the (3)Institute for Biology and Chemistry of Proteins, CNRS, UPR 412, Lyon, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Type XIV collagen is a newly described member of the fibril-associated collagens with interrupted triple helices (FACITs). Expression of this collagen has been localized to various embryonic tissues, suggesting that it has a functional role in development. All FACITs thus far described (types IX, XII, XIV, and XVI) contain a highly homologous carboxyl-terminal triple helical domain designated COL1. We have studied the capacity of various matrix metalloproteinases (interstitial collagenase, stromelysin, matrilysin, and 92-kDa gelatinase) to degrade the COL1 domain of collagen XIV. We found that only 92-kDa gelatinase cleaves COL1. Furthermore, digestion of whole native collagen XIV by the 92-kDa gelatinase indicates that this enzyme specifically attacks the carboxyl-terminal triple helix-containing region of the molecule. COL1 is cleaved by 92-kDa gelatinase at 30 °C, a full 5-6 °C below the melting temperature (T) of this domain; native collagen XIV is also degraded at 30 °C. In comparison to interstitial collagenase degradation of its physiologic native type I collagen substrate, the 92-kDa enzyme cleaved COL1 (XIV) with comparable catalytic efficacy. Interestingly, following thermal denaturation of the COL1 fragment, its susceptibility to 92-kDa gelatinase increases, but only to a degree that leaves it several orders of magnitude less sensitive to degradation than denatured collagens I and III. These data indicate that native COL1 and collagen XIV are readily and specifically cleaved by 92-kDa gelatinase. They also suggest a role for 92-kDa gelatinase activity in the structural tissue remodeling of the developing embryo.


INTRODUCTION

The collagen family of extracellular matrix proteins can be divided into several groups, each with distinct structural and functional characteristics(1) . The historically oldest group, the fibrillar collagens (types I, II, III, V, and XI), contains members that form banded collagen fibrils. Type XIV collagen (2, 3) belongs to the more recently recognized and consequently less understood group of fibril-associated collagens with interrupted triple helices (FACITs). (^1)FACITs share regions of high sequence homology (3, 4, 5) and distinct structural characteristics. By definition, molecules in this class contain more than one triple helical domain separated by nontriple helical segments(6, 7) . FACITs do not require proteolytic processing from a procollagen form as the fibrillar collagens do. Furthermore, the FACITs do not form homopolymers but rather are associated with collagen fibrils. In fact, their multidomain structure and tissue distribution suggest that FACITs constitute molecular bridges linking together the fibrils of a tissue (8, 9) . Four FACITs are known at present, types IX, XII, XIV, and XVI, the best characterized being type IX, associated with type II collagen fibrils(10, 11) , and a major constituent of hyaline cartilage. Type XII collagen is found in dense connective tissues such as tendons, ligaments, and perichondrium(12, 13, 14) . The homology between types IX and XII collagens is highest in the short carboxyl-terminal triple helical domain designated COL1(15) . This triple helical domain has been used as a ``signature'' to define FACIT molecules(16) . The structure of COL1 is unique to FACITs and is characterized by the presence of a precisely conserved pair of cysteine residues and by imperfections in the triple helix(3) . While cysteine residues and helix imperfections have been found in other collagen types (e.g. type IV collagen(17) ; type VI collagen(18) , and type XIII collagen(19) ), their locations are completely different from those observed in FACITs.

Recently described FACITs with COL1-like domains include types XIV and XVI collagens(3, 20, 21, 22) . Type XIV collagen was isolated from tissues containing predominantly type I collagen, such as skin and tendon(3) . The biologic function of type XIV collagen is not yet clearly understood. However, aside from a role in linking together fibrils of a tissue, collagen XIV has been shown to interact with heparin sulfate proteoglycan, collagen VI and the dermatan sulfate side chain of decorin and may be important in cell-matrix interactions(23, 24) . As determined by cDNA analysis, collagen XIV has a number of sequences in its nonhelical domains that are homologous to other structural proteins and has two RGD potential cell recognition sequences in its COL2 domain (7, 25) . Collagen XIV has also been localized around growing hair follicles, which suggests a role for this molecule in the formation of skin appendages(26) . Interactions between epidermis and dermis are vital for hair follicle initiation and development(27) . The prominent location of collagen XIV in developing hair follicle mesenchyme may be important for such epidermal-mesenchymal interactions(26) .

