[8] Catabolism and turnover of collagens: Collagenases

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MAJOR COMPONENTS OF THE EXTRACELLULAR MATRIX

[8] C a t a b o l i s m

and Turnover By HENNING

[8]

of Collagens: Collagenases 1

BIRKEDAL-HANSEN

Introduction Vertebrate collagenases are endopeptidases capable of cleaving the helical domain o f native collagen molecules under physiological conditions. Collagen types I, II, and III are cleaved, although at different rates, 2 by the same "interstitial" or " t y p e I - I I - I I I collagenase." Scission of the c o m p o n e n t a-chains of the collagen triple helix occurs at a single characteristic site, a Gly-Ile or Gly-Leu bond, located about one-fourth of the distance from the COOH-terminus. 3,4 Denatured c~-chains in random coil configuration are also cleaved but at a much slower rate than native triple-helical molecules. 5 Recent data suggest that there are two immunologically 6 and kinetically 7 distinct interstitial collagenases, one secreted by fibroblasts, epithelial cells, and macrophages and another stored exclusively in p o l y m o r p h o n u c l e a r leukocyte granules and released in the degranulation process. Types IV and V collagens are resistant to interstitial collagenases, 8,9 and there is mounting evidence that these collagens are degraded by an altogether different set o f proteases ( " t y p e IV collagen a s e , " " t y p e V collagenase"). " T y p e IV collagenase" cleaves type IV collagen and procollagen into two major fragments which share some resemblance with the 3/4-1/4 cleavage products of interstitial collagenases. Z°,11 T y p e V collagen is susceptible to a variety of neutral endopeptidases including several metalloproteases with gelatinolytic activity This work w a s supported by National Institutes of Health Grants D E 02670, DE 05817, and D E 6028. 2 H. G. W e l g u s , J. J. Jeffrey, and A. Z. Eisen, J. Biol. Chem. 256, 9511 (1981). 3 p. p. Fietzek and K. K 0 h n , Int. Rev. Connect. Tissue Res. 7, 1 (1976).

4 E. J. Miller, E. D. Harris, E. Chung, J. E. Finch, P. A. McCroskery, and W. T. Butler, Biochemistry 15, 787 (1976). H. G. Welgus, J. J. Jeffrey, G. P. Stricklin, and A. Z. Eisen, J. Biol. Chem. 2,57, 11534 (1982). 6 K. A. Hasty, M. S. Hibbs, A. H. Kang, and C. L. Mainardi, J. Exp. Med. 159, 1455 (1984). 7 A. L. Horwitz, A. J. Hance, and R. G. Crystal, Proc. Natl. Acad. Sci. U.S.A. 74, 897 (1977). 8 H. Sage, H. G. Woodbury, and P. Bornstein, J. Biol. Chem. 254, 9893 (1979). 9 H. Sage and P. Bornstein, Biochemistry 18, 3815 (1979). i0 L. I. Fessler, K. G. Duncan, J. H. Fessler, T. Salo, and K. Tryggvason, J. Biol. Chem. 259, 9783 (1984). 11T. Salo, T. Turpeenniemi-Hujanen, and K. Tryggvason, J. Biol. Chem. 260, 8526 (1985). METHODS IN ENZYMOLOOY, VOL. 144

Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.

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which may possibly qualify for "type V collagenases. ''12 Although the biologic function of neither collagenase has yet been established beyond doubt there is considerable substantial evidence to suggest that interstitial coUagenases are involved in the extraceUular dissolution and metabolic degradation of type I and, possibly, types II and III collagens. There is considerably less evidence yet to support the role of types IV and V collagenases inasmuch as the corresponding substrate collagens are susceptible to a number of general proteases. Since very little is known yet of their biologic degradation, collagen types VI-XI will not be included in this review. Detection and Measurement of Collagenolytic Activity The initial TC A (3/4) and TC B (1/4) fragments formed by cleavage of native type I collagen with interstitial collagenase denature at temperatures several degrees lower than the intact molecules (TC, 40-41°; TC A, 32°; TC 8, 28o).~3The fragments, therefore, may or may not retain a native triple helical configuration depending on the assay temperature. Some of the assays outlined in the following are based on production of stable, native TC A and TC s fragments while others utilize their spontaneous denaturation above 28-32 ° . In either case, the specificity of the assay is dependent on retention of the intact, uncleaved collagen molecules in native triple-helical form. In this context, it is important to realize that local segments of the triple helix reversibly unfold at temperatures as much as 10° below the nominal denaturation temperature (Tin)TM and that Tm for fibriUar substrates is substantially higher (6-8 °) than for collagen in solution (Table I), yet not as high as for authentic collagen fibrils. ~5,16 Techniques for isolation of collagen types I-V were treated extensively in the previous volume. 17,18Likewise, a variety of assays utilizing collagen in solution or in fibrillar form as well as assays based on hydrolysis of the synthetic DNP-III peptide were reviewed in detail in the same volume) 9 The present review, therefore, includes mainly new methods developed in the intervening period together with a limited number of 12 C. L. Mainardi, M. S, Hibbs, K. A. Hasty, and J. M. Seyer, Collagen Rel. Res. 4, 479 (1984). ~3T. Sakai and J. Gross, Biochemistry 6, 518 (1967). ~4 p. L. Privalov, E. I, Tiktopulo, and V. M. Tischenko, J. Mol. Biol. 127, 203 (1979). ~5 H. Birkedal-Hansen, R. E. Taylor, A. S, Bhown, J. Katz, H.-Y. Lin, and B. R. Wells, J. Biol. Chem. 2,60, 16411 (1985). ~6H. Birkedal-Hansen and K. Dano, Anal. Biochem. 115, 18 (1981). z7 E. J. Miller and R.K. Rhodes, this series, Vol. 82, p. 33. ~8 H. Sage and P. Bornstein, this series, Vol. 82, p. 96. ~9 E. D. Harris and C. Vater, this series, Vol. 82, p. 423.

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TABLE I MIDPOINT MELTING TEMPERATURE OF INTERSTITIAL COLLAGENS IN SOLUTION AND IN FIBRILS Collagen Source

Type

Form

Tm

Rat tail tendon Rat tail tendon Calf skin Calf skin Calf skin Calf skin

I I I I III Ili

Solution Fibrils Solution Fibrils Solution Fibrils

39.5°6 47.0°'~ 41.0°b 48.7°b 40.2°b 47.0°b

From Birkedal-Hansen and Dano.~6 b From Birkedal-Hansen et al. j5 general and standardized techniques for the measurement of collagenolytic activities.

Extraction of Type I Collagen Extraction of Salt-Soluble Type I Collagen. Salt-soluble type I collagen is obtained from skins and tendons of rats, guinea pigs, and calves. The yield is relatively low, but can be improved by rendering the animals lathyritic by a dietary supplement of fl-aminopropionitrile. The skins are processed through a meat grinder and washed with several changes of cold distilled water to r e m o v e plasma proteins. The residue is extracted by constant stirring overnight at 4 ° with 0.5 M NaC1 in 50 m M Tris-HCl, p H 7.5, supplemented with protease inhibitors (20 m M EDTA, 10 m M PMSF, I0 m M PMCB). The supernatant is decanted and the residue reextracted one or two more times. The collagen is purified from this extract by fractional salt precipitation as briefly outlined below and described elsewhere in detail. ~7,18 Extraction of Acid Soluble Type I Collagen. The residue left after repeated extraction with neutral 0.5 M NaC1 is washed with several changes o f cold distilled water to remove the salt and then extracted overnight at 4 ° with 0.5 M H O A c with constant stirring. The suspension is strained through cheese cloth and the supernatant clarified by centrifugation at 20,000 rpm for 20 min. The residue is extracted at least one more time. Filtration through celite reduces the lipid content and is advantageous when processing skins. Collagen is purified from this preparation as described below.

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Extraction of Type 1 Collagen by Limited Digestion with Pepsin. The insoluble residue (from nonlathyritic animals) still contains most of the type I collagen in addition to substantial amounts of collagen types III and V which can be solubilized in native form by limited proteolysis with pepsin (1 mg/ml in 0.5 M HOAc, 4° 24 hr). The residue is removed by centrifugation at 40,000 g for 20 min and the procedure repeated as needed. Pepsin cleaves in the nonhelical COOH- and NH2-terminal crosslink bearing regions and solubilizes truncated native molecules with intact helical domains. Collagen types I, III, and V can be purified from this extract as described below. Purification of Type 1 Collagen The purification of type I collagen is greatly facilitated by choosing tissue sources with little or no contaminant structures. Rat tail tendon is a particularly clean source which has the added advantage that virtually all of the collagen is soluble in acid with a considerable yield (200-400 mg/rat tail). Normal or lathyritic skins are good alternatives for most purposes. Salt-soluble collagen is first precipitated with 25% saturated (NH4)2SO4 and redissolved in 0.5 M HOAc. This material, and the acidand pepsin-solubilized collagen preparations described earlier are then precipitated in the cold by addition of solid NaCI to 0.9 M. The precipitate is harvested by centrifugation at 40,000 g for 20 min and redissolved in 0.05 M HOAc and dialyzed against several changes of 0.02 M Na2HPO4 over a 24- to 48-hr period. The white, fluffy precipitate which forms at neutral and slightly alkaline pH is harvested by centrifugation at 40,000 g for 20 min. The phosphate precipitation can be repeated as necessary, but one cycle is usually enough to purify the acid-soluble type I collagen fraction from rat tail tendons. The precipitated material is finally dissolved in 50 mM HOAc, dialyzed against the same buffer, and lyophilized.

Chemical Labeling of Type I Collagen Among the many procedures available for radioactive labeling of collagens by chemical reaction two have gained wide acceptance, namely reductive 3H/~4C-methylation2° and reaction with 3H/~4C-labeled acetic anhydride. 2~,22Both methods permit incorporation of 3H or 14C to high specific activity with minimal change of physicochemical properties, includ20 K. Otsuka, J. Sodek, and H. Limeback, Eur. J. Biochem. 145, 123 (1984L zl B. Johnson-Wint, Anal. Biochem. 104, 175 (1980). zz T. E. Cawston and A. J. Barrett, Anal. Biochem. 99, 340 (1979).

