THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 264, No. 7, Issue of March 5, pp. 3822-3826,1989 Printed in U. S. A.

Cleavage of Type VI1 Collagen by Interstitial Collagenase and Type IV Collagenase (Gelatinase) Derived from Human Skin*

(Received for publication, September 21, 1987)

Jo Louise Seltzer$, Arthur Z. EisenS, Eugene A. Bauers, Nicholas P. Morris711 , Robert W. GlanvillellII , and Robert E. BurgesonT))**$$ From the $Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, the §Department of Dermatology, Stanford University School of Medicine, Stanford, California 94305, and the Whriners Hospital for Crippled Children and the Departments of )I Biochemistry, **Cell Biology and Anatomy, and $$Dermatology, Oregon Health Sciences University, Portland, Oregon 97201

Type VI1 collagen is the major structural protein of anchoring fibrils, which are believed to be critical for epidermal-dermal adhesion in the basement membrane zone of the skin. To elucidate possible mechanisms for the turnover of this protein, we examined the capaci- ties of two proteases, human skin collagenase, which degrades interstitial collagens, and a protease with gelatinolytic and type IV collagenase activities, to cleave type VI1 collagen. At temperatures below the denaturation temperature, pepsin cleaves type VI1 col- lagen into products of -95 and -75 kDa. Human skin collagenase cleaved type VI1 collagen into two stable fragments of -83 and -80 kDa, and the type IV colla- genase (gelatinase) produced a broad band of -80 kDa as determined by sodium dodecyl sulfate-polyacryl- amide gel electrophoresis. Cleavage of type VI1 colla- gen was linear with time and enzyme concentration for both enzymes. Although the K,,, values were similar for both enzymes, the catalytic rate of cleavage by type IV collagenase is much faster than by interstitial col- lagenase, and shows a greater rate of increase with increasing temperature. Sequence analysis of the cleavage products from both enzymes showed typical collagenous sequences, indicating a relaxation in the helical part of the type VI1 collagen molecule at phys- iological temperature which makes it susceptible to gelatinolytic degradation.

Interstitial collagenase from both normal skin cells and cells from patients with recessive dystrophic epi- dermolysis bullosa, a severe hereditary blistering dis- ease in which both an anchoring fibril defect and ex- cessive production of collagenase can be observed, pro- duced identical cleavage products from type VI1 collagen. These data suggest a pathophysiological link between increased enzyme levels and the observed de- crease or absence of anchoring fibrils.

Type VI1 collagen was shown to be a unique gene product following purification from human amnion (1, 2). Each of the three identical a-chains/molecule consists of a 150-kDa glob- ular domain at the carboxyl terminus attached to a helical

Grants AM 12129, AR 19537, AM 35532, and AM 35689, by the * This work was supported by United States Public Health Service

Shriners Hospital for Crippled Children, the Washington University/ DEBRA Center for Research and Therapy of Epidermolysis Bullosa, and the Washington University-Monsanto Biomedical Research Agreement. 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.

section of 170 kDa. In tissue, type VI1 collagen is found arranged as antiparallel dimers, with the helical portions disulfide-bonded in a short overlap, and with the COOH- terminal globular positions at either end (3).

Immunologic methods have identified type VI1 collagen as the principal component of anchoring fibrils, structures which are believed to be important in attaching the epidermis to the dermis (4). Segment-long spacing crystallites of type VI1 collagen suggest that the triple helical domain can fully ac- count for the banded pattern observed in the anchoring fibrils (2). Ultrastructurally, these fibrils are seen to insert directly into the lamina densa and either arch and reinsert into that structure, or extend perpendicularly from the lamina densa and insert into anchoring plaques in the dermis which contain the 150-kDa globular domain. Additional anchoring fibrils bridge adjacent anchoring plaques to form an extended net- work of fibrils, entrapping large numbers of dermal fibrous components (5-8).

Anchoring fibrils appear to be absent and/or altered in several disease states, most notably recessive dystrophic epi- dermolysis bullosa (RDEB),’ a hereditary blistering disorder (9-12). It is not known whether this is due to a genetic defect in the expression of the anchoring fibril complex or to its degradation by excessive proteolysis in the dermis (13, 14).

Prolonged proteolysis of the helical portion of type VI1 collagen by pepsin results in two fragments, P1 and P2, of mass 95 and 75 kDa, respectively (1). Trypsin and chymo- trypsin also cleave this molecule in the central region, giving products of slightly different sizes.