The matrix metalloproteinases (^2)are a gene family of enzymes that contribute to the degradation and remodeling of connective tissues(28) . These enzymes are important in normal physiologic remodeling such as wound healing (29) and uterine involution(30, 31) , but they have also been implicated in destructive pathologic processes including rheumatoid arthritis (32) and tumor invasion(33) . Interstitial collagenase, the most thoroughly studied member of this gene family, catalyzes the initial and rate-limiting step in the cleavage of native types I, II, and III collagens(34, 35, 36) . The 92-kDa and 72-kDa gelatinases are structurally related enzymes that are produced by different cell types. In addition to denatured collagens (gelatins) of all genetic types, both gelatinases degrade native types IV and V collagens, fibronectin, and insoluble elastin (37, 38) . Stromelysins-1 and -2 are highly related metalloproteinases with the capacity to attack a broad range of matrix substrates including laminin, fibronectin, proteoglycans, and types IV and IX collagens in their nonhelical domains(28, 37, 39) . Matrilysin is the smallest metalloproteinase, but it exhibits potent catalytic activity against noncollagenous matrix components including proteoglycans, fibronectin, laminin, entactin, and elastin(37, 40, 41) .

The susceptibility of most FACITs to the matrix metalloproteinases has not been studied. Yet, these collagens must be degraded during the turnover of fibrillar collagens with which they are so closely associated. The purpose of this study was to examine the susceptibility of one such FACIT, collagen XIV, and in particular its COL1 domain, to cleavage by matrix metalloproteinases.


MATERIALS AND METHODS

Reagents

4-20% polyacrylamide gradient gels, acrylamide and prestained molecular weight standards were purchased from Bio-Rad. Bisacrylamide was from Eastman Kodak Company. SDS, 99% pure, was obtained from BDH Chemicals Ltd. (Poole, United Kingdom). Tris base and p-aminophenylmercuric acetate were purchased from Sigma. All other chemicals were reagent grade.

Preparation of Types I, III, and XIV Collagens

Native rat tendon type I collagen was prepared by acid extraction followed by salt and ethanol precipitations as described previously(42) . Human placental type III collagen, purified by pepsin treatment and differential salt precipitation, was kindly provided by Robert Burgeson (Massachusetts General Hospital, Boston, MA). Native bovine type XIV collagen was purified by the method of Aubert-Foucher et al.(4) . Briefly, a fetal bovine tendon NaCl extract was batch-absorbed to carboxymethyl-cellulose. After extensive washing with 10 mM Tris-HCl, pH 7.4, containing 0.1 M NaCl and 1% isopropyl alcohol, elution was carried out with the same buffer in 0.5 M NaCl. At this point, the eluted material contained mainly a mixture of type I and type XIV collagens. Final purification was achieved using concanavalin A-Sepharose chromatography. As neither denaturing agents nor proteolytic enzymes were present during these procedures, the resultant collagen is native and intact.

Purification of COL1 Domain

The purification of the COL1 domain of collagen XIV was performed as described previously(4) . Fetal calves (estimated age, 6-7 months) were obtained at a local slaughterhouse. Skins (hairless) were cut in small pieces at 4 °C, frozen in liquid nitrogen, and milled in a freezer mill (SPEX Industries, Metuchen, NJ). The powders were suspended in a 0.5 M acetic acid, 0.2 M NaCl solution (15 ml/g of fresh tissue) and treated with pepsin (0.5 mg/ml) (Sigma) for 24 h at 4 °C. The digestion was stopped by bringing the pH to 8.5 by the addition of 5 M NaOH. After centrifugation (12,000 times g for 30 min) the supernatant was salt fractionated by dialysis against increasing concentrations of NaCl (0.9 M, 1.2 M, and 2.0 M) in 0.5 M acetic acid. The material was collected at each step by centrifugation (12,000 times g for 45 min) and dialyzed against 0.5 M acetic acid.

High Performance Liquid Chromatography (HPLC)

The 2.0 M NaCl precipitate was chromatographed by HPLC as described previously (4) on a reverse phase column using an aqueous acetonitrile gradient (16-56%, 60 min) in the presence of 10 mM heptafluorobutyric acid as an ion pairing agent. The equipment used was from Waters and consisted of a model 625 LC chromatograph and a model 484 tunable absorbance detector adjusted at a wavelength of 206 nm. The column used was a C(18) Waters Delta Pak (3.9 times 300) protected with a Waters Guard-Pak precolumn.