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ing ability to form reconstituted fibrils, provided the reactions are carried out under mild conditions. Rat tail tendon type I collagen is dissolved at 3 mg/ml in 13 mM HCI and centrifuged at 40,000 g for 1 hr. The isotope, 25 mCi of 3H-labeled acetic anhydride (Amersham TRK 2), is condensed in the lower end of the ampule by submersion in dry ice/ethanol. The ampule is opened in a fume hood and 500/zl of p-dioxane, dried by addition of NazSO4 crystals, is added. The collagen solution (20 ml) is rapidly mixed with 5 ml of a 5-fold phosphate buffer concentrate (described in "Assays Using Type I Collagen Fibrils") to yield a pH of 7.5 and final concentrations of 0.2 M NaCI 18 m M Na2HPO4/NaH2PO4. A total of 5 mCi of 3H-labeled acetic anhydride is added with vigorous stirring over a period of 5-10 rain (2-3 aliquots) and the reaction is allowed to proceed for 30 min at 4 ° in the fume hood. The solution is then dialyzed against 13 mM HCI with changes every or every other day until dialyzable radioactivity falls below 1000 cpm/ml. The collagen is stored in this fashion at 4 ° for several months. Reaction with 3H/14C-labeled acetic anhydride readily yields activities of 1-5 × 106 cprn/mg without measurably affecting the fibril properties as long as the reaction is carried out at a pH not above 7.5. Under optimal conditions, only 8-10% of the radioactivity is located in the nonhelical domains as evidenced by its susceptibility to limited digestion with pepsin. Labeling at higher pH (pH 8.5-9.0) results in extensive blockage of lysine side chains necessary for fibril formation. "Overlabeled" type I collagen, however, may still be an excellent substrate in soluble form as outlined below. The same method can be used to label collagen types II, III, IV, and V. Reductive methylation of collagen using the method of Jentoft and Dearborn 23 was described by Otsuka et al. z° Two hundred microcuries of [~4C]formaldehyde is added to 0.2 mg collagen in 0.2 ml 40 mM Tris-HC1 buffer, pH 7.2, containing 1.2 mg NaBH3CN and the reaction is continued for 24 hr at 4 °. The sample is then diluted with reaction buffer and dialyzed against the same buffer and finally against 0.5 M HOAc. The yield is about 800,000 dpm/mg.

Measurement o f Enzymatic Hydrolysis of Collagen Cleavage of interstitial and basement membrane collagens in solution can be monitored by viscosimetric methods as reviewed in detail in a previous volume in this series. 19The collagen is dissolved at 0.3-1.0 mg/ ml in neutral buffer and the temperature set in the 15-28 ° range, well 23 N. Jentoft and D. G. Dearborn, J. Biol. Chem. 254, 4359 (1979).

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below Tm (39-42°). Preparations heavily contaminated with other protease activities must be assayed in the lower end of this interval to prevent further digestion of the initial reaction products. Under nondenaturing conditions complete cleavage yields a 55-60% drop of relative viscosity. The reaction is stopped by EDTA which instantly inactivates collagenase and, if necessary, by addition of other protease inhibitors to prevent digestion of fragments and intact molecules during the subsequent heating in electrophoretic sample buffer. Electrophoresis can be performed either with Laemmli's 24 or Neville's 25 systems and the resultant gels stained either with coomassie blue (50/.~g collagen per lane) or with silver stains z6 (1/xg collagen per lane). To reduce the contamination of the sample with protein bands which originate from the enzyme preparation rather than the substrate, native collagenous components can be precipitated with 50% p-dioxane. Alternately, the substrate can be radiolabeled and the reaction products visualized by fluorography.

Assay of Type I Collagen in Solution Dioxane-Based Assays. 27,28 Acid- or salt-soluble ~4C- or 3H-labeled type I collagen (5000 cpm per sample) is brought to neutral pH either by dialysis or by mixing with a neutral buffer concentrate. Final concentrations are 50 mM Tris-HC1, pH 7.4, 0.2 M NaCI, 5 mM CaCI2. Glucose (0.5 M) or arginine (50 raM) is added to prevent fibril formation if the collagen concentration exceeds I00/xg/ml. The enzyme is added in the same buffer and the sample incubated for 1-16 hr at temperatures in the 22-30 ° range depending on the purpose of the experiment and the purity of the enzyme. The reaction is stopped by addition of EDTA (20 raM) or 1,10-phenanthroline (5 mM) which instantly inactivate collagenase and other metalloproteases. However, contaminant serine and thiol proteases are not inhibited. This is a crucial point when assaying unknown samples since the initial reaction is followed by a 30 min incubation at 30 or 35 ° to denature TC A and TC B fragments. During this period uninhibited proteases will degrade the native collagen at a substantial rate (see "Specificity of the Enzymatic Hydrolysis of Collagen"). After addition of inhibitors the samples are mixed with one volume ofp-dioxane and chilled on ice for 10 min. The solutions are centrifuged at 10,000 g for 10 min at 4° and 24 U. Laernmli, Nature (London) 227, 680 (1970). 25 D. M. Neville, J. Biol. Chem, 246, 6328 (1971). 26 W. Wray, T. Boulikas, V. P. Wray, and R. Hancock, Anal. Biochem. 118, 197(1980). 27 K. Terato, Y. Nagai, K. Kawanishi, and S. Yamamoto, Biochim. Biophys. Acta 445,753 (1976). ,_8T. lshikawa and M. E. Nimni, Anal. Biochem. 92, 136 (1979).

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MAJOR COMPONENTS OF THE EXTRACELLULAR MATRIX

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aliquots of supernatant counted by liquid scintillation spectrometry. With minor changes this assay can be adapted to types II, III, and V collagen. Electrophoretic Assays o f Collagenase. Detailed protocols for the electrophoretic resolution of intact collagen chains and peptides generated by vertebrate collagenase have been published elsewhere. 17 Good separation of individual o~-chains and their major cleavage products is obtained with the Laemmli system 24 using a polyacrylamide concentration of 7.5-9%. The o~2(I) chain is completely separated from the al(I) chain and from the 3/4 al(I) fragment but the al(I) chain is not resolved from the reduced al(III) chain. In Neville's discontinuous buffer system, 25 using 7% polyacrylamide gels, the al(I) and a2(I) chains are separated by a larger margin than in the Laemmli system and the al(III) and al(I) chains are completely resolved. However, the 3/4 al(I) fragment overlaps with, and is only partially resolved from, the intact o~2(I) chain. Perhaps the most sensitive assay available in this category is that developed by Otsuka et al. 2° based on fluorography of biosynthetically labeled, cartier-free collagen produced by fibroblasts in culture. Confluent cultures of rat gingival fibroblasts are incubated for 24 hr with 5/zCi each of [14C]proline and [14C]glycine in proline- and glycine-deficient medium supplemented with 2% fetal calf serum and 50/xg/ml ascorbate. The medium is harvested, dialyzed against 0.5 M HOAc and digested with 1 rag/ ml pepsin dissolved in 0.5 M HOAc for 5 hr at 15° and then for 24 hr at 4° in order to convert procollagen to collagen and degrade contaminant proteins. Native, pepsin-resistant collagen is precipitated by addition of NaCI to 1.7 M and the precipitate collected by centrifugation at 22,000 g for 60 min at 4 °. The pellet is redissolved in 1.5 ml 0.5 M HOAc and dialyzed for 24 hr against 0.0 1 M HOAc. The supernatant which contains the pepsindigested type I collagen is collected following centrifugation at 30,000 g for 6 hr. The final solution is diluted to 3,000 dpm/10/.d with I0 mM HOAc and stored frozen at - 2 0 °. The assay is performed by mixing 4/zl of enzyme preparation with 26 /.tl assay buffer (50 mM Tris-HCl, pH 7.6, containing 5 mM CaCI2, 0.05% Tween-20, and 0.02% NAN3). If desired, the enzyme sample is activated by addition of 4/zl of 10 mM APMA for 30 rain at room temperature. Finally, 10/,d of ~4C-labeled collagen solution is added and the samples incubated for 20 hr either at 22 or at 28°. Incubation is terminated by addition of 20/zl of a 4-fold concentrated electrophoresis sample buffer containing 8 M urea and 8% SDS in stacking gel buffer. The samples are heated to 65 ° for 20 rain and resolved by electrophoresis in 7.5% polyacrylamide gels (16 hr 7.5 mA per gel) with a 5% stacker according to Laemmli. 24 Fluorograms are prepared by the method of Bonnet and Laskey 29 29 W. M. Bonner and R. A. Laskey, Eur. J. Biochem. 46, 83 (1974).

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using a Kodak SB-5 X-ray film. The cleavage pattern is quantified by a scanning densitometer at 555 nm coupled to a digital integrator.

Assays Using Type I Collagen Fibrils (Gels) as Substrate The advent of highly efficient chemical labeling techniques has permitted the development of fibrillar assays using relatively small amounts (2550 t~g) of fibrils (gels) per sample. The assay outlined below is developed by a combination of methods published by several laboratories.15,zl A 2.0 mg/ml solution of rat tail tendon type I collagen diluted with unlabeled collagen to a specific activity of 200,000 cpm/ml is rapidly mixed with onefourth volume of a phosphate buffer concentrate formed by mixing 40 ml of 0.2 M NaHzPO4/NazHP04 buffer adjusted to pH 7.4 with 40 ml 0.1 M NaOH and 8.3 ml 5 M NaCI. The collagen solution is kept on ice throughout the procedure and 20/xl aliquots (30/zg collagen) are dispensed in 96well flat bottom microtest plates. Each collagen droplet is dispensed in the bottom "corner" of the well leaving about one-third of the bottom free from collagen to facilitate washing of the resultant gel as proposed by Johnson-Wint. 21 The plate is incubated at 37° for 2 hr to allow for heat gelation and each gel is covered with 200 tzl distilled H20 and incubated overnight at 35° . The following day, the liquid is removed by means of an aspirator bottle equipped with a trap and connected to the house vacuum and 200 tzl/well of assay buffer (50 mM Tris-HCl, pH 7.4, 0.2 M NaCI, 5 mM CaCI2 15 m M NAN3) is added. The plates are stored in this fashion until used. The enzyme is added in a total volume of 100/xl and incubated at 35 ° for 4-16 hr after repeated washing of the gels with assay buffer. Dissolution of the collagen fibrils is measured by counting of 50tzl aliquots of the assay fluid in a liquid scintillation counter. Controls include gels incubated with medium alone or with 10 tzg/ml of TPCK-trypsin. As a variation of this method, Johnson-Wint 2~ proposed the use of airdried films of reconstituted collagen fibrils. Conventional hydrated gels are prepared as just described and washed with distilled HzO to remove buffer and salts. The gels are then drained and dried in a cold airstream. Alternatively, the gels can be dried first and then washed free from salts. "Dried fibril films" give slightly higher trypsin counts than hydrated gels but are somewhat simpler to handle. They can be stored in assay buffer for extended periods of time, but become incre~ingly susceptible to trypsin when stored dry. Trypsin (100/xg/ml, 16 hr) releases an average of 1020% of the radioactivity from freshly prepared hydrated gels and as much as 20-30% from airdried films. At this concentration, however, the substrate is no longer in excess; rather there is a severalfold molar excess of enzyme. The trypsin susceptibility is always higher when the gels are freshly made than after storage for several days. It is interesting to note

