Proc. Natl. Acad. Sci. USA Vol. 87, pp. 5888-5892, August 1990 Biochemistry

Generation of collagenase-resistant collagen by site-directed mutagenesis of murine proal(I) collagen gene (collagenase resistance/Mov]13/in vitro mutagenesis/imutant type I collagen)

HONG WUt, MICHAEL H. BYRNEt, ALEX STACEYt§, MARY B. GOLDRINGt, JAMES R. BIRKHEADt, RUDOLF JAENISCHt, AND STEPHEN M. KRANE* tWhitehead Institute for Biomedical Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142; and tDepartment of Medicine, Harvard Medical School and Medical Services (Arthritis Unit), Massachusetts General Hospital, Boston, MA 02114 Communicated by Jerome Gross, April 30, 1990 (received for review March 9, 1990)

a2(I) collagen chain (16). The introduction of in vitro mutagenized Collal genes into Movl3 cells is, therefore, a suitable approach to evaluate the effect of specific Collal mutations on type I collagen synthesis and degradation. The collagenases cleave native type I, II, and III collagens by hydrolyzing the peptide bond between residues Gly-Ile (or Leu) located at residues 775 and 776 of the helical portion of the al(I) chain to yield a larger three-quarter length fragment (TCA) and a smaller one-quarter length fragment (TCB) (1, 4, 17). The region around the cleavage site is more hydrophobic than other parts of the collagen molecule and deficient in the hydroxyproline and proline residues that stabilize the triple helix. Our strategy was to generate mutations around the collagenase cleavage site in a mouse Collal genomic clone, transfect the mutant gene into fibroblasts derived from homozygous Movl3 mice, and then analyze the secreted collagen molecules for susceptibility to collagenase cleavage. These studies showed that type I collagen molecules containing wild-type a2(I) and mutant al(I) chains with amino acid substitutions of Pro for Ile at position 776 or for Gln and Ala at positions 774 and 777 were completely resistant to enzyme digestion.

Collagenase (matrix metalloproteinase 1) ABSTRACT cleaves type I, II, and III collagen helices at a specific site between Gly-Ile or Gly-Leu bonds (residues 775 and 776, Pj-P1'). To understand the mechanism of collagen processing, mutations around the cleavage site have been introduced into the cloned murine proal(I) collagen (Collal) gene. These mutant constructs have been transfected into homozygous Movl3 fibroblasts that do not express the endogenous Collal gene due to a retroviral insertion. Secreted triple-helical type I collagens containing substitutions of Pro for Be (position 776) (P1') were not cleaved by human rheumatoid synovial collagenase, whereas those containing substitutions of Met for BIe (position 776) were cleaved. Type I collagens containing double substitutions of Pro for Gln-774 (P2) and Ala-777 (P2') were not cleaved regardless of whether they contained the wild-type residue Ile at position 776 or the substitution of Met for He at position 776. The wild-type a2(I) chains derived from the endogenous Colla2 gene were also resistant to enzyme digestion when they were complexed with the mutant al(I) chains, indicating that the presence of normal al(I) sequences is critical for cleavage of the a2(I) chains in the type I heterotrimer.

MATERIALS AND METHODS

Collagenases are the only enzymes characterized so far that are capable of degrading undenatured type I, II, and III collagens extracellularly at neutral pH (1-5). A genetic approach to study the role of collagenases in physiological and pathological processes of connective tissue remodeling has been hampered by the lack of existing mutations in the collagenase genes. An alternative approach would be to introduce mutations into the collagen molecule that would render it resistant to cleavage by collagenase. Many inherited or spontaneous mutations in humans that lead to alterations in the structure and function of collagen have been described (6-11), but none has been reported that results in altered susceptibility to collagenase cleavage. We have used the Movl3 mutant to analyze sequence requirements for collagenase cleavage of the proal(I) collagen chain by introducing amino acid substitutions into the Collal gene. In the Movl3 mouse strain, the Collal gene is inactivated due to a proviral insertion while transcription of the Colla2 gene is not affected (12-14). Fibroblast cell lines derived from Movl3 homozygous embryos are deficient not only in the production of al(I) chains but also in the production of stable a2(I) collagen chains because triple helices are not formed in the absence of proal(I) chains (15). Transfection of human or mouse Collal genes into homozygous Movl3 cells, however, results in the synthesis and secretion of triple-helical type I collagen containing the endogenous