Fast Protein Liquid Chromatography

The HPLC system described above was used for fast protein liquid chromatography. The fractions from the HPLC containing the COL1 domain were dialyzed against 50 mM sodium acetate buffer, pH 4.8, and chromatographed on a Mono S column (5 mm times 5 cm) (Pharmacia Biotech Inc.). The COL1 domain was eluted by a NaCl gradient (0-1 M, 20 min, 2 ml/min).

Production and Purification of Metalloproteinases

Human interstitial procollagenase was purified from the serum-containing conditioned media of phorbol-treated U937 cells, as described previously(43, 44) . Briefly, media was subjected to sequential CM-52 followed by DEAE/red-Sepharose chromatography, resulting in fully pure enzyme maintained in a zymogen state. Recombinant 92-kDa gelatinase was isolated from E1A-transfected HT-1080 cells as described by Wilhelm et al.(45) . Purification was achieved by sequential gelatin-agarose and red-Sepharose chromatography and resulted in 92-kDa enzyme free of associated tissue inhibitor of metalloproteinases (TIMP). Matrilysin was expressed in non-secretor zero mouse myeloma cells and the native recombinant protein purified as described previously(37) . Stromelysin was purified from the conditioned media of interleukin-1-stimulated human skin fibroblasts by subjecting the material to zinc-chelate chromatography (Sigma) using a 0.05-0.8 M glycine gradient. Stromelysin-containing fractions were then placed over a DEAE-Sepharose (Pharmacia) column linked to reactive red 120-agarose (Sigma). The enzyme was eluted with a 0.05-2.0 M NaCl gradient, and stromelysin-containing fractions were collected and pooled. 72-kDa progelatinase was purified as described (46) from human fibroblast conditioned media. This 72-kDa enzyme was isolated as a complex with TIMP-2. 72-kDa gelatinase, largely free of TIMP-2, was kindly provided by Jo Seltzer (47) (Washington University, St. Louis, MO).

Activation of Metalloproteinase Zymogens

Interstitial procollagenase, 92-kDa progelatinase, 72-kDa progelatinase, prostromelysin, and promatrilysin were generally all activated by exposure to 1 mMp-aminophenylmercuric acetate at 37 °C for 2 h. In the sole case of experiments designed to compare the catalytic efficacy of interstitial collagenase against type I collagen to 92-kDa gelatinase against COL1 (XIV), the following methodology was employed to ensure maximal and irreversible activation. Procollagenase was activated by sequential exposure to trypsin and soybean trypsin inhibitor (8-fold molar excess to the serine protease) as reported previously(34) . 92-kDa progelatinase was activated by treatment with a 1:20 ratio (w/w) of trypsin-activated stromelysin at 37 °C for 1 h(48) . The stromelysin was then removed by adsorption to antistromelysin-Sepharose. No stromelysin remained by enzyme-linked immunosorbent assay (detection limit, 1 ng).

Verification of Catalytic Activity for Each Metalloproteinase

Proteolytic activity of each of the purified metalloenzymes was established by assay against known susceptible substrates as follows: 1) 92-kDa gelatinase and matrilysin activities were tested by measuring the solubilization of [^3H]elastin at 37 °C(38) ; 2) collagenase activity was determined by degradation of native type I collagen at 25 °C and quantification of three-quarter-length tropocollagen^A products using scanning densitometry(34) ; and 3) stromelysin activity was measured using a proteoglycan bead assay(49) .

Degradation of the COL1 Domain of Type XIV Collagen by Matrix Metalloproteinases

COL1 (2.8 µg) was incubated for 18 h at 30 °C with 6.5 times 10M each of matrilysin, interstitial collagenase, 92-kDa gelatinase, or stromelysin in a final volume of 34 µl (0.05 M Tris, pH 7.5, 0.01 M CaCl(2), 0.15 M NaCl, and 0.02% Brij). The reactions were stopped with SDS sample buffer containing dithiothreitol, boiled, and subjected to polyacrylamide gel electrophoresis(50) . The gels were then stained with 1% Coomassie Brilliant Blue and densitometrically scanned with a Gilford spectrophotometer set at 560 nm.

Denaturation of Types I and III Collagens and the COL1 Domain of Type XIV Collagen

Native collagens were heated to 60 °C for 15 min and then used immediately in reactions performed at 30 °C.