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MAJOR COMPONENTS OF THE EXTRACELLULAR MATRIX

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that the sensitivity to collagenase and the solubility in 0.5 M HOAc at 4 ° closely parallel the decline in trypsin susceptibility upon storage. Assay of Fibrillar Type I Collagen at Acidic pH. Reconstituted collagen fibrils rapidly dissolve below pH 5 as hydrogen ions catalyze the hydrolysis of the intermolecular aldolimine cross-links ( - - C H - - N - - ) . It is, therefore, impossible with ordinary reconstituted fibril assays to measure collagen fibril breakdown in the pH range where lysosomal cathepsins are optimally active (pH 3-5). Reduction of the cross-link to the corresponding acid-stable dihydro form ( - - C H 2 - - N H - - ) , however, permits development of a reconstituted fibril substrate which is resistant to acid dissolution and suitable for measurement of collagen breakdown at acidic pH. 3° Using the method outlined below, 60% of all a-chains are cross-linked whereas the remaining 40% escape cross-linking and are released upon denaturation of the component collagen molecules. 3° Substrate gels are prepared in 96-well microtest plates as described above and dried in a cold airstream. The fibril films are then incubated with a freshly prepared solution of 10 mM KBH4 in distilled H20 for two consecutive l-hr periods and washed extensively with 50 mM Tris--HCl, pH 7.5, to remove traces of borohydride. The films are then incubated with 50 mM HOAc for 2 hr at 35 ° to remove uncross-linked collagen and washed with several changes of assay buffer (50 mM NaOAc, pH 4.0). Enzymes are added in a total volume of 100 txl 50 mM NaOAc, pH 4.0, 10 mM EDTA, 10 m M cysteine and incubated with the substrate for 16 hr at 35 °. The release of radioactivity is measured by liquid scintillation counting of 50/.d aliquots. Borohydride-reduced fibril films are completely dissolved by pepsin as the cross-links are located in the protease-sensitive nonhelical domains. At 4 ° pepsin yields native predominantly monomeric collagen but at 35 ° the collagen is completely degraded to small peptides. Pepsin is, therefore, used to determine the total radioactivity of the substrate films. Other acidic proteases which cleave between the cross-link site and the triple helix (cathepsin B, papain, ficin, bromelain) also dissolve the fibrils and subsequently degrade the solubilized collagen to small peptides. Overnight incubation at 35° with 50 mM NaOAc, pH 4.0 (buffer blank), releases approximately 5% of the radioactivity. The susceptibility to trypsin (at pH 7.5) is not affected by the KBH4 reduction and remains at the level characteristic of airdried fibril films.

Preparation of Type III Collagen A small amount of salt-soluble type III collagen (15-20 mg per skin) can be extracted from fetal calf skin without pepsin treatment and further 30 j. K a t z and H. Birkedal-Hansen, unpublished results.

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B

,81,1_ /31,2--

_ pN-al(III)

tll-t12--

-- a l(III)

1234 FIG. 1. Reconstituted fibrils of fetal bovine skin type l l l / p N - l l l collagen. (A) Scanning electron micrograph of reconstituted fibrils formed by heat gelation at 35° of a 2.4 mg/ml solution of type I l I / p N - l l I collagen in 18 mM phosphate buffer, pH 7.5, 0.15 M NaCI. The gel was air dried at room temperature, washed with distilled H20, fixed with 2% glutaraldehyde, critical point dried, and coated with gold-palladium. White bar measures 1000 nm. (B) Composition of reconstituted fibrils. The samples were resolved by electrophoresis in 7% polyacrylamide with a 3.5% stacker using Neville's system} 5 Lane 1: Control fetal bovine skin type 1 collagen. Lane 2: type III/pN-IlI collagen stock solution (7.5/xg) before heat gelation; the sample contained 8.8% type I collagen. Lane 3: Fibrils reconstituted from 7.5 /~g type l l l / p N - l l l collagen by heat gelation. The fibrils were dispersed, washed twice with 0.2 M NaCI in 50 mM Tris-HCl buffer, pH 7.4, and harvested by centrifugation. The pellet was dissolved by heating in electrophoretic sample buffer under reducing conditions. The ratio of pN-al(llI) to cd(IlI) chains (1.3) was similar to that of the stock solution. Lane 4: The supernatant which remained after removal of the fibrils by centrifugation still contained 17% of the total collagen, predominantly molecules composed of pN-~l(III) chains. (C) Transmission electron micrograph of reconstituted fibril formed from type l l l / p N - I I I collagen. Bar measures 100 nm. Reproduced from Ref, 31 with the permission of the publisher.

purified from acidic contaminants by chromatography in the native state on DEAE-cellulose to yield a preparation which consists of almost equal amounts of intact type III collagen and pN-III collagen 3t (Fig. 1, lane 2). Extraction is carried out in the presence of protease inhibitors to avoid excision of the pN peptides and the nonhelical domains. A substantially larger yield of type III collagen is obtained by limited pepsin digestion of fetal calf skin followed by differential salt precipitation as outlined by Chung and Miller. 32 Type III collagen precipitates at 1.5-1.7 M NaCI (pH 7.5) whereas most of the contaminant type I collagen first precipitates at 2.5 M NaCI. Nevertheless, the 1.5 M NaCI precipitate almost invariably contains 5-10% type I collagen which is not readily eliminated even by 3t R. Timpl, R. W. Glanville, H. Nowack, H. Wiedemann, P. P. Fietzek, and K. Kiihn, Hoppe-Seyler's Z. Physiol. Chem. 356, 1783 (1975). n E. Chung and E. J. Miller, Science 183, 1200 (1974).

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MAJOR COMPONENTS OF THE EXTRACELLULAR MATRIX

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repeated salt precipitation. The contaminant type I collagen, however, does not measureably affect the outcome of the assay described below. Studies by Henkel and Glanville 33 have suggested that types I and III collagen may form heteropolymeric fibrils so that it may not be possible to completely eliminate the residual type I collagen from these preparations. Procollagen type III can be purified free of contaminants from bovine fetal skin as outlined in a previous volume in this series.18

Fibrillar Assays Using Type 1II Collagen Both salt-soluble type III/pN-III collagen and type III collagen prepared by pepsin digestion form fibrils of sufficient rigidity to withstand mechanical agitation and washing. Since studies by Fleischmajer et al. 34 have indicated that authentic type III collagen fibrils consist of partially processed molecules with intact pN peptides, we have utilized pN-III-rich salt soluble collagen, extracted from fetal bovine skin and purified by passage over DEAE-cellulose, as substrate 35 (Fig. 1). Type III/pN-III collagen dissolved at 3 mg/ml in 13 mM HCI is rapidly mixed with a phosphate buffer concentrate designed to yield 2.4 mg/ml collagen in 18 mM phosphate buffer, pH 7.5, 0.15 M NaC1 (final concentrations). The solution is kept on ice and dispensed in 96-well microtest plates as described above. The plates are then incubated overnight at 35° to allow for heat gelation. The gels are washed overnight with distilled H20 and equilibrated with assay buffer (50 mM Tris-HCl, pH 7.5, 0.2 M NaC1, 5 mM CaClz). Enzyme is added in a total volume of 100/xl assay buffer and incubated with the gels for 4-16 hr at 35°. Cleavage is measured by the release of radioactivity to the buffer. It is of note that fibrillar type III collagen is quite resistant to trypsin, thermolysin, pronase, and PMN elastase which rapidly cleave this collagen in solution. 15,31,36

Preparation and Labeling of Type IV Collagen There are relatively few good sources of type IV collagerdprocollagen. Metabolically labeled type IV procollagen can be isolated from organ cultures of EHS tumors maintained in mice by the procedure originally 33 W. Henkel and R. W. Glanville, Eur. J. Biochem. 122, 205 (1982). 34 R. Fleischmajer, R. Timpl, L. Tuderman, L. Raisher, M. Wiestner, J. S. Perlish, and P. N. Graves, Proc. Natl. Acad. Sci. U.S.A. 78, 7360 (1981). 35 p. H. Byers, K. H. McKenney, J. R. Liechtenstein, and G. Martin, Biochemistry 13, 5234 (1974). 36 H. G. Welgus, R. E. Burgeson, J. A. M. Wootton, R. R. Minor, C. Fliszar, and J. J. Jeffrey, J. Biol. Chem. 260, 1052 (1985).

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outlined by Tryggvason et a/. 37'38 and purified in a simple one step procedure by precipitation with 1.71 M NaCI. Bovine lens capsules contain a relatively high proportion of type IV collagen which can be brought in solution in native form by limited digestion with pepsin. The procedure is somewhat difficult to control and often results in generation of a multitude of dissimilar fragments which yield rather complex cleavage patterns when used as substrate for collagenolytic proteases. For preparation of radiolabeled type IV procollagen,~°,37 EHS tumors maintained in D57BL/6J mice are harvested 18 days after inoculation. The minced tissue is preincubated with proline-free medium for 30 min at 37° and then for 5 hr with the same medium supplemented with 10% dialyzed fetal calf serum, 75/zg/ml ascorbate, 50/zg/rnl/3-aminopropionitrile, and 40/zCi/ml [3H]proline (100 Ci/mmol, Amersham Corp.). Following incubation, the tissue is homogenized and extracted with 0.5 M HOAc containing 20 mM EDTA and 4 mM NEM. The supernatant is clarified by centrifugation, adjusted to pH 7.4, and the collagen precipitated with 1.71 M NaCI in 50 mM Tris-HCl buffer, pH 7.4. The precipitate (type IV procollagen) is redissolved in 50 mM Tris-HCl buffer, pH 7.4, 0.2 M NaCI, and 10 mM CaC12. Purity is assessed by SDS-PAGE followed by fluorography. Isolation of type IV collagen from intact bovine lens capsules requires limited digestion with pepsin which severs the dimeric globular cross-link region and also separates the tetrameric (7 S) crosslink domain from the major helical portion of the individual molecules. Bovine lens capsules are first extracted with 0.5 M NaCI (50 mM TrisHCI, pH 7.4) and with 0.5 M HOAc for two consecutive 24-hr periods. The solution is decanted and the capsules blotted dry and resuspended in 0.01-1.0 mg/ml pepsin at 4° for 24 hr during which most of the capsule matrix goes into solution. The solution is clarified by centrifugation (20,000 g; 20 min) and collagenous components precipitated by 1.2 M NaC1. The precipitate is harvested by centrifugation and redissolved in 50 mM Tris-HC1, pH 7.5, and applied in the native state to a DEAE-ceUulose column equilibrated with 50 mM Tris-HC1, pH 7.4, 50 mM NaCI, 2 M urea. Type IV collagen emerges in the unabsorbed fraction. The collagen is then dialyzed against 0.5 M HOAc or desalted in the same solvent and lyophilized or stored frozen in small aliquots. Type IV collagen isolated in this manner can subsequently be labeled with 3H or lac to high specific activity either by reductive methylation or by reaction with acetic anhydride as previously outlined. Intact lens capsules can also be radiolabeled by acetylation before 37 K. Tryggvason, P. G. Robey, and G. R. Martin, Biochemistry 19, 1284 (1980). 3s T. Salo, L. A. Liotta, and K. Tryggvason, J. Biol. Chem. 258, 3058 (1983).