Construction of Mutant Collal Collagen Gene. All constructs were prepared as described by Stacey et al. (18) using derivatives of the murine Collal genomic clone 10D (16, 37). The nucleotide and amino acid sequences of the wild type and each mutation are given in Fig. 1. To facilitate mutagenesis in or around the collagenase cleavage, a Kpn I-Sac II "cassette" was constructed in which the Kpn I site at position 2026 (within an intron) was destroyed, rendering the other Kpn I site (at position 1809) unique, and amino acids Val-Val (782 and 783) were replaced by Ala-Ala to create a new Sac II site (mutants II-V). S1 Nuclease Protection Assay. Mutant constructs were transfected along with the selective markers, pAG60 and pSV2-neo, which contain the neomycin phosphotransferase gene conferring resistance to the drug G418 into the Movl3 cells or NIH 3T3 cells as described (19, 20) at a molar ratio of 7:1. NIH 3T3 cells were selected in G418 at 1 mg/ml (GIBCO) and Movl3 cells were selected in G418 at 0.3 mg/ml because they had different sensitivities for optimal selection. Total RNA from individual clones was prepared according to the method of Auffray and Rougeon (21). S1 nuclease analysis was performed to test the presence of mutant transcripts as well as the a2(I) collagen transcripts by established procedures (18, 22, 23). Five micrograms of RNA from each clone was ethanol precipitated with a mixture of the S1 probes that were empirically determined to be in excess by

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§Present address: Department of Physiology, Colorado State University, Fort Collins, CO 80523. 5888

Biochemistry: Wu et al.

Proc. Natl. Acad. Sci. USA 87 (1990)

region (16, 18). The mutant constructs encoded various amino acid substitutions around the collagenase cleavage site spanning amino acid residues between positions 774 and 783 (Fig. 1). The presence of mutant proal(I) and wild-type proa2(I) collagen transcripts was determined by S1 nuclease protection analysis. As expected, the 196-base fragment protected by the mutant transcript was detected in transfected Movl3 cells (Fig. 2A), whereas the 196-base and the 95-base protected fragments were seen in transfected NIH 3T3 clones (Fig. 2B). No 95-base protected fragment was seen in transfected Movl3 cells (not shown in this particular gel), as shown previously (18). The 122-base fragment characteristic of the wild-type proa2(I) collagen transcripts was detected in all clones. The relative levels of these protected fragments differed among clones, indicating that different amounts of proal(I) collagen transcripts were produced from the mutant constructs and that the ratio of al(I):a2(I) varied from clone to clone. Type I Collagen Containing Mutant al(l) Chains Is Resistant to Collagenase Cleavage. Type I collagen containing mutant al(I) chains was prepared by limited digestion of proteins with pepsin after labeling the cells with [3H]proline. Whereas untransfected Movl3 cells did not produce detectable type I collagen, in agreement with previous reports (13, 15, 16) (Fig. 3A, lane 3), Movl3 cells transfected with the mutant constructs produced type I collagen consisting of the mutant al(I) chains and the wild-type a2(I) chains (Fig. 3A, lanes 5, 7, and 9). Different amounts of mutant al(I) chains were produced by individual transfected clones, and the amount of type I collagen as well as the al(I):a2(I) ratio varied from clone to clone (Fig. 3A), consistent with the variations in collagen transcriptional levels. To test the susceptibility of mutant type I collagen to collagenase digestion, the labeled collagens were exposed to partially purified rheumatoid synovial fibroblast collagenase. An excess of unlabeled rat tail collagen was added as carrier. Digestion conditions (18-24 hr at 20°C) were chosen to allow complete cleavage of the carrier collagen (Fig. 3B) as well as labeled type I and III collagens synthesized by control NIH 3T3 cells (Fig. 3A), indicating that the enzyme was in excess. As shown in Fig. 3A, lanes 1, 2, 5, and 6, wild-type type I and type III collagens were completely digested by collagenase, as indicated by the presence of the expected cleavage products TCAal(I), TCBal(I), TCAa2(I), TCBa2(I), TCAal(III), and TCBal(III). Although the TCAal(l) and TCBal(III) migrated similarly under nonreducing conditions (lanes 2 and 6), both cleavage products were well separated under reducing conditions (data not shown). The type V collagen chains were not cleaved by collagenase under this condition, consistent with previous observations (29). In contrast to wild-type collagen produced