TIMP Inhibition of 92-kDa Gelatinase

TIMP was purified from human skin fibroblast-conditioned serum-free medium as described previously(51) . 92-kDa gelatinase (2.4 µg) was incubated either alone or with TIMP (1.62 or 8.10 µg) for 15 min at room temperature. The enzyme and enzyme/TIMP mixtures were then added to COL1 for 18h at 37 °C, and degradation products were resolved by polyacrylamide gel electrophoresis.

Determination of the Melting Temperature of the COL1 Domain

The COL1 domain purified by fast protein liquid chromatography was treated with trypsin as described by Bruckner and Eikenberry(52) . The samples were heated from 30 to 39 °C at a rate of 12 °C/h. At the indicated temperatures, the samples were rapidly cooled in an ice bath. After adding trypsin to 0.01 mg/ml, the samples were incubated for 2 min at 20 °C to digest nontriple helical protein. The samples were then prepared for analysis by SDS-polyacrylamide gel electrophoresis.

Comparison of the Catalytic Efficacy of Interstitial Collagenase against Type I Collagen to 92-kDa Gelatinase Against COL1 (XIV)

The concentrations of type I collagen and COL1 (XIV) were determined by hydroxyproline assay as described by Bergmann and Loxley (53) . Equimolar concentrations of type I collagen and COL1 (XIV) were used in all assays (4 times 10M). Reaction mixture volume was 30 µl, and the buffer used was 0.05 M Tris, pH 7.5, containing 0.01 M CaCl(2), 0.4 M NaCl, and 0.02% Brij. The 0.4 M NaCl was used to retard any aggregation of type I collagen during the incubation period. Reactions were performed at 30 °C and 32 °C for 1 h, as indicated. Molarities of 92-kDa gelatinase used to degrade COL1 at 30 °C were 6.6 times 10M, 3.3 times 10M, and 1.6 times 10M; molarities at 32 °C were 5.3 times 10M, 1.3 times 10M, and 4.4 times 10M. Molarities of collagenase used to degrade type I collagen at 30 °C were 3 times 10M, 1.5 times 10M, and 7.5 times 10M; molarities at 32 °C were 1.0 times 10M, 3.3 times 10M, and 1.1 times 10M. Degradation was quantified by scanning the gels densitometrically with a Beckman spectrophotometer equipped with a linear scanning device.


RESULTS

T Determination for the COL1 Domain of Type XIV Collagen

Prior to studying the degradation of COL1 by matrix metalloproteinases, the melting temperature of this helical fragment was established by determining its trypsin sensitivity after incubation over the temperature range of 30-39 °C (Fig. 1). As indicated, this helical fragment exhibited no trypsin sensitivity whatsoever until 35 °C, and the T(m) was found to be 35-36 °C. In view of this result, we performed all subsequent experiments with matrix metalloproteinases at 30 °C, a full 5 °C below the melting temperature of COL1, thereby ensuring that this collagenous fragment would be maintained in a native state.


Figure 1: Trypsin susceptibility of the COL1 domain of type XIV collagen as a function of temperature. COL1 (2 µg) was incubated at the temperatures indicated at the top of the figure and treated with trypsin as described under ``Materials and Methods.'' The reaction mixtures were resolved by nonreducing SDS-polyacrylamide gel electrophoresis (15% gel), and protein bands were stained with Coomassie Blue. Electrophoretic mobility of CNBr fragments from type I collagen are indicated as standards on the left of the figure. Electrophoretic mobility of the COL1 domain is shown by the arrow on the right of the figure.



Degradation of the COL1 Domain of Type XIV Collagen by Matrix Metalloproteinases

The capacity of four different matrix metalloproteinases (92-kDa gelatinase, stromelysin, interstitial collagenase, and matrilysin) to degrade the COL1 domain of type XIV collagen was examined. In Fig. 2, COL1 (2.8 µg) was incubated with equimolar concentrations (6.5 times 10M) of 92-kDa gelatinase, stromelysin, collagenase, and matrilysin for 18 h at 30 °C. Under these conditions, the only enzyme that exhibited capacity to degrade the COL1 domain was the 92-kDa gelatinase. There were no distinct cleavage products identifiable from the action of 92-kDa gelatinase, which apparently degraded the COL1 domain into small peptides. The highly related 72-kDa gelatinase, which exhibits virtually identical substrate specificity to 92-kDa gelatinase(37) , also degraded COL1 at 30 °C (data not shown). The COL1 domain appears as a doublet in these denaturing electrophoretic gels because two different amino-terminal sequences of COL1, VRTIQGPP and IQGPP, are produced during the purification of this domain. The resultant difference in molecular mass is approximately 1 kDa as the three chains are identical. Also, since COL1 is prepared by pepsin cleavage and the specificity of this enzyme is wide, the carboxyl terminus is likely heterogeneous as well.