152

MAJOR COMPONENTS OF THE EXTRACELLULAR MATRIX

[8]

dissolution of the component molecules. The technique described below is a modification of the method of Smith et al. 39 Bovine lens capsules are extracted sequentially with NaCI and with HOAc as already described. The capsules are then washed several times with 50 mM Tris-HC1, pH 8. I, and vigorously stirred while 3H-labeled acetic anhydride in dry dioxane (5 mCi per 20-50 capsules) is added dropwise over a 5-min period. The capsules are left stirring for 30 min at 4 °. Unincorporated isotope is then decanted and the lens capsules washed extensively with 50 m M HOAc and stored in this solvent. The percentage of radioactivity incorporated into collagen is determined by hydrolysis of the intact lens capsule with protease-free clostridial collagenase (Advance Biofactures). The substrate is either aliquoted after homogenization in a hand-held glass homogenizer or used intact to study the ability of live cells to degrade basement membranes as outlined in a later section. Assays Using Type IV Collagen The assay described below 38 is based on separation of larger cleavage fragments and intact molecules from small, TCA-tannic acid-soluble peptides. The substrate (3000 cpm) is incubated with enzyme and assay buffer (50 m M Tris-HC1, pH 7.5, 0.2 M NaCI, 10 mM CaC12) in a total volume of 650/zl either at 37° for 5 hr or at 30° for 18 hr. The assay is terminated by addition, in sequence, of 20/zl bovine serum albumin (1 mg/ml) and 100 t.d of a solution of 10% TCA, 0.5% tannic acid. The mixture is incubated on ice for 30 min and centrifuged at 5000 g for 15 min. A sample of 600/.d of the supernatant is withdrawn for liquid scintillation counting. The assay measures complete degradation of the substrate as larger cleavage products are precipitated by the TCA-tannic acid mixture along with intact molecules. It is, therefore, most useful in combination with analysis by SDS-PAGE/fluorography of reaction products formed under nondenaturing conditions (15-25°). The specificity of the assay remains to be determined. The use of rather high temperatures (30-37 °) is a point of concern as it is still uncertain to what extent this substrate is also cleaved by less specific proteases (gelatinases, cathepsins). Preparation and Labeling of Type V Collagen Type V collagen can be obtained from human and animal skin and uterus by the methods of Burgeson et al. 4° and Rhodes and Miller. 41 The 39 C. Smith, H. E. Van Wart, and D, E. Schwartz, Anal. Biochem. 139, 448 (1984). 40 R. E. Burgeson, F. A. El Adli, I. 1. Kaitila, and D. W. Hollister, Proc. Natl. Acad. Sci U.S.A. 73, 2579 (1976). 4J R. K. Rhodes and E. J. Miller, Biochemistry 17, 3442 (1978).

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pepsin digest obtained after sequential extraction with NaC1 and HOAc is adjusted to 0.9 M NaC1 and the precipitate (types I and III collagen) removed by centrifugation. Type V collagen is then selectively precipitated by raising the NaC1 concentration to 1.2 M. Type V collagen is further purified by CM-cellulose chromatography in the native state Iv and labeled either by reductive methylation or by acetylation with acetic anhydride as already described.

Assays Using Type V Collagen Type V collagen does not form fibrils and is assayed in soluble form by methods based on dioxane precipitation. 42 The substrate (5000 cpm) is dispensed in Eppendorf tubes and mixed with enzyme in a total volume of 200 tzl assay buffer (50 m M Tris-HCl, pH 7.5, 0.5 M NaCI) and incubated at 32.5 ° for 4-16 hr. The assay is terminated by addition of 100/xl of a solution of 50 mM EDTA, 3 mM PMSF, and incubated for another 30 min at 35° before 300-tzl p-dioxane is added. The samples are left for 15 min at room temperature and centrifuged at 10,000 g for 5 rain. Aliquots of 300 ~1 are withdrawn for liquid scintillation counting. The substrate is also cleaved by leukocyte gelatinase 43 and thrombin. 44 Specificity of the Enzymatic Hydrolysis of Collagen Native collagen molecules become increasingly susceptible to nonspecific proteolysis in an interval from about 10° below the denaturation temperature (Tin) to Tin. This is illustrated in Fig. 2 by incubation of rat tail tendon collagen in solution (Tin = 39.5 °) with trypsin. The substrate is quite resistant at 15-20 ° but becomes increasingly susceptible above 2930° and is completely cleaved at 35-37 °. This is often overlooked in assays utilizing soluble type I collagens in the temperature range 30-35 °. The substrate is still cleaved faster by collagenase than by any other protease (trypsin and gelatinase) but the assay is not specific for coUagenase in this temperature range. Cleavage of collagen in solution by crude complex mixtures of unknown composition, therefore, cannot be taken as evidence of true vertebrate collagenase activity above 28-29 ° and statements to the effect that type I collagen preparations in solution are resistant to trypsin above 30° are erroneous. Moreover, several studies 45,46have indi4z C, L. Mainardi, J. M. Seyer, and A. H. Kang, Biochem. Biophys. Res. Commun. 97, 1108 (1980). 43 G. Murphy, J. J. Reynolds, U. Bretz, and M. Baggiolini, Biochem. J. 203, 209 (1982). 44 H. Sage, P. Pritzl, and P. Bornstein, Biochemistry 20, 3778 (1981). 45 B. R. Olsen, Z. Zellforsch. Mikrosk. Anat. 61, 913 (1964). 46 L. Ryhanen, E. J. Zaragoza, and J. Uitto, Arch. Biochem. Biophys. 223, 562 (1983).

154

MAJOR COMPONENTS OF THE EXTRACELLULAR MATRIX

[8]

I00

! j.o 60 ~40

o,F

~o

zo

°o ~ ~o"r,~(m~ ~ ~o ~o,o

24

z8

7"-

o/

32

0

4b

Temperotum (*C) FIG. 2. Trypsin susceptibility of native rat tail tendon type I collagen. The cleavage of3H labeled type I collagen in solution (0.5 mg/ml, 30 ~ Tris-HCl, pH 7.4, 0.2 M NaCI, 5 mM CaCI2, 1.0 Mglucose) by trypsin (500/zg/ml) was monitored by the dioxane method. Incubation was for 2 hr at the temperature indicated. The reaction was then stopped by addition of a 2-fold molar excess of soybean trypsin inhibitor and the samples cooled to 22°. Undigested, native collagen was then precipitated with 50% dioxane and after centrifugation (10,000 g, I0 rain) the 3H-activity in the supernatant was measured by liquid scintillation spectrometry. Under these conditions, control collagen (O) denatured with a midpoint melting temperature of 39.5 °. In the presence of trypsin (e), type I collagen remained highly resistant to cleavage up to 26-28 ° but became increasingly susceptible at higher temperatures. Trypsin in this manner effectively lowered T~ by approximately 8°. Inset shows susceptibility of collagen solution at 34° to increasing trypsin concentrations. Fifty microgram samples of 3H-labeled type I collagen were incubated with 2.5 to 25/~g of trypsin in a total volume of 50 ttl Tris-HCl buffer for the times indicated. Excess soybean trypsin inhibitor was then added and the sample rapidly cooled to 22 °. Undigested and native collagen was precipitated with 50% dioxane. Reproduced from Ref. 16 with the permission of the publisher.

cared that the collagenase-sensitive region in type I collagen, and even more so in type III collagen, ~5,47is a locus of minor resistance which under certain conditions is cleaved by trypsin near the collagenase cleavage site with the resultant formation of 3/4 fragments deceivingly similar to those produced by vertebrate collagenase. Formation of TC A and TC B fragments from type I collagen in solution, therefore, cannot be taken as absolute proof of vertebrate collagenase activity although it probably still is one of the best indications. 47 E. J. Miller, J. E. Finch, E. Chung, W. T. Butler, and P. B. Robertson, Arch. Biochem. Biophys. 173, 631 (1976).

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CATABOLISM AND TURNOVER OF COLLAGENS

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Reconstituted fibrils are considerably more resistant to proteolysis than monomeric, soluble forms of the same collagen ~5,~6largely because of the greater thermostability of the fibrillar form (Table I). It is, therefore, possible to conduct assays with fibrillar substrates as higher temperatures (35-37 °) than with collagens in solution without compromising specificity as long as the temperature is below the 10° interval where the helix reversibly unfolds. Authentic fibrils of type I collagen in the tissues (Tin = 5560°) denature at even higher temperatures than reconstituted fibrils and are even more resistant to proteolysis. Consequently, cleavage of reconstituted fibrils is not necessarily indicative of ability to cleave authentic fibrils, and cleavage of collagen in solution is not necessarily indicative of ability to dissolve any of the fibriUar forms. The recently discovered resistance of fibrils of type III collagen to proteases which rapidly cleave in solution illustrates this point rather well, and shows that results obtained with one substrate form cannot be freely extrapolated to other forms. 15A6'36 Moreover, a large body of evidence now suggests that the resistance of type I collagen to general proteases, even in the fibrillar form, is not absolute but is a function of temperature and enzyme/substrate ratio. The microbial proteases thermolysin and pronase, for example, at 1 mg/ml solubilize 80-90% of reconstituted fibrils (30 txg) during overnight incubation at 35 °. Authentic fibrils are also to some extent susceptible to these enzymes inasmuch as pronase (0.5 mg/ml) degrades 60%, and thermolysin (0.5 mg/ml) 40%, of intact rat tail tendon collagen (1 mg/ml dry weight) at 37° during a 24-hr incubation period. The proper classification of an enzymatic activity as that of a genuine vertebrate interstitial collagenase therefore minimally requires satisfaction of the following criteria: (1) the ability to cleave native type I collagen in solution in the collagenase-sensitive region at temperatures more than 10° below the denaturation temperature for that particular collagen, (2) the ability to completely dissolve reconstituted fibrils of type I collagen at neutral pH at temperatures more than 10° below the fibrillar denaturation temperature and at relatively high substrate to enzyme ratios, and (3) the ability to completely dissolve authentic collagen fibrils such as those of intact rat tail tendon at neutral pH. The requirement for complete dissolution of the substrate is important since a number of proteolytic enzymes have some limited activity on collagenous matrices as outlined in the following but fail to completely dissolve the structural integrity of the substrate. Widely accepted criteria to distinguish true types IV and V collagenases from gelatinases and general proteases do not exist. A number of serine proteases (chymotrypsin, elastase) cleave native type I collagen molecules at neutral pH between the major telopeptide cross-link sites and the helical domain. It is therefore theoretically possi-