Human skin fibroblast collagenase cleaves within the triple helix of interstitial collagens types I, 11, and 111, although it does not cleave two basement membrane collagens, types IV and V (15, 16). In this paper we present evidence that human skin collagenase, as well as a protease derived from human skin which displays both gelatinolytic (17) and types IV and V collagenolytic activities, currently referred to as type IV collagenase (see Ref. 18), can at physiologic temperature degrade the type VI1 collagen helix into products similar in size to the pepsin cleavage products. Unless prolonged incu- bation at greater than physiologic temperature occurs, these products retain helicity and are stable to further proteolysis.

MATERIALS AND METHODS

Preparation of Collagens-Type VI1 collagen was purified from human chorioamniotic membranes as previously described (1) and upon solubilization was stored frozen in 0.05 M Tris-HCI, pH 7.5, 0.4

The abbreviations used are: RDEB, recessive dystrophic epider- molysis bullosa; NaDodSOr-PAGE, sodium dodecyl sulfate-polyacryl- amide gel electrophoresis.

3822

Metalloprotease Degradation of Type VII Collagen 3823

M NaC1. "C-Labeled type I collagen was extracted from guinea pig skin by the method of Gross and Lapiere (15).

Purification of Enzymes-Collagenase was purified to homogeneity as the proenzyme from serum-containing culture medium of normal and recessive dystrophic epidermolysis bullosa human skin fibro- blasts, using a modification of the method of Stricklin et al. (19, 20). In this procedure, a concentration step on a Znz+/chelate affinity column was interposed between elution from CM-cellulose and gel filtration on Sephadex G-100. Type IV collagenase (gelatinase) was purified to homogeneity from medium of human skin in explant cultures by the method of Seltzer et al. (21).

Enzyme Assays-Procollagenase was fully activated by incubation with 1 mM phenyl mercuric chloride for 90 min at 37 "C (22). Type I collagenase activity was assayed by incubating the active enzyme with 25 p1 of a 2 mg/ml "C-labeled native reconstituted guinea pig collagen substrate gel (specific activity = 25,000 cpm/mg) at 37 "C as previ- ously described (23).

Type IV collagenase (gelatinase), which is fully activated when purified from skin explant culture medium, was assayed as described previously (17). Briefly, 25 pl of 14C-labeled guinea pig type I collagen was denatured by heating at 100 "C for 10 min. 75 pl of the purified enzyme was added, and after incubation at 37 "C, the reaction was terminated with 25 11 of 100% trichloroacetic acid. Product peptides soluble in 20% trichloroacetic acid were counted in a liquid scintilla- tion spectrometer.

The cleavage of type VI1 collagen by interstitial collagenase or type IV collagenase was carried out at varying temperatures and under varying conditions of enzyme concentration and times of incubation. Cleavage products were analyzed by separation of total reaction mixtures on 8% NaDodS04-PAGE according to the method of King and Laemmli (24), assuming molecular weights of -170,000 for the intact oll(VII) chains and -75,000 and -95,000 for the two pepsin fragments (1). The digestion of the starting material and the appear- ance of product peptides were quantitated by scanning of the Coo- massie Blue-stained gels on an LKB 2202 Ultroscan laser densitomer, using the Gelscan computer program for quantitation of the peaks.

Electroblotting and Sequencing-Protein sequencing was per- formed on an Applied Biosystems 470A gas-phase Sequenator. Sam- ples were electroblotted onto polyvinylidene difluoride membrane for NaDodS04-polyacrylamide gels and loaded directly onto the sequen- ator by the method of Aebersold et al. (25). Identification of the phenylthiohydantoin-derivatives was performed by high pressure liq- uid chromatography as previously described (26).

Other Assays-The protein concentration of enzyme preparations was determined spectrophotometrically by the method of Groves et 01. (27) using bovine serum albumin to establish a standard curve. Immunoreactive fibroblast collagenase was measured by enzyme- linked immunosorbent assay using the method of Cooper et al. (28).

RESULTS

We initially incubated the 510-kDa triple helical portion of type VI1 collagen with human skin collagenase at 28 "C. Product peptides of approximately half the size of the a-chain were observed. Increasing the incubation temperature to as high as 37 "C revealed more extensive cleavage without the formation of additional products (see below). Fig. 1 shows the human skin collagenase cleavage products of M, -83,000 and -80,000 as determined by gel electrophoresis. It is of interest that the collagenase produced by recessive dystrophic epider- molysis bullosa fibroblasts makes a cleavage that appears to be qualitatively identical to that of the normal collagenase.