Figure 2: Degradation of the COL1 domain of type XIV collagen by matrix metalloproteinases. The COL1 domain of type XIV collagen (2.8 µg) was incubated with the indicated metalloproteinases at 30 °C for 18 h. The enzymes were all used at a final concentration of 6.5 times 10M. Reactions were stopped by boiling in SDS sample buffer containing dithiothreitol and then applied to a 15% polyacrylamide gel.



Degradation of the COL1 domain of type XIV collagen demonstrated significant temperature dependence. As shown in Fig. 3, at 32 °C and pH 7.4, 92-kDa gelatinase (6.3 times 10M) degraded approximately 90% of the COL1 fragment. However, at 25 °C, an identical amount of enzyme cleaved only a trivial proportion of the substrate.


Figure 3: Temperature dependence of the degradation of the COL1 domain of type XIV collagen by 92-kDa gelatinase. COL1 (3.0 µg) was incubated with 92-kDa gelatinase (6.3 times 10M) at the various temperatures indicated for 24 h. The enzyme-free control was incubated at 25 °C. Reactions were stopped by boiling in SDS sample buffer containing dithiothreitol and then applied to a 4-20% gradient gel.



Comparison of the Catalytic Efficacy of 92-kDa Gelatinase Degradation of COL1 (XIV) with Interstitial Collagenase Cleavage of Type I Collagen

To assess the potential relevance of 92-kDa gelatinase degradation of COL1, its efficacy of catalysis was compared with that of interstitial collagenase degradation of native type I collagen, the latter generally accepted to be of high physiologic importance. Incubations were performed at 30 and 32 °C for 1 h as indicated. As shown in Fig. 4, at 30 °C, approximately 50% degradation of type I collagen required 0.15 µg of interstitial collagenase versus 0.625 µg of 92-kDa gelatinase to achieve similar cleavage of COL1. Normalizing for differences in molecular mass, 92-kDa gelatinase cleavage of COL1 occurred at approximately half the rate of collagenase cleavage of type I collagen. At 32 °C, approximately 50% degradation of type I collagen required 0.08 µg of interstitial collagenase versus 0.08 µg of 92-kDa gelatinase to achieve similar cleavage of COL1. On a molar basis, 92-kDa gelatinase cleavage of COL1 occurred twice as readily as collagenase cleavage of type I collagen. Thus, rates of 92-kDa gelatinase degradation of COL1 are of quite similar magnitude to those of interstitial collagenase degradation of type I collagen.


Figure 4: Comparison of 92-kDa gelatinase degradation of COL1 with interstitial collagenase cleavage of native type I collagen. Incubation of equimolar concentrations of COL1 (XIV) and type I collagen (4 times 10M) were performed with the target enzymes at 30 and 32 °C for 1 h, as described under ``Materials and Methods.'' Molarities of 92-kDa gelatinase used to degrade COL1 at 30 °C were 6.6 times 10M, 3.3 times 10M, and 1.6 times 10M. Molarities at 32 °C were 5.3 times 10M, 1.3 times 10M, and 4.4 times 10M. Molarities of collagenase used to degrade type I collagen at 30 °C were 3 times 10M, 1.5 times 10M, and 7.5 times 10M. Molarities at 32 °C were 1.0 times 10M, 3.3 times 10M, and 1.1 times 10M. Reaction mixtures using COL1 were separated on a 23% polyacrylamide gel. Type I collagen products were separated on an 8% polyacrylamide gel. Degradation was determined by densitometric scanning of substrate disappearance.