156

MAJOR COMPONENTS OF THE EXTRACELLULAR MATRIX

[8J

hie that these enzymes may dissolve native triple-helical molecules from authentic or reconstituted fibrils and subsequently attack and degrade the helical domain as the transition from fibrillar to soluble form effectively lowers the denaturation temperature by 7-8 ° or more. In reality, however, these proteases have little activity on intact rat tail tendon collagen and, even at 50-100 gg/ml, solubilize only 10-20% of the radioactivity of reconstituted fibrils. Trypsin does not cleave between the cross-link site and the helical domain. It is, therefore, ideally suited as a control for nonspecific attacks on the helical domain of fibrillar collagens. Trypsin does not dissolve intact rat tail tendon but, as already shown, cleaves type I collagen in solution in a 10° temperature interval below Tin. The susceptibility of reconstituted fibrils to trypsin varies with the species of the collagen source, the temperature, the trypsin concentration, and the position of the label. Claims of 5% release of radioactivity from reconstituted fibrils (gels) are unrealistic unless very large substrate/enzyme ratios are used. With the labeling procedure described earlier about 8-10% of the radioactivity is incorporated into the nonhelical domains. Examination of the amino acid sequence of the al(I) chain 3 reveals that trypsin can remove only 4 residues from the COOH-terminal telopeptide and none from the NH2-terminus when the cross-link site is engaged as it is in 60% of all achains in reconstituted fibrils. 3° It is likely, however, that labeling (Nacetylation, N-methylation) of lysyl side chains at position 9 N or 16c48 results in blockage of the cross-link sites so that the corresponding achains are among those which escape cross-linking. In that case, an additional stretch of 9 amino acids from I N to 9N and the corresponding COOH-terminal stretch of 11 amino acids from 16c to 27C may also be excised by trypsin. It was originally thought that, in addition to the telopeptides, trypsin cleaved only denatured molecules in the substrate preparation but this is probably an oversimplification since there is mounting evidence that trypsin can cleave the collagenase-sensitive domain at least at rather high enzyme to substrate ratios. 45,46The advent of labeling techniques which permit production of substrates with very high specific activity has made it technically possible to incubate, for instance, 1 /zg of collagen (10,000-20,000 cpm) with 1 mg of enzyme and to create conditions where the enzyme is present in 103--104molar excess. Experiments such as these, although of little physiological relevance, have shown that the resistance to proteolysis by proteases other than collagenase is only relative. 4s Nomenclature according to K. Kiihn, in "lmmunochemistry of the Extracellular Matrix" (H. Furthmayer, ed.), Vol. I, p. 1. CRC Press, Boca Raton, Florida, 1982.

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CATABOLISM AND TURNOVER OF COLLAGENS

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Lysosomal thiol proteases (cathepsin B, N, L, and S) and several plant thiol proteases (papain, bromelain, ficin) partially solubilize certain acidinsoluble skin and tendon collagen preparations. At low temperature (415°), cleavage occurs predominately or exclusively in the cross-link-bearing nonhelical domains and the low pH of the reaction mixture (pH 3-5) results in dissolution of the truncated triple-helical molecules from the polymeric matrix. A similar set of reactions (catalyzed by lysosomal thiol proteases) could conceivably occur at physiologic temperatures and ultimately lead to complete degradation of the less resistant solubilized collagen molecules. The collagenous matrix of bone which is cross-linked somewhat differently from that of tendon, as evidenced by its inability to swell in acids, is considerably more resistant to dissolution by animal and plant thiol proteases. Pepsin and cathepsin D also cleave in the nonhelical domains and effectively solubilize skin and tendon collagens in native form at 4-10 °. At higher temperatures (35-37 °) pepsin completely degrades this collagen to small peptides. Appropriate Controls. The reversible local unfolding of the collagen triple helix in the temperature interval T~ to T m - 10° permits attack on the helical domain of native type I collagen in the native state by a multitide of dissimilar proteases. It is, therefore, necessary to include in each assay a set of controls to determine the level of susceptibility to standard proteases. Trypsin is widely used already and is clearly the best choice for fibrillar collagen substrates since it does not attack the short nonhelical sequences between the engaged cross-link sites and the helical domain whereas most other general proteases do. Plasmin which shares this property with trypsin may also qualify. The higher the enzyme/substrate ratio, the higher trypsin susceptibility the assay registers. A concentration of 100/~g/ml trypsin used by several laboratories in the past is clearly too high with some of the newer assay methods which employ very small amounts (1-50 ~g) of collagen substrate. A 10- to 100-fold molar excess of substrate (based on active site titration of the enzyme) are probably more reasonable. In the fibril assay described on page 147 (30 t~g collagen gels) a 10- to 100-fold excess of substrate requires 40-400 ng of TPCK-trypsin in a total volume of 100 ~1 or final trypsin concentrations of 0.4 to 4.0 /~g/ml whereas 100 /zg/ml of trypsin represents a 2.5-fold molar excess of enzyme ooer substrate so that the enzyme itself becomes the major substrate. Specificity considerations become even more complex when dealing with collagen in solution which often requires rather high assay temperatures to generate sufficient reaction rates. Under these conditions the assay is no longer specific for collagenase and it becomes even more important to incorporate in each assay a set of standard controls. Solution

158

MAJOR COMPONENTS OF THE EXTRACELLULAR MATRIX

[8l

assays can still be utilized with pure enzyme preparations and have great advantages for kinetic measurements since the reaction is entirely homogeneous. As a means of screening unknowns and complex biologic fluids, however, soluble collagen assays in the 30-35 ° temperature range are not all that useful. Specificity considerations unquestionably are equally important for collagen types I I - V but the current state of knowledge does not yet permit a detailed account.

Zymography Using Type I Collagen Fibrils as Substrate The recent development a zymographic overlay technique has made it possible to visualize collagenolytic activity associated with individual electrophoretic bands. 49 Collagenase/procollagenase is incubated with 2% SDS at room temperature for 1 hr and then resolved by electrophoresis (25 mA for 2.5 hr) in a 1.5 mm 11% polyacrylamide gel using Neville's 25 discontinuous buffer system. Preincubation with SDS activates procollagenase without any change of molecular weight by an as yet unknown mechanism but does not denature the enzyme. 49 The resultant activation is always more complete with crude harvest media than with pure procollagenase and, therefore, may involve a third, yet unidentified component. After electrophoresis, the gel is placed on an orbital shaker (20-30 rpm) at room temperature and washed in succession with 200 ml 2.5% Triton X-100 in distilled water (40 min), 200 ml 2.5% Triton X-100 in 50 mM TrisHCI, pH 7.5 (40 min), and 200 ml 50 m M Tris-HCl, pH 7.5, 5 m M CaCI2, 1 /zM ZnClz (40 min). The gel is briefly rinsed with distilled water and placed on top of a dried film of reconstituted collagen fibrils prepared as described below. A sheet of I0 x 14 cm gelbond film (hydrophilic side up) is attached to a leveled glass plate with a few drops of water and the edges taped down with 2-ram-thick 3M masking tape. A stock solution of 3 mg/ ml rat tail tendon type I collagen in 13 m M HC1 is rapidly mixed at 4 ° with one fourth-volume of a HEPES-based "neutralizing" buffer, formed by adjusting 0.25 M HEPES, 0.75 M NaCI to pH 7.5 and then adding 5 N NaOH to a final concentration of 65 mN. The composition of the final neutralized collagen solution is 2.4 mg/ml collagen, 0.05 M HEPES, pH 7.5, 0.16 M NaCI. Ten milliliters of this solution is poured onto the gelbond film and distributed evenly over the surface with a pipet tip. Bubbles are removed by capillary action using a pasteur pipet. The preparation is transferred to a humidified incubator, leveled, and incubated at 37° for 2 hr. The film is then dried overnight in a laminar flow hood, 4~ H. Birkedal-Hansen and R. E. Taylor, Biochem. Biophys. Res. Cornmun. 107, 1173

(1982).

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CATABOLISM AND TURNOVER OF COLLAGENS

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washed extensively to remove salt precipitates, and either used immediately or stored in buffer. Incubation is at 37° for 16 hr. The sandwich is then sprayed with distilled water to allow separation of the polyacrylamide gel and the collagen fibril film. Each is stained separately with 0.05% Coomassie blue in 30% MeOH, 7% HOAc and destained with the solvent. During the incubation the enzyme diffuses out of the gel and gives rise to lytic zones in the fibril film as shown in Fig. 3. Note that both the 45/47K collagenase and the 65/60K procollagenase bands are active at this stage (Fig. 3, lane c). The reaction requires in the range from 0.1 to 0.5/,tg/lane of human fibroblast collagenase/procollagenase to generate clearly visible lytic bands. When proper trypsin controls are included, the sensitivity can be improved somewhat by raising the temperature to 39-

-150K -lOOK - 75K - 60K - 50K -40K - 3OK

a

b

c

FIG. 3. Collagenase zymogram. The samples were preincubated with 2% SDS at room temperature for 1 hr and resolved by electrophoresis in 11% polyacrylamide gels by the method of Neville. 25 The gels were then washed in with 2.5% Triton X-100 as described in detail in the text and incubaed at 37° for 16 hr in contact with an air-dried film of reconstituted collagen fibrils. The fibril film was finally stained with Coomassie blue to visualize zones of collagenolytic activity. Lane a: 0.5/xg clostridial collagenase; lane b: 5 U of crude bovine gingival collagenase; lane c: 10 U of crude human fibroblast collagenase. Reproduced from Ref. 49 with the permission of the publisher.