The existence of two primary collagenase cleavage products of a size similar to those of the two pepsin products suggested a possible relaxation of the triple helix in the mid-portion of the type VI1 monomer. If this were the case, we reasoned that type VI1 collagen might also be susceptible to human skin gelatinase, a protease which has recently been shown to have type IV collagenolytic activity (18). Indeed this enzyme cleaved type VI1 collagen, yielding a broad band of -80 kDa (Fig. 1). A minor cleavage product of slightly smaller size was also produced (Fig. 2).

Circular dichroism (at 221 nm) of disulfide-bonded type VI1 collagen indicates that a biphasic denaturation occurs with

I

Normal Collagenase

0 I I 1 I I I L C

a - RDEB 1 Collagenase

2

a - C IViG

I Type IV

Gelatinase 6 -

2 -

4 -

Collagenase

0 . I I I I I I I I L 10 20 30 40

Posit ion Number

FIG. 1. Densitometric scan of type VI1 collagen cleavage by various human skin proteases. Type VI1 collagen (3 pg) was incubated at 37 "C for 18 h with purified normal human skin colla- genase (0.1 pg), RDEB collagenase (0.05 pg), or human skin type IV collagenase (gelatinase) (0.12 pg). The procollagenase was fully acti- vated by preincubation with 1 mM phenyl mercuric chloride at 37 "C for 90 min as described (21). Densitometric scan of type VI1 collagen cleavage products: Control, intact type VI1 collagen substrate; C, principal collagenase cleavage products; CEB, RDEB collagenase; C W I G , principal type IV collagenase (gelatinase) cleavage products.

values of T,,, a t 41 and 55 "C. The amplitudes of the transitions suggest that the major portion of the triple-helix melts at the lower temperature while the disulfide bond-stabilized 60-nm overlap exhibits a higher thermal stability.' To examine the possible effects of temperature on proteolytic susceptibility, we incubated type VI1 collagen with interstitial collagenase or type IV collagenase a t varying temperatures. Breakdown of type VI1 collagen by both enzymes was extremely temper- ature-dependent. By using a high ratio of substrate to enzyme, the same cleavage products as shown in Fig. 1 were obtained at 28 and 32 "C (data not shown). Fig. 2 demonstrates the dramatic increase in cleavage rates with small temperature increments. At 34 "C, interstitial collagenase, at an en- zymembstrate ratio of 1:5, cleaved 4 0 % of the type VI1 collagen in a 20-h incubation (Fig. 2A). With an increase in incubation temperature of only three degrees (Fig. 2B), the substrate was completely cleaved, with very little further breakdown of the products. Breakdown of the reaction prod- ucts from interstitial collagenase was somewhat more pro- nounced at 39 "C (Fig. 2C), a temperature approaching the

H. P. Bachinger, N. P. Morris, and R. E. Burgeson, unpublished observations.

3824 Metalloprotease Degradation of Type VII Collagen

95 kDa + 7 5 kDa,

95 kDa _I

75 kDa4 II) - -94kDa - 67 kDa - 43 kDa - 30 kDa

4 67 kDa

-43 kDa

-30kDa

- 94 kDa

m! "67 kDa

a43 kDa

e30 kDa

FIG. 2. Effect of temperature on the cleavage of type VI1 collagen by various proteases. In each case type VI1 collagen (9 pg) was incubated in buffer alone (VU) , with previously activated normal human skin collagenase (CN = 2 pg), or with normal human skin type IV collagenase (gelatinase) (C IV/C = 0.2 pg), at the indicated temperature prior to examination using NaDodS0,-PAGE. The M, scales from collagen helical (left) and globular (right) peptides are indicated. The collagen helical scale was determined using type VI1 collagen monomer and the pepsin cleavage fragments P1 and P2. A, 34; B, 37; C, 39 'C.

denaturation temperature of the helix. The reaction rate of type VI1 collagen breakdown with type

IV collagenase, also shown in Fig. 2, A, B, and C, clearly demonstrates a relaxation in the helicity of the central portion of the type VI1 monomer with increasing temperature. It has been shown that type IV collagenase is unable to cleave the helical type I collagen molecule (17). Although this enzyme can apparently cleave within the helix of types IV and V collagen (18), the reaction rate is more than 2 orders of magnitude slower than with denatured type I collagen (or indeed with any denatured collagen). Since even a t 39 "C the product peptide produced by type IV collagenase is fairly resistant to further breakdown, and since the NH2-terminal sequence of the product is characteristic of collagen (see below), it is highly probable that susceptibility to cleavage is a t least partially determined by some loosening of the collagen helix.