Degradation of Native versus Denatured COL1 by 92-kDa Gelatinase

Since 92-kDa gelatinase exhibits high efficiency in the degradation of denatured collagens (i.e. gelatin) of all genetic types, we compared the capacity of this enzyme to cleave native versus denatured COL1 fragment of type XIV collagen and also the corresponding forms of types I and III collagens. In Fig. 5, the different native collagens were incubated with 2.6 times 10M 92-kDa gelatinase, and the respective denatured collagens were incubated with 10-fold decreasing amounts of enzyme starting from 2.6 times 10M (lane1) to 2.6 times 10M (lane5). The native and denatured collagens were exposed to enzyme at 30 °C for 18 h. As shown in Fig. 5A, native type I collagen was completely resistant to cleavage by 92-kDa gelatinase, but, after thermal denaturation, it became exquisitely sensitive to degradation by this enzyme. Fig. 5B shows very similar results with type III collagen. Native type III was not cleaved by the highest concentration of 92-kDa gelatinase employed (2.6 times 10M), whereas after denaturation, it was degraded even more readily than type I gelatin (to completion by 2.6 times 10M enzyme). Interestingly, the COL1 fragment of type XIV collagen, as shown in Fig. 5C, exhibited a very different pattern of susceptibility to 92-kDa gelatinase than types I and III collagen. Consistent with results we have shown earlier ( Fig. 2and Fig. 3), native COL1 was degraded by 92-kDa gelatinase, with approximately 50% of the substrate cleaved by 2.6 times 10M enzyme. Upon thermal denaturation of COL1, this fragment became approximately 50-fold more susceptible to 92-kDa gelatinase. However, as compared with types I and III gelatins, denatured COL1 was a surprisingly poor substrate. In fact, denatured COL1 was several orders of magnitude more resistant than its denatured types I or III collagen counterparts to proteolytic scission by the 92-kDa enzyme.


Figure 5: Degradation of native and denatured COL1 and of native and denatured types I and III collagens by 92-kDa gelatinase. A, native type I collagen (3.0 µg) was incubated at 30 °C for 18 h with either no(-) or 2.6 times 10M 92-kDa gelatinase. Denatured type I collagen was incubated with either no(-) or 10-fold dilutions of 92-kDa gelatinase starting in lane1 with 2.6 times 10M enzyme to 2.6 times 10M enzyme in lane5. Products were separated on a 10% polyacrylamide gel. B, native type III collagen (3.0 µg) was incubated at 30 °C for 18 h with either no (-) or 2.6 times 10M 92-kDa gelatinase. Denatured type III collagen was incubated with either no(-) or 10-fold dilutions of 92-kDa gelatinase starting in lane1 with 2.6 times 10M enzyme to 2.6 times 10M enzyme in lane5. Products were separated on a 10% polyacrylamide gel. C, native COL1 fragment of type XIV collagen (3.0 µg) was incubated at 30 °C for 18 h with either no(-) or 2.6 times 10M 92-kDa gelatinase. Denatured COL1 (XIV) was incubated with either no(-) or 10-fold dilutions 92-kDa gelatinase starting in lane1 with 2.6 times 10M enzyme to 2.6 times 10M enyzme in lane5. Products were separated on a 20% polyacrylamide gel.



TIMP Can Inhibit 92-kDa Gelatinase Degradation of the COL1 Fragment

TIMP is a physiologic inhibitor of 92-kDa gelatinase, which binds noncovalently but with high affinity to the enzyme and inhibits its catalytic activity. In Fig. 6, the COL1 domain of type XIV collagen was incubated with 92-kDa gelatinase for 24 h at 30 °C, resulting in approximately 90% substrate degradation. When the enzyme was incubated with COL1 in the presence of increasing amounts of TIMP, substrate degradation was inhibited dramatically.


Figure 6: TIMP inhibits degradation of the COL1 domain of type XIV collagen by 92-kDa gelatinase. COL1 (3.0 µg) was incubated with 92-kDa gelatinase (2.6 times 10M) at 30 °C for 24 h. In the reaction mixtures indicated, TIMP (1.6 or 8.1 µg) was added to the gelatinase as described under ``Materials and Methods.'' Products were resolved on a 4-20% polyacrylamide gradient gel.



Degradation of Whole Native Type XIV Collagen by 92-kDa Gelatinase

We next addressed whether and how the whole native type XIV collagen molecule is attacked by 92-kDa gelatinase. Type XIV collagen consists of two triple helical domains (COL1 and COL2) interspersing nontriple helical domains (NC1, NC2, and NC3). In Fig. 7, it is apparent that incubation of native type XIV collagen with 92-kDa gelatinase for 18 h at 30 °C leads to accumulation of the NC3 domain as a final product. This indicates that the ``tail'' region of the molecule consisting of (NC1 + COL1 + NC2 + COL2) has been removed and degraded. Furthermore, the arrowheads show two bands that accumulate as early cleavage products and whose electrophoretic mobility is intermediate between their undigested counterparts (alpha1[XIV] monomer and dimer) and the NC3 domain of type XIV collagen. To exhibit this electrophoretic behavior, these species must retain the reduction-resistant disulfide bridge present in the NC2 domain of collagen XIV ((4) ). (^3)Therefore, cleavage of COL1 likely represents the initial point of attack of 92-kDa gelatinase against the native type XIV molecule.