160

MAJOR COMPONENTS OF THE EXTRACELLULAR MATRIX

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40°. Instead of the reconstituted collagen fibril film an agarose-collagen gel prepared by the method of Yankelow et al. 5° can also be used. Mainardi et al.12 suggested yet another modification of the zymographic technique. After electrophoretic resolution of the samples the Triton X-100-washed polyacrylamide gels are sliced into l-ram segments which are assayed individually for collagenolytic activity by the fibril assay. Gelatinase activity can be demonstrated in electrophoretograms by a similar and even simpler technique based on copolymerization of heatdenatured type I collagen with the polyacrylamide gel. 51 The gelatin chains are cleaved to small peptides, which diffuse out of the gel and give rise to lytic zones when stained. Samples are prepared as described above by incubation with 2-5% SDS and then resolved by electrophoresis in the gelatin-acrylamide gel formed by addition of 1 mg/ml heat-denatured rat tail tendon type I collagen to the polyacrylamide solution. After electrophoresis, the gel is washed in succession in 2.5% Triton X-100 in distilled water and in Triton X-100 in 50 m M Tris-HC1, pH 7.5. For metalloproteases, 5 m M CaC12, I/zM ZnC12 is added. Incubation is at 37° for 2-4 hr before the gel is stained with Coomassie blue. Vertebrate collagenase does not generate small diffusible peptides and does not give rise to visible lytic bands with this method although it does possess some limited activity on denatured a-chains. 5 Microbial collagenases, however, cleave denatured o~-chains as well as native collagen and are readily visualized by this method. Cellular Collagen B r e a k d o w n A s s a y

Live cells seeded directly on a film of reconstituted collagen fibrils under certain conditions dissolve and degrade this substrate. This system offers several advantages for the study of cellular regulation of collagen breakdown and has provided some rather unexpected findings. The method described here is a modification of the techniques developed by Werb et al. 5z and Huybrechts-Godin et al. 53 A stock solution of rat tail tendon type I collagen (3 mg/ml) in 13 mM HCI is rapidly mixed with HEPES-based neutralizing buffer described above and dispensed in 150/~1 aliquots (20,000 cpm) in 24-well cluster dishes. The plates are incubated at 37° for 2 hr to allow for heat gelation and then placed overnight in the laminar flow hood to dry. Ultraviolet light must be turned off as the 5o j. A. Yankelow, W. B. Wacker, and M. M. Schweri, Biochim. Biophys. Acta 482, 159 (1977). 51 C. Heussen and E. B. Dowdle, Anal. Biochem. 102, 196 (1980). 52 Z. Werb, C. Mainardi, C. A. Vater, and E. D. Harris, N. Engl. J. Med. 296, 1017 (1977). 53 G. Huybrechts-Godin, P. Hauser, and G. Vaes, Biochem. J. 184, 643 (1979).

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collagen will denature if exposed to this radiation. The dried plates are washed repeatedly with distilled water to remove salt precipitates and, if necessary, stored in PBS with antibiotics. Immediately before use, the plates are equilibrated with 2-3 changes of culture medium. The cells are seeded at a suitable density: 5,000-50,000 cells per cm 2 for fibroblasts (human, rat, mouse skin) and 50,000-200,000 cm 2 for epithelial cells (rat tongue keratinocytes, human skin, and mammary carcinomas). The cells are allowed to attach overnight in the presence of serum which completely blocks collagen breakdown. The plates are then washed several times with PBS and incubated with MEM supplemented with 5 mg/ml bovine serum albumin (MEM-BSA) in addition to activators or inhibitors to be tested in a total volume of 500/.d per well. To monitor the release of radioactivity, 50- to 100-/~1 aliquots are removed for counting at suitable intervals (every hour, every 2 hr, every day), and replaced by fresh medium to maintain volume and composition. An example of this assay is shown in Fig. 4. Secretion of procollagenase is a necessary but not sufficient requirement for collagen breakdown inasmuch as the procollagenase remains in latent form throughout the incubation period unless a (proteolytic) activating agent (trypsin, chymotrypsin, plasmin) is added. 54 The susceptibility of the substrate film to trypsin is concentration dependent and varies from 10 to 12% at 1 /xg/ml to as much as 25% at 100/xg/ml (Fig. 4). The cellular collagen breakdown assay can be performed only with collagens which form fibrils in vitro such as those of types I and III and, with some modification, type II. Collagens absorbed onto plastic culture dishes from neutral or acidic solution are highly susceptible to a range of proteases and, therefore, not useful as substrates. Intact bovine lens capsules, however, can be dried onto the surface of plastic culture dishes in a cold airstream and used as a type IV collagen-enriched substrate for live cells. The capsules are dissected and thoroughly cleaned for contaminant tissue structures and sequentially extracted with 1 M NaC1, 50 mM TrisHCI, pH 7.5 (2 x 24 hr) and with 0.5 M HOAc (2 x 24 hr). The intact lens capsules are then radiolabeled by the method of Smith et al. 39 (see "Preparation and Labeling of Type IV Collagen") and washed in sequence in neutral and acidic buffers and finally suspended in 5 mM HOAc. The labeling procedure may result in incorporation of radioactivity into noncollagenous basement membrane proteins which are also released by cellular proteolysis but visual inspection of the dish after the experiment is usually sufficient to verify dissolution of the structural integrity of the capsule and thereby attack on the collagenous framework. Residual type .~4 H. Birkedal-Hansen, u n p u b l i s h e d results.

162

MAJOR COMPONENTS OF THE EXTRACELLULAR MATRIX

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,-,/REPCLONES IOu[MEM'BSA /

CCL-fO/ /

/

g

I00 #g/ml

1 /zq/ml CCL-IO

CCL-4 I

2

3

INCUBATION TIME {days) FIG. 4. Collagen breakdown by live rat mucosal keratinocytes. Cells from two collagenase-secreting keratinocyte clones (CCL-4, CCL-10) derived from rat tongue mucosa were seeded at a density of 100,000 cells per cm2 in 24-well cluster dishes coated with reconstituted [3H]acetyl collagen fibrils. The cells were allowed to attach overnight in the presence of serum and then washed with PBS and incubated in serum-free MEM-BSA. The dissolution of the collagen coating was monitored by the re/ease of radioactivity to the medium. Clones CCL-4 and CCL-10 released only 4-6% of the radioactivity in MEM-BSA, but when stimulated by addition of 1.0 #g/ml trypsin, the cells completely dissolved the collagen coating in 1-2 days. Controls included wells incubated either with MEM-BSA alone or with trypsin at 1.0, 10.0, and 100.0 p.g/ml trypsin dissolved in MEM-BSA. In the absence of cells, trypsin released from 10 to 25% of the radioactivity in a dose-dependent manner during a 3-day incubation period. Less than 2% of the radioactivity went into solution in MEMBSA alone. I V collagen is digested b y 2 0 / z g / m l clostridial collagenase to d e t e r m i n e the total r a d i o a c t i v i t y o f e a c h sample. I n the e x a m p l e s h o w n in Fig. 5, rat t o n g u e k e r a t i n o c y t e s w e r e s e e d e d at 12,000 and 50,000 p e r c m 2 and incub a t e d with s e r u m - f r e e M E M with o r w i t h o u t 1 /.,g/ml plasmin. T h e cells (50,000 p e r c m 2) r e l e a s e d 60% o f the r a d i o a c t i v i t y (5 days) w h e n stimulated b y p l a s m i n , w h e r e a s o n l y 1 0 - 1 5 % w a s released b y either plasmin or cells alone.

Isolation of Fibroblast Procollagenase M e t h o d s f o r p r o d u c t i o n o f v e r t e b r a t e c o l l a g e n a s e s / p r o c o l l a g e n a s e s in culture and f o r e x t r a c t i o n o f the e n z y m e s f r o m cells and tissues h a v e b e e n

[8]

CATABOLISM AND TURNOVER OF COLLAGENS

163

80

50 ~ io3 + P L . j

,,o"

/if "I" /./"

j./"

4O i /'/

g

2O

12 x 105 + PLN

/,, .d/" d"

I0.

Time (doys) FIG. 5. Dissolution of bovine lens capsule by live rat muscosal keratinocytes. Cells of clone CCL-4 were seeded at 12,000 and 50,000 per cm 2 on 3H-labeled intact bovine lens capsules dried onto 35-ram plastic petri dishes. The cells were allowed to attach overnight in the presence of serum and were then incubated for 5 days in serum-free MEM. Dissolution of the labeled lens capsules was measured by the release of radioactivity. At the conclusion of the experiment residual radioactivity was dissolved by clostridial collagenase and the cumulated daily release expressed as a percentage of total, The cells alone (C; El, ©) released only slightly more of the radioactivity than the medium by itself (M; A). When stimulated by addition of 1 /~g/ml human plasmin, the cells dissolved up to 60% of the incorporated label (11, O). Plasmin alone (&) dissolved 15% of the radioactivity during the 5day incubation period.

comprehensively reviewed by Harris and Vater 19in a previous volume of this series. The outcome of any purification sequence is highly dependent on the quality of the starting material and the amount of total activity available. Human skin fibroblasts, rabbit synovial fibroblasts, and fibroblasts from several other tissues and organs are rich sources of procollagenase/collagenase inasmuch as collagenase is a major secretory component which accounts for 2-6% of total secreted protein in unstimulated and 20-25% in TPA-induced cultures. 55,56By comparison, PMN collagenase constitutes in the order of 0.1% of the total protein of PMN extracts. Purification methods developed by Stricklin e t al. 57 based on chroma55 K. J. Valle and E. A. Bauer, J. Biol. Chem. 254, 10115 (1979). 56 j. Aggeler, S. M. Frisch, and Z. Werb, J. Cell Biol. 98, 1622 (1984), ~7 G. P. StrickEn, E. A. Bauer, J. J. Jeffrey, and A. Z. Eisen, Biochemistry 16, 1607 (1977).