To determine the nature of the cleavage specificity the NH2-terminal sequence for both the -83-kDa interstitial col- lagenase cleavage peptide and the type IV collagenase cleavage product was obtained. Under the experimental conditions used, the NH2 terminus of the type VI1 collagen monomer was blocked. The data suggest very little loss of Coomassie staining material in the conversion to the product peptides (Fig. 3); we assume that the single sequence obtained for both product peptide bands represents both the peptide from which sequence was obtained and another peptide of approximately equal length beginning at the original blocked NH2 terminus. Fig. 4 demonstrates schematically the approximate sites of proteolytic cleavage within the type VI1 collagen monomer. As shown in Table I the product sequences, while different, were both indicative of cleavage within the collagenous por- tion of the molecule. About 10% of the amino acid sequence of type VI1 collagen has been determined, including the pepsin cleavage site and the NH2 terminus of P2." The sites of interstitial collagenase and type IV collagenase cleavage are different from portions of the molecule heretofore sequenced.

Breakdown of type VI1 collagen catalyzed by human skin collagenase is a linear function of both enzyme concentration (Fig. 3A) and time (Fig. 3B), as is the case for type IV collagenase (Fig. 3C). At 37 "C cleavage of the type VI1 trimer by type IV collagenase is much faster than by interstitial

R. Burgeson and N. P. Morris, unpublished data.

collagenase. The K, for type VI1 trimer for each enzyme is about 0.3 p ~ , but kat (moles of substrate degraded per mol of enzyme/h) for type IV collagenase is 160 uersus 0.05 for collagenase. Since the relative effectiveness of interstitial collagenase to type IV collagenase was greater a t lower tem- peratures, the increased kat for the gelatinolytic enzyme a t 37 "C is presumably due to partial melting of the helix.

DISCUSSION

From the data presented, it is clear that human skin inter- stitial collagenase and type IV collagenase (gelatinase) can cleave type VI1 collagen monomer. Both extracellular neutral proteases are specific for collagenous substrates. Even when enzyme and substrate are present in nearly equimolar amounts, the cleavage fragments maintain the helicity pre- dicted from the amino acid composition (Fig. 2). Any compro- mise in the helical stability through heat denaturation would have resulted in degradation into small peptides by the gela- tinolytic activity of the type IV collagenase. The significant temperature dependence of proteolysis supports the hypoth- esis that the middle region of the intact type VI1 monomer, which is susceptible to pepsin cleavage at 4 "C with prolonged incubation, has a relaxed helical character. The >3000-fold difference in rate between type IV collagenase and interstitial collagenase, reflecting the difference between the capacities of the two enzymes to cleave type I gelatin (18, 291, also supports this hypothesis. I t should be emphasized that while the size of the product peptides from both enzymes indicates cleavage near the middle of the 170-kDa monomer, our data do not preclude the possibility of additional cleavages.

The slight size differences between interstitial collagenase and type IV collagenase cleavage products reflected by gel electrophoresis (Fig. 1) are almost certainly related to differ- ences in the respective enzyme specificities. It has been shown that collagenase cleaves only at Gly-Ile and Gly-Leu bonds (30), while type IV collagenase (gelatinase) also cleaves Gly- Phe, Gly-Val, and Gly-Ala bonds at similar rates (31). Thus, the slightly smaller size of type IV collagenase cleavage prod- ucts may reflect more cleavages in the middle region of the type VI1 molecule, producing small peptides which are not readily visible under the conditions of NaDodS04-PAGE. Nevertheless, the slightly smaller sizes of the apparent initial type IV collagenase products could alternatively be due to

NORMAL COLLAGENAY

0 0.25 050 1.00 . .

A 100 L

Metalloprotease Degradation of Type VIZ Collagen

""

- "

Collagenase Added (119)

B

XU 0' 110' 220' 330'

"" - -- - -

100 - NORMAL COLLAGENASE

80 - -

2 0

60 -

LL "

n

Time (min)

3825

HUMAN SKIN IPE ECOLLAGENASElGELATlNASE

S T d 120' 240' 360'

I I

120 240 360

Time (min)

FIG. 3. Effect of protease concentration and time of incubation on the cleavage of type VI1 collagen. A, effect of interstitial collagenase concentration. Type VI1 collagen (8.5 pg) was incubated with varying concen- trations of activated normal human skin collagenase for 330 min a t 37 "C. Reaction products were analyzed by NaDodS0,-PAGE and product peaks were quantitated using an LKB 2202 Ultroscan laser densitometer and the Gelscan computer program. B, effect of time of incubation with interstitial collagenase. Type VI1 collagen (8.5 pg) was incubated with activated normal human skin collagenase (1 pg) a t 37 "C. At the indicated times, one-fourth of the total reaction mixture was removed and stopped by the addition of 30 pg/ml EDTA. Products were analyzed as indicated in A above. C, effect of time of incubation with type IV collagenase (gelatinase). Type VI1 collagen (5 pg) was incubated with human skin gelatinase (0.4 pg) for the indicated times. Portions of the mixture were removed and analyzed as described in I? above.