Figure 7: Cleavage of whole native type XIV collagen by 92-kDa gelatinase. Two preparations of type XIV collagen (4.0 µg/lane) were used in this experiment. Lanes1-3 contain a mixture of type I and type XIV collagens from the carboxymethyl-cellulose pool obtained as described under ``Materials and Methods.'' Lanes4-7 contain pure bovine tendon type XIV collagen, which was obtained following concanavalin A-Sepharose chromatography as described under ``Materials and Methods.'' Lanes1 and 4 represent the respective collagen preparations that were not incubated. Lanes2 and 5 were incubated with buffer alone at 30 °C for 18 h. Lanes3 and 6 were incubated with 92-kDa gelatinase (0.5 µg) at 30 °C for 18 h. Lane7 contains the pure NC3 domain of the type XIV collagen, obtained as described (4) . The arrowheads mark partially digested forms of type XIV collagen, which must retain the disulfide bridge in the NC2 domain to exhibit this pattern of electrophoretic migration. After incubation, the samples were acetone-precipitated before being analyzed by 5% SDS-polyacrylamide gel electrophoresis.




DISCUSSION

In this study, we have examined the capacity of several human matrix metalloproteinases to degrade the COL1 fragment of type XIV collagen. Our data indicate that this important helical component of FACITs is susceptible only to 92- and 72-kDa gelatinases. The structure of COL1 is unique to FACITs and is characterized by the presence of a pair of cysteines, one at the carboxyl-terminal end of the triple helix and one 5 residues inside the amino-terminal NC1 domain. The COL1 domain of all FACITs has two imperfections of triple helical structure located at similar positions relative to the cysteines, one Gly-X-Gly-X-Y imperfection and one Gly-X-Y-X-Y imperfection. Disulfide bridges and helix imperfections have been found in other collagen types such as type IV collagen(17) , type VI collagen(18) , and type XIII collagen(19) ; of these collagens, only type IV is susceptible to 92-kDa gelatinase. In addition to type IV collagen, 92-kDa gelatinase is known to degrade a number of other matrix proteins including native collagen types V, VII, and X, as well as denatured collagens of all genetic types, fibronectin, and insoluble elastin(38, 46, 54) .

Helix imperfections of the COL1 domain may represent the target of attack of 92-kDa gelatinase. In this regard, we have previously shown that type X collagen is susceptible at two loci to interstitial collagenase and 72/92-kDa gelatinases, both cleavage sites constituting the Gly-X bonds of Gly-X-Y-X-Y sequences. Since 92-kDa gelatinase is known to be an efficient gelatinase, it was critical for us to determine the T(m) of the COL1 fragment of type XIV collagen. Using trypsin susceptibility as a probe, this T(m) was found to be 35-36 °C (Fig. 1). Therefore, we performed all experiments at 30 °C, a full 5 °C less than the T(m). Nevertheless, we found that 92-kDa cleavage of COL1 at 30 °C (or even at 28 °C, data not shown) failed to produce electrophoretically identifiable fragments (Fig. 2), suggesting that the products of initial helical scission(s) rapidly denature and are subsequently degraded to completion by the enzyme, therefore making the determination of a turnover number unfeasible. To place the rate of catalysis of the COL1 fragment by 92-kDa gelatinase into biological perspective, we compared it to the rate of cleavage of native type I collagen by interstitial collagenase. We found that at 30 °C, 92-kDa gelatinase degraded COL1 with approximately 50% the efficacy, on a molar basis, of interstitial collagenase cleavage of native type I collagen (Fig. 4). At 32 °C, 92-kDa gelatinase degraded COL1 approximately 2-fold as rapidly as collagenase degrades type I collagen. Therefore, 92-kDa gelatinase cleavage of the COL1 domain of type XIV collagen is approximately equivalent in rate to that of the physiologically accepted degradation of type I collagen by collagenase. It is important to note, however, that the specific activity of stromelysin-activated 92-kDa gelatinase (used in Fig. 4) is severalfold greater than that of p-aminophenylmercuric acetate-activated 92-kDa gelatinase (used in other figures of the manuscript). (^4)Furthermore, the degradation of COL1 shown in Fig. 2, Fig. 3, Fig. 5, and Fig. 6exceeded 75% and therefore greatly underestimates initial velocity. In any event, the importance of 92-kDa gelatinase cleavage of native COL1 is underscored by digestions of whole native type XIV collagen (Fig. 7), which strongly suggest that this helical domain represents a physiologic point of attack of the enzyme upon this entire native collagen molecule.