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tography on CM-cellulose at pH 7.5 followed by gel filtration on Ultrogel AcA 44 were reviewed in a previous volume in this series. 19Variations of this method which utilize either phosphocellulose: 7 or sulfopropylSephadex 58 at neutral or slightly acidic pH are also highly effective since very few contaminants bind to the cation-exchange columns under these conditions. The alternative method described below 59 which is composed of purification steps already reported individually in the literature permits isolation of milligram quantities of collagenase from conditioned culture medium in one continuous process without bringing the enzyme out of solution. The method is based on the observation that the binding of collagenase to heparin-Sepharose 4B and Zn 2÷-chelate-Sepharose 4B takes place under somewhat different conditions so that the eluate from one column can be applied directly to the other without further processing (precipitation, dialysis). Conditioned serum-free culture medium (5-10 liter) is diluted 1 : 1 with 50 m M Tris-HC1, pH 7.4, and pumped onto a 5 × 10 cm column of heparin-Sepharose 4B at 4 ° at maximal speed (450-500 ml/hr). The column is washed with 50 mM Tris, pH 7.5, 0. I0 M NaCI and bound protein, including all of the collagenase, is eluted in a single peak by 0.5 M NaCI in 50 mM Tris-HCl, pH 7.5. The eluate is then immediately applied to a 1.5 x 15 cm column of iminodiacetate-Sepharose 4B saturated with ZnCI2. The column is washed with 0.5 M NaCI in 50 mM Tris, pH 8.1, and bound protein, including all of the collagenase activity, is eluted in a single peak with 50 m M imidazole, 0.5 M NaC1, 50 mM TrisHCI, pH 8.1. The eluate, about 60-80 ml, is then concentrated to 10-15 ml by passage over a small 1 × I0 cm heparin-Sepharose 4B column after dilution with 3 volumes of 50 mM Tris-HCl, pH 7.5, to reduce the salt concentration. Bound material is again eluted with 0.5 M NaC1, 50 mM Tris-HCl, pH 7.5, and immediately applied to a 2.5 × 95 cm column of Ultrogel AcA 44 equilibrated with 50 mM Tris-HCl, pH 7.5, 0.2 M NaC1, 5 m M CaCI~. Collagenase constitutes about 50% of the UV280 absorbing material before the gel filtration step and elutes in pure form as a symmetrical, included peak clearly separated from contaminants which emerge in the void volume. The yield is about 1 mg/liter culture medium.

Isolation of PMN Collagenase PMN collagenase can be isolated from human peripheral blood with a yield of 0.5-1.0 mg/101°cells?2'6°'6~ The purification procedure given be58 T. A. Bicsak and E. Harper, J. Biol. Chem. 259, 13145 (1984). 59 H. Birkedal-Hansen, unpublished results. 6o p. Christner, D. Damato, M. Reinhart, and W. Abrams, Biochemistry 21, 6005 (1982). 6i H. W. Macartney and H. Tschesche, Eur. J. Biochem. 130, 71 (1983).

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low is developed by Christner et al. 6° An alternative procedure has been published by Macartney and Tschesche. 61 The source of the enzyme may be either whole PMN lysates, PMN granule fractions, or material released from the cells by brief stimulation with 10-6-10 -7 M TPA. Christner et al. 6° isolated PMN granules from dextran and Ficoll-Hypaque-purified cells lysed with 0.34 M sucrose, 50 mM NaCI. Debris and nuclei are removed by centrifugation at 27,000 g for 20 min. The pH of the supernatant is adjusted to 7,0 with a 2 M Tris stock solution and the granules lysed in a sonifier at 0° by 30-sec pulses. The pellet is removed and again subjected to sonication two more times. The granule extract from 10 units of blood is applied in 10 ml to a 1.8 × 140 cm Sephadex G-200 column equlibrated with 50 mM Tris-HCl, pH 7.5, 0.4 M NaCI, 1 mM CaCI2. Active fractions are pooled and dialyzed against 0.1 mM CaC12, lyophilized and dissolved as a 10-fold concentrate in 10 mM Tris-HCl, pH 8.0, and applied to a 1.2 x 22 cm column of Trasylol-Sepharose 4B prepared by coupling 300 mg Trasylol to 100 ml Sepharose 4B by the CNBr procedure. The absorbed protein, including the collagenase activity, is eluted with a 100 ml linear gradient from 0.05 to 0.4 M NaCI in 10 mM TrisHCI, pH 8.0. Collagenase elutes as the first peak in the gradient with an overall recovery of approximately 77%. Isolation o f Type 1V Collagenase

The method described below is that developed by Salo et al. 38 using organ cultures of the murine PMT sarcoma as a source of enzyme. The culture medium is first fractionated by 25-60% saturated (NH4)2SO4precipitation. The pellet (60%) is then redissolved in 50 mM Tris-HCl, pH 7.4, 0.2 M NaC1, I0 m M CaC12 and applied to a 1.5 × 10 cm concanavalin A-Sepharose 4B column equilibrated with the same buffer. Bound protein, including type IV collagenolytic activity, is eluted with 1 M ot-methylglucoside. The fractions with the highest activity are pooled, dialyzed against CaCiz-free buffer (50 m M Tris-HCl, pH 7.4, 0.2 M NaCI), and concentrated by Amicon filtration. The sample is then passed slowly over a 1 ml Type IV coUagen-agarose column equilibrated with the same CaCl2-free buffer and bound protein is eluted with 1 M NaCI, 50% ethylene glycol, 50 m M Tris-HCl, pH 7.4. The fractions with the highest activity are pooled and finally passed over a 0.5 x 90 cm BioGel A-0.5 m molecular sieve column in 50 mM Tris-HCl, pH 7.4, 0.2 M NaC1, 10 mM CaClz. This procedure results in a 4200-fold purification relative to the (NH4)2SO4cut but the recovery is low (5%) and the yield from a total of 405 mg (NH4)2SO4 precipitable protein is only 5/~g. The enzyme consists of a Mr 68,000/62,000 doublet.

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Isolation o f Type V Collagenase Mainardi et al. ~z described the isolation from rabbit alveolar macrophages of a neutral secretory protease which cleaves type V collagen in addition to types I and V gelatin. The enzyme does not cleave native type I collagen. Twenty liter batches of culture medium from Freund's adjuvant activated rabbit alveolar macrophages are concentrated 200-fold by an Amicon hollow fiber device with a 10,000 Da exclusion limit and dialyzed against 0. I M Tris-HC1, pH 8.3, 0.05 M NaCI, 5 mM CaCI2, 0.02% NaN3 and cleared by centrifugation. The sample is applied to a 2 x 30 cm DEAE-Affigel blue column equilibrated with the same buffer and eluted with a 200 ml linear gradient from 0.05 to 0.2 M NaC1. Active fractions are pooled and dialyzed against 1 mM Tris-HCl, pH 8.3, 5 mM CaClz, 0.02% NAN3, and applied to a 2 x 10 cm column of type V collagen-Sepharose 4B equilibrated with the same buffer. Collagenase is eluted with a 200 ml linear gradient from 0.0 to 0.5 M NaCI. Active fractions are again pooled and applied to a 2 × 100 cm column of Sephadex G-200 and later to a column of Ultrogel AcA 34 in 50 mM Tris-HCl, pH 7.6, 0.5 M NaCI, 5 mM CaClz, and 0.02% NAN3. Recovery and yield are not reported but the peak eluted from the final molecular sieve column contains type V collagenase at a concentration of 1.6/zg/ml. Immunologic Techniques

Production o f Polyclonal Antibody to Interstitial Collagenase A number of laboratories have produced monospecific polyclonal antibodies to interstitial procollagenase/collagenase by conventional techniques. The following methods3 uses procollagenase prepared as described in "Isolation of Fibroblast Procollagenase" and subjected to an additional purification step by electrophoresis in 11% polyacrylamide, as the antigen. Human fibroblast procollagenase (500-1000/xg) is resolved by electrophoresis in a 3 mm 11% polyacrylamide gel equipped with a single well. A portion of the sample (200/zl; 100-200/xg) is reacted with fluorescamine (20/zl; 1 mg/ml) dissolved in dry dioxane for 10 min at 22° so that the migration of the sample can be monitored with a UV lamp. After electrophoresis, the procollagenase bands are excised from the gel under the guidance of the UV light and homogenized to a thick paste with PBS using a ice-cooled Polytron homogenizer operated at full speed for several consecutive bursts. The preparation is finally strained in succession through 26-, 23-, and 21-gauge needles, mixed with Freund's complete

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(first injection) or incomplete adjuvant (booster injections), and injected (2 ml, 100 t~g) at multiple sites into the back and side skin of 3-4 kg white New Zealand rabbits. A sample of 40 ml of blood is collected from the marginal ear vein before immunization. Booster injections are given every 2 weeks and 40 ml blood samples collected 1 week after each injection. IgG is isolated either by passage over protein A-Sepharose 4B or by the method of Harboe and Ingild62 by (NH4)2SO4 precipitation and chromatography on DEAE-Sephadex at pH 5.0. Production of Monoclonal Antibodies to Interstitial Procollagenase

Human fibroblast procollagenase purified as described above is mixed with complete Freund' s adjuvant and injected into the rear food pads and the inguinal region of two BALB/c mice (75/zg/mouse). Booster injections (5) are given every third day according to the method of Kearney et al. 63 The animals are sacrificed and the regional lymph nodes removed I day after the last injection. Cells teased from the nodes are washed twice with PBS, mixed 10:1 with mouse myeloma cells (X63-Ag8.653), and again washed in PBS. The cell pellet is shaken loose and a solution of 40% PEG 4000 in PBS preheated to 37 ° is added dropwise with shaking. After 30 sec, 5 ml of RPMI 1840 is added dropwise over a 5-min period. The cells are again pelleted by centrifugation and resuspended in selective medium (RPMI 1640 containing 15% fetal calf serum, 100/xM hypoxanthine, 0.4 /xM aminopterin, 1.6/zM thymidine, 50/~M 2-mercaptoethanol, 2 mM glutamine) and seeded in 24-well plates at approximately 0.5 x 106 cells per well. The medium is supplemented at a I : 100 v/v ratio with a feeder cell suspension produced by peritoneal washing of one or two mice with 5 ml each of RPM 1640. After 10-14 days of incubation, wells with growth (50-70%) are screened for immunoreactivity either by ELISA or by inhibition of coUagenase activity. In the latter case fetal calf serum is replaced either by serum-free medium or by serum in which o~2Mhas been inactivated by acid treatment (pH 2.3, 1 hr, 37°) or by reaction with 50 mM methylamine (pH 8.1, 1 hr, 22°). Reactive hybridomas are cloned by limiting dilution and recloned as needed either by limiting dilution or by growth in soft agar. Immunoglobulin is isolated by passage of the culture medium (50-500 ml) over a 1 x 5 cm protein A-Sepharose 4B column. 62 N. Harboe and A. Ingild, in " A Manual of Quantitative Immunoelectrophoresis. Methods and Applications" ( N. H. Axelsen, J. Kroll, and B. Weeke, eds.), p. 161. Universitetsforlaget, Oslo, Norway, 1973. 63 j. F. Kearney, in "Fundamental Immunology" (W. E. Paul, ed.), p. 751. Raven, New York, 1984.