using increasing amounts of RNA. After denaturation at 80'C, the hybridization was carried out at 50'C for 16-20 hr. Collagenase Digestion. Preparation of labeled procollagen was performed as described (24). Samples were dissolved in collagenase buffer (0.15 M NaCl/0.05 M Tris HCl, pH 7.4/ 0.005 M CaCl2/0.25 M glucose) and incubated at 20'C with trypsin-activated procollagenase for various periods up to 18 hr. Procollagenase was obtained from medium conditioned by primary cultures of rheumatoid synovial fibroblasts, precipitated with 50% saturated (NH4)2SO4, and chromatographed on columns of Ultrogel AcA-54 (LKB, Bromm, Sweden) (25). The peak containing procollagenase, which was assayed after activation with trypsin (TRPCK; Cooper Biomedical), was collected and used. After incubation with collagenase, the collagen samples were mixed with loading buffer with or without 0.1% 2-mercaptoethanol and fractionated by electrophoresis on 7% polyacrylamide SDS/PAGE gels (24). Fluorography was performed as described (24). Determination of Denaturation Temperature of Collagens. Denaturation (melting) temperature was determined by measuring the susceptibility of mutant collagens to trypsin digestion over a range of temperatures by a modification of the method of Constantinou et al. (26). Cyanogen Bromide (CNBr) Digestion. Labeled type I collagen chains or collagenase cleavage products of these chains were first separated by SDS/PAGE as described. To identify bands in wet gels, proteins were first fluoresceinated (27). Following SDS/PAGE, bands identified under UV light were cut out of the wet gels, solubilized using an electroeluter (model 422, Bio-Rad) according to the manufacturer's instructions, and freeze-dried. For CNBr cleavage samples were then redissolved in CNBr in 70%o (vol/vol) formic acid and maintained at 15-20°C for 3 hr. The CNBr/formic acid was then removed by freeze-drying. In other experiments, appropriate bands were identified in the slab gels, excised, incubated directly in 5% CNBr in 70% formic acid, and incubated for 2 hr at 15-20°C followed by water washes according to the procedure of Byers et al. (9). The resulting peptides were separated by SDS/PAGE in 10%o or 12% acrylamide or 10-20% gradient gels (Integrated Separation Systems, Newton, MA) and analyzed by fluorography.

RESULTS Mutagenesis of Collal Gene and Derivation of Cell Clones Producing Mutant mRNAs. Five different mutations were introduced into a murine Collal genomic clone that includes about 17 kilobases (kb) of coding region, 3.7 kb of 5' and 3 kb of 3' flanking sequences, and a 21-base-pair (bp) polylinker fragment inserted into the Xba I site in the 5' untranslated

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Biochemistry: Wu et al.

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Proc. Natl. Acad. Sci. USA 87 (1990) 3T3 1 2

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FIG. 2. S1 nuclease analysis of the expression of wild-type (endogenous) and mutant (exogenous) Collal genes in Movl3 and NIH 3T3 (3T3) cells. Five micrograms of total RNA from each transfected clone was used. Hybridization was carried out at 50TC with a mixture of mouse al(I) (depicted in C) and a2(I) S1 probes. The a2(1) probe, derived from pAZ1002 (28), functions as an internal control by giving rise to a 122-base protected fragment. (A) Mutant gene expression in Movl3 cells. Lane 1, untransfected Movl3 cells; lanes 2-5, Movl3 cells transfected with mutants II, I, IV, and V, respectively. (B) mRNA from mutant IV transfected NIH 3T3 cells. Lanes 1-3, mRNA extracted from clones 4, 8, and 11, respectively. (C) Scheme of the S1 nuclease analysis used to distinguish endogenous and exogenous al(I) transcripts. The probe was 600 bases in length and included the 21-base insert as shown (triangle). The exogenous transcripts protect a 196-base fragment, whereas the endogenous transcripts protect a 95-base fragment.