Thermal denaturation of COL1 increased its sensitivity to degradation by 92-kDa gelatinase approximately 50-fold (Fig. 5), supporting the concept that the initial cleavage of this collagen at 30 °C indeed represents catalytic activity against a native helical substrate. However, cleavage of denatured COL1 by 92-kDa gelatinase was surprisingly poor as compared with this enzyme's degradation of denatured types I and III collagens (Fig. 5). The explanation for this interesting finding is not at all clear. While the presence of intramolecular disulfide bonds might be expected to preserve tertiary structure following denaturation or perhaps even enhance its reformation, type III collagen, as well as COL1, contains these stabilizing elements. Nevertheless, this resistance of denatured COL1 to proteolysis as compared with denatured types I and III collagens was maintained when trypsin was substituted for 92-kDa gelatinase (data not shown).

By performing our studies with recombinantly expressed 92-kDa gelatinase and thereby using enzyme without associated TIMP, we were able to avoid the problem of TIMP-inhibited enzyme, which necessarily accompanies the isolation of 92-kDa progelatinase from normal mammalian cell cultures(55, 56) . Since we have shown such enzyme to be almost 10-fold as active as 92-kDa gelatinase purified as a complex with TIMP (55) , this maneuver was critical to our studies. As expected, however, the addition of exogenous TIMP was able to largely ablate the capacity of 92-kDa gelatinase to degrade the COL1 fragment of type XIV collagen (Fig. 6).

Type XIV collagen mRNA has been found in many tissues including lung, heart, muscle, skin, stomach, tendon, sternum, and calvaria(57) . It has also been localized to various embryonic tissues, indicating a role in development. For example, using monoclonal antibodies, type XIV collagen has been found in the embryo in large bundles of collagen fibrils within tendons and, interestingly enough, also around growing hair follicles in basket-like structures(26) . The specific localization of type XIV collagen around hair follicles suggests a function for this protein during the formation of skin appendages. During the invagination of epithelial cells forming the hair follicle there is extensive association with underlying dermis, and this extracellular matrix is probably a site of protein turnover. In fact, TIMP gene expression has been shown in the sheath of follicles that form vibrissae(58) . Furthermore, epithelial cells from hair follicles have been reported to express 72-kDa gelatinase (59) as well as the 92-kDa gelatinase. It may therefore be speculated that these gelatinases play an important role in basement membrane remodeling in the embryo, especially during the morphogenesis of hair follicles. Finally, since FACITs link interstitial collagens, the involvement of more than one matrix metalloproteinase (e.g. interstitial collagenase and a gelatinase) may be required to effectively degrade collagen fibrils otherwise composed largely of types I, II, or III collagens.


FOOTNOTES

*
This work was supported by Grants AR35805, HL47328, T32-AR07284, and HL29594 from the National Institutes of Health and by a grant from the CNRS. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Div. of Dermatology, Jewish Hospital, 216 South Kingshighway, St. Louis, MO 63110. Tel.: 314-454-8290; Fax: 314-454-8293.

(^1)
The abbreviations used are: FACITs, fibril-associated collagens with interrupted triple helices; TIMP, tissue inhibitor of metalloproteinases; HPLC, high performance liquid chromatography.

(^2)
EC numbers of the matrix metalloproteinases used are: EC 3.4.24.7, interstitial collagenase; EC 3.4.24.17, stromelysin-1; EC 3.4.24.35, 92-kDa gelatinase (gelatinase B); EC 3.4.24.24, 72-kDa gelatinase (gelatinase A); EC 3.4.24.23, matrilysin.

(^3)
E. Aubert-Foucher, unpublished observations.

(^4)
H. G. Welgus, unpublished observations.


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