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The column is washed with PBS and bound Ig eluted at 4° with 0.1 M HOAC, 0.2 M NaCI. The eluate is immediately neutralized with a 2 M Tris-HCl stock solution adjusted to pH 8.1. Alternatively, ascites is produced by intraperitoneal injection of 107 hybridoma cells into mice primed by ip injection of 1 ml pristane oil 1-3 weeks before inoculation of cells. Growth of tumors is monitored visually and ascites is tapped every 3-6 days as needed and purified by precipitation with 50% saturated ( N H 4 ) 2 S O 4 followed by gel filtration using an Ultrogel AcA 44 column equilibrated with PBS. Using similar methods, Hasty et al. 6 raised a monoclonal antibody to PMN collagenase from a partially purified enzyme preparation. R e a c t i v i t y o f P r o c o l l a g e n a s e / C o l l a g e n a s e on W e s t e r n Bolts

Screening of monoclonal antibodies and determination of polyclonal antisera specificity are greatly facilitated by immunoperoxidase staining of Western blots. The method below is essentially that described by Larsson et al. 64 Whole conditioned culture media or cell extracts are resolved by S D S - P A G E and transferred to a 12 × 14 cm nitrocellulose sheet which allows for cutting of about 30 lanes each 4.5-5.0 mm wide. The samples are prepared either by incubation for 1 hr at room temperature with 2-5% SDS in Neville's sample buffer under nondenaturing conditions or by heating to boiling in the presence or absence of reducive agents. The antigen mixture is then resolved by electrophoresis in 11% polyacrylamide using Neville's 25 discontinuous buffer system and 25 mA per gel for 2.5 hr and transferred to nitrocellulose paper by the method of Towbin et al. 65 and Burnette. 66 The paper is dried and stored in this fashion. Individual lanes, 4.5-5.0 mm wide, are cut with a paper cutter and blocked for 1 hr at 37 ° with 1% BSA in borate-saline (0.125 M Na2B407, pH 8.4, 0.125 M NaC1) (BS-BSA). One to three strips are placed in a Vshaped trough (buffer reservoir for multichannel pipetter marketed by Finpipet), and covered with 1500-2000 tzl antibody solution. Polyclonal antisera are diluted 1 : 200 to 1 : 10,000 and hybridoma media 1 : 1-1 : 50 with 50 m M Tris-HC1, pH 7.5, 0.2 M NaCI, 1% Triton X-100 (TBSTriton). Incubation is overnight at 4 ° followed by 1 hr at room temperature to allow for temperature equilibration. The antibody solution is then removed and the strips washed in three changes of TBS-Triton, 10 min each, at room temperature and incubated for 1.5 hr at room temperature L. I. Larsson, L. Skriver, L. S. Nielsen, J. Grondahl-Hansen, P. Kristensen, and K. Dano, J. Cell Biol. 98, 894 (1984). 65 H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350, (1979). 66 W. N. Burnette, Anal. Bioehem. 112, 195 (1981).

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with the second antibody, either HPO-conjugated swine anti-rabbit Ig (DAKO) or HPO-conjugated rabbit anti-mouse Ig both at 1 : 200 dilution in TBS-Triton, 1% BSA (TBS-Triton-BSA). The strips are washed in succession with TBS-Triton (2 x 10 min) and with 50 mM Tris-HCl, pH 7.5 (10 min). During the last wash the substrate is prepared by dissolution of 3,3-diaminobenzidine in distilled water (25 mg/50 ml). After vigorous stirring for 10 min the suspension is filtered to remove undissolved material and 15 /xl of 30% H202 is added to the filtrate. The strips are then submerged in the substrate solution and the development of brown bands monitored visually. With strong reactivity the image develops within 5-10 sec; weakly reacting antibodies require 1 min or more. The strips should be removed from the solution as quickly as possible. Exposure to the substrate beyond 2 min increases the background unacceptably without improving the staining pattern. The reaction is stopped by rinsing with 23 changes of distilled water.

Competition ELISA of Human Fibroblast Collagenase This a s s a y 67 follows standard methods in the field and utilizes a polyclonal rabbit antiserum raised against human fibroblast procollagenase purified from conditioned culture medium. Polystyrene microtest plates (EIA, Flow Laboratories) are coated overnight at 4° with 200/.d/well of a 0.5 /.tg/ml solution of human fibroblast procollagenase in borate-saline buffer, pH 8.4. Standards and unknowns are serially diluted in PBS, 0.05% Tween-20, 1% BSA, and 200/xl aliquots are incubated overnight at 4° with 200/~1 of a 1 : 8,000 dilution of the polyclonal antibody in the same buffer. The antigen-antibody mixtures are centrifuged the next day at 10,000 g for 8 min and 2000/xl of the supernates is incubated with the coated plates for 45 min at 37°. The plates are then washed 3 times with PBS-Tween and incubated for 1 hr at 37° with a 1 : 1,000 dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG in PBS-Tween. After another washing cycle in PBS-Tween, 200/xl of substrate solution (1 rag! ml p-nitrophenyl phosphate in 10% diethanolamine buffer, pH 9.8, 0.2 mM MgCI2) is added to each well and the plates incubated for 30 min at room temperature. The detection limit of this assay is around 0.2 ng (2 ng/ ml) which makes it 10-fold more sensitive than the previously published radioimmunoassays68,69 and probably 10 to 50-fold more sensitive than the best radiofibril assays. 67 T. W. Cooper, E. A. Bauer, and A. Z. Eisen, Collagen Rel. Res. 3, 205 (1983). 6s E. A. Bauer, A. Z. Eisen, and J. J. Jeffrey, J. Biol. Chem. 247, 6679 (1972). 69 A. Z. Eisen, J. Nepute, G. P. Stricklin, E. A. Bauer, and J. J. Jeffrey, Biochim. Biophys. Acta 350, 442 (1974).

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Immunoprecipitation

The protocol outlined in the following is based on the method described by Rosenfeld et al. 7° for immunoprecipitation of lysosomal enzymes in cellular extracts and culture media. Subconftuent or early confluent human skin fibroblast cultures are washed free of serum with PBS and incubated for 6-24 hr with 50 ~Ci/ml [35S]methionine (Amersham) in methionine-free MEM. The medium is harvested and dialyzed against 50 mM Tris-HCl, pH 7.4, 0.2 M NaCI, 5 mM CaCI2 whereas the cell layer is washed several times to remove unincorporated isotope and scraped off with a rubber policeman. Cell extract and medium are then adjusted to 1% Triton X-100, 0.8% SDS and boiled for 2 min and diluted with an equal volume 50 mM Tris-HC1, pH 7.4, 0.19 M NaCI, 6 mM EDTA, 2.5% Triton X-100, 0.02% NAN3, and 100 U/ml Trasylol. Ig from immune or preimmune sera is added and the reaction mixtures incubated for 1 hr at 22° or overnight at 4°. Immune complexes are precipitated by addition of protein A-Sepharose 4B (3 mg/tube) followed by incubation for 3 hr at room temperature. The precipitates are harvested by centrifugation at 10,000 g for 5 min and washed three times with 0.2% SDS in 50 mM TrisHCI, pH 7.5, 0.19 M NaC1, 6 mM EDTA, 2.5% Triton X-100, 0.02% NAN3, I00 U/ml Trasylol and boiled for 2 min in 10% SDS, I M DTT, 2 mM EDTA. Both protein A-Sepharose 4B and formalin-fixed S. aureus also bind a high-molecular-weight labeled contaminant, possibly fibronectin. This contaminant can be somewhat reduced by preincubation of the S. aureus preparation with serum or by passage of the [35S]methioninelabeled sample over protein A-Sepharose 4B before immunoprecipitation. Isolation o f RNA and in Vitro Translation o f Collagenase Messenger

The method described below is developed in Dr. Z. Werb's laboratory56,71 as a modification of the method of Monson 72 detailed in the previous volume. Diethyl pyrocarbonate (DEP)-water (Milli Q filtered water stirred with 0.1% DEP for 30 min and autoclaved for 3 hr) is used throughout for making solutions. The cells are dissolved by agitation in solution A (5 M guanidine thiocyanate, 50 mM EDTA, 2% sodium sarkosyl, 0.1% 2-mercaptoethanol, 0.2% antifoam A) and either used immediately or frozen in liquid nitrogen and stored at -70 °. The lysates are homogenized by 3 × 30 70 M. G. Rosenfeld, G. Kreibich, D. Popov, K. Kato, and D. D. Sabatini, J. Cell Biol. 93, 135 (1982). 7~ Z. Werb, personal communication. 72 j. Monson, this series, Vol. 82, p. 218.

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sec bursts in a Polytron homogenizer or by passage through a 25-gauge needle and cleared by centrifugation for 20 rain at 17,000 rpm in a Beckman JA 20 rotor. After addition of 1/4 volume of solution B (5.7 M CsCI, 25 mM EDTA), the lysate is overlayered onto a 5 ml cushion of solution B in a Beckman SW 27 cellulose nitrate tube and centrifuged 24 hr at 26,000 rpm at 20 ° to pellet RNA. Supernatants above the cushion are aspirated, tube walls washed three times with solution A, and, after inversion of the tubes and draining, R N A pellets are redissolved in 1.5 ml per tube of 10 mM EDTA, pH 8.0. The solution is heated briefly to 60 ° to dissolve RNA, and RNA is precipitated with 3 volumes of ethanol, 0.1 volume of 0.2 M lithium or potassium acetate, pH 6. RNA is pelleted at 17,000 rpm in a Beckman JA20 rotor at - 1 0 °, redissolved in 10 mM EDTA, pH 8.0, and extracted with an equal volume of chloroform-butanol (4:1) with two back-extractions of the organic phase. The combined aquaeous phases are then LiOAc or KOAc-ethanol precipitated three times to remove residual guanidine and cesium chloride, dried, and redissolved in D E P water at 1.0 mg/ml \~,1.~260~g' ~lx' 0l%nm 25). RNA is translated in the micrococcal nuclease-treated lysate system of Pelham and Jackson 73 using components obtained from Bethesda Research Labs. Reaction volumes are typically 15-30/~l containing 1-5/.~g of total RNA. Translations are reacted for 60 min at 30° and stopped by digestion with 0.1 mg/ml ribonuclease A for I5 min at 30°. This digestion eliminates background bands seen in gels due to charged tRNA and its degradation products. A maximum of 5/~l per lane is boiled in Laemmli sample buffer 24 and analyzed by fluorography. Alternative methods were published by Brinckerhoff et al. TM and by Bauer et al. 75 =

73 H. Pelham and R. Jackson, Eur. J. Biochern. 67, 247 (1975). 74 C. E. Brinckerhoff, R. H. Gross, H. Nagase, L. Sheldon, R. C. Jackson, and E. D. Harris, Biochemistry 21, 2675 (1982). 75 E. A. Bauer, T. W. Cooper, J. S. Huang, J, Altman, and T. F. Deuel, Proc. Natl. Acad. Sci. U.S.A. 82, 4132 (1985).