by NIH 3T3 cells (Fig. 3A, lane 2), the al(I) collagen chains produced by Movl3 cells transfected with mutants I and II (see Fig. 1) were completely resistant to collagenase digestion (Fig. 3 A and B). Importantly, the wild-type a2(I) chains in the heterotrimeric collagen molecules were not cleaved by the enzyme, suggesting that the correct sequences of both al(I) and a2(I) chains in register are required for the enzyme activity. Under the same conditions, -31-39% of type I collagens produced by Movl3 cells transfected with mutants III and V were cleaved by collagenase (data not shown). These findings suggest that the enzyme recognizes sequences beyond the highly conserved region defined previously (17). To examine whether the secreted mutant collagen was in the native helical conformation, the melting temperatures of the mutant as well as wild-type collagens were compared by digestion with trypsin at different temperatures (see Materials and Methods). All mutant type I collagens tested had a melting temperature of -380C, indistinguishable from that of the wild-type collagen (data not shown). Mixed Collagen Type I Heterotrimers Containing Mutant and Wild-Type al(I) Chains Are Sensitive to Collagenase Cleavage. The results described above indicated that heterotrimers consisting of two mutant al(I) chains and one wildtype a2(I) chain were resistant to cleavage by collagenase. The following experiments were designed to examine whether a single mutant al(I) chain in the triple helix would be sufficient to render the heterotrimer resistant to collagenase cleavage. Mutants IV and V carrying a Met at position 776 had been engineered from mutants II and III. Since Met and Ile have similar hydrophobicity, we expected that mutants IV and V would behave similarly to their parental constructs. This was

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27 times in type I, II, III, and IV collagens from different species, suggesting that sequences flanking the cleavage site are important for enzyme binding or catalytic action. To understand the function of surrounding sequences that underlie specificity at the cleavage site, we generated mutants II and IV. In these mutants, Pro was substituted in the -Yaa- position of the Gly-Xaa-Yaatriplet. Such substitutions are predicted to stabilize the triple helix (31, 33), particularly because Pro residues in the -Yaaposition are substrates for prolyl 4-hydroxylase (34). Although we were unable to detect a change in denaturation temperature of the native mutant collagen, the Pro substitutions rendered the protein completely resistant to collagenase digestion. These results are consistent with the hypothesis that local helix instability may be a factor that determines the ability of collagenase to cleave native collagens. Val-782,783 -* Ala. These substitutions were introduced into the gene in order to generate a cassette that would facilitate the introduction of oligonucleotides containing the various point mutations into the Collal gene. Contrary to our expectations, however, the rate of collagenase cleavage of type I collagens containing mutation III and V was slower than that of the wild-type collagen. The P7'-P8' sites in collagens I, II, and III contain one or two hydrophobic residues such as Val, Ile, Leu, or Phe. Because Ala residues are more hydrophilic than Val, and substitutions of Val782,783 Ala are not conservative, our results suggest that hydrophobicity in this domain may be important for efficient cleavage. Our results also suggest that cleavage of the a2(I) collagen chain in the type I heterotrimer is dependent on the presence of cleavable sequences in the al(I) chains. The data described in Fig. 4 suggest, however, that a single mutant al(I) chain in a mixed heterotrimer does not prevent cleavage of all three chains of the triple helix. Nevertheless, resistance of a single mutant chain would probably prevent dissociation of partially digested wild-type chains in the heterotrimer. We cannot predict what effect the expression of the mutant genes might have on development when introduced into transgenic animals. It is possible that type I collagen that is resistant to cleavage by collagenase would interfere with tissue remodeling during early morphogenesis. We do not know, however, whether a collagenase-resistant Collal gene would act in a dominant negative manner (18, 35, 36), resulting in disturbance of embryonic development of a wild-type embryo carrying a mutant transgene. Alternatively, the collagenase-resistant Collal gene may cause a recessive phenotype. This could be analyzed in vivo by crossing a transgenic mouse carrying the mutant gene with Movl3 mice, thus eliminating the wild-type Collal genes and revealing the phenotype of the recessive mutation. If the developmental consequences of collagenase-resistant Collal genes are not severe and live mice can be obtained, they should provide useful models for human inflammatory diseases in which remodeling of collagen in connective tissues is disturbed.