D7 II I PI)

type IV col lagenase J 1 L pepsln

interst i t ia l collagenase

FIG. 4. This schematic representation of the type VI1 col- lagen triple-helical domain illustrates the most likely rela- tionships between the cleavage sites of type IV collagenase, interstitial collagenase, and pepsin. Both type IV and interstitial collagenase cleave within helical sequences while the pepsin site is within a non-helical disruption that presumably contributes increased flexibility to this region. Additional sites may occur a t both the NH, or COOH termini.

additional cleavages near the NH2- or COOH-terminal por- tion of the helix.

Anchoring fibrils, thought to be important for the integrity of the epidermal-dermal junction (2, 4), are conspicuously

absent in certain diseases. Thus, the ability of interstitial collagenase and type IV collagenase to cleave the major pro- tein component of anchoring fibrils may have clinical signif- icance. For example, areas invaded by the basal cell carcino- mas often show disruption of basement membrane, and con- comitant loss of anchoring fibrils can be demonstrated immunologically (32). Likewise, although reports differ in the extent to which a defect in anchoring fibrils is present, all RDEB patients show a t least some alteration or decrease in the number of anchoring fibrils (9-12). Although it has been suggested that the primary defect causing absence of anchor- ing fibrils might be failure to express the type VI1 collagen gene, in a recent report four of four RDEB patients examined were shown to have the COOH-terminal globular domain of type VI1 collagen in their basement membrane, even though no anchoring fibrils were present (33). Since it has also been suggested that the blistering and skin fragility exhibited by patients with recessive dystrophic epidermolysis bullosa may

TABLE I Comparison of interstitial collagenase and type IV collagenuse (gelntinase) cleavage sites

with known type VII collagen sequence 40 pg of type VI1 collagen was incubated for 20 h a t 36 "C with 9 pg of interstitial collagenase and 0.4 pg of type

IV collagenase (gelatinase). The reactions were terminated with EDTA, concentrated, subjected to NaDodS0,- PAGE, and sequenced as described under "Materials and Methods." X, cysteine or tryptophan; Z, hydroxyproline; - , unidentified residue; ( ), ambiguity in sequencing (letter within parentheses is most likely residue).

NH2-terminal sequence

Interstitial collagenase cleavage peptide GX-G-"-Z-G-K--ZcG--I Type IV collagenease cleavage peptide G7-G-P-Z-G-P-Z-G-P-K P1-pepsin cleavage site, type VI1 monomer A-(T)-Q-P-R-P-E-P-G-P-V

P2-pepsin cleavage site --E-A-G-D-K-K-GG-P CB5

"Y---G(K)"G-Q-Q-G-E

G 7 A - P - V - G - F ~ Z - G - ~ A -

3826 Metalloprotease Degradation of Type VII Collagen

be related to excessive production of collagenase (13, 14, 20) our findings may provide a pathophysiologic link between the increased levels of the enzyme and the decrease in anchoring fibrils. Furthermore, patients with RDEB frequently have a marked increase in blistering following a febrile episode. The significant temperature dependence of the cleavage of type VI1 collagen by these enzymes (Fig. 2) could also provide a plausible explanation for this phenomenon.

The fact that extracellular neutral metalloproteases known to be present in connective tissue are capable of degrading anchoring fibril components seems significant, especially in light of the known elevations of collagenase in some disease situations. However, our current in vitro findings do not exclude the possibility that other proteases may play a role in type VI1 collagen turnover. Because the globular portions of type VI1 collagen also contribute to the structure of anchoring fibrils, anchoring plaques, and basement membranes (5 ,7) , it is probable that proteases secreted by inflammatory infiltrates could also be destructive to anchoring fibrils, especially through attack on the globular domains of the molecule.

Acknowledgements-We thank M. L. Eschbach, K. Akers, and S. White for excellent technical help. We thank Dr. Gregory Grant, Director of the Washington University Protein Chemistry Labora- tory, for performing amino acid sequence analyses.

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