Proc. Natl. Acad. Sci. USA 87 (1990) This work was supported by National Institutes of Health Grants 5R35 CA44339 and P01 HL41484 to R.J. and National Institutes of Health Grants AR03564 and AR07258 to S.M.K. This study was performed as part of the Ph.D. thesis requirement of H.W. at the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School. 1. Gross, J. (1981) in Cell Biology of the Extracellular Matrix, ed. Hay, E. D. (Plenum, New York), pp. 217-258. 2. Harris, E. D., Jr., & Vater, C. A. (1982) Methods Enzymol. 82, 423-452. 3. Harris, E. D., Jr., Welgus, H. G. & Krane, S. M. (1984) Collagen Relat. Res. 4, 493-512. 4. Woolley, D. E. (1984) in Extracellular Matrix Biochemistry, eds. Piez, K. A. & Reddi, A. H. (Elsevier, New York), pp. 119-157. 5. Birkedal-Hansen, H. (1987) Methods Enzymol. 144, 140-171. 6. Prockop, D. J. & Kivirikko, K. I. (1984) N. Engl. J. Med. 311, 376-386. 7. Cheah, K. S. E. (1985) Biochem. J. 229, 287-303. 8. Prockop, D. J. (1985) J. Clin. Invest. 75, 783-787. 9. Byers, P. H., Tsipouras, P., Bonadio, J. F., Starman, B. J. & Schwartz, R. C. (1988) Am. J. Hum. Genet. 42, 237-248. 10. Cetta, G., Ramirez, F. & Tsipouras, P. (1988) Ann. N. Y. Acad. Sci. 543, 1-187. 11. Cole, W. B., Jaenisch, R. & Bateman, J. R. (1989) Q. J. Med. 70, 1-4. 12. Jaenisch, R., Harbers, K., Schnieke, A., Lohler, J., Chumakov, I., Jahner, D., Grotkopp, D. & Hoffmann, E. (1983) Cell 32, 209-216. 13. Schnieke, A., Harbers, K. & Jaenisch, R. (1983) Nature (London) 304, 315-320. 14. Lohler, J., Timpl, R. & Jaenisch, R. (1984) Cell 38, 597-607. 15. Dziadek, M., Timpl, R. & Jaenisch, R. (1987) Biochem. J. 244, 375-379. 16. Schnieke, A., Dziadek, M., Bateman, J.,-Mascara, T., Harbers, K., Gelinas, R. & Jaenisch, R. (1987) Proc. Natl. Acad. Sci. USA 84, 764-768. 17. Fields, G. B., Van Wart, H. E. & Birkedal-Hansen, H. (1987) J. Biol. Chem. 262, 6221-6226. 18. Stacey, A., Bateman, J., Choi, T., Mascara, T., Cole, W. & Jaenisch, R. (1988) Nature (London) 332, 131-136. 19. Graham, R. & Van der Eb, A. (1973) Virology 52, 456-457. 20. Colbbre-Garapin, F., Horodniceanu, F., Kourilsky, P. & Garapin, A.-C. (1981) J. Mol. Biol. 150, 1-14. 21. Auffray, C. & Rougeon, F. (1980) Eur. J. Biochem. 107, 303-314. 22. Berk, A. J. & Sharp, P. A. (1977) Cell 12, 721-732. 23. Wu, H., Bateman, J., Schnieke, A., Sharpe, A., Barker, D., Mascara, T., Eyre, D., Bruns, R., Krimpenfort, P., Berns, A. & Jaenisch, R. (1990) Mol. Cell. Biol. 10, 1452-1460. 24. Goldring, M. B. & Krane, S. M. (1987) J. Biol. Chem. 262, 16724-16729. 25. Dayer, J.-M., Stephenson, M. L., Schmidt, E., Karge, W. & Krane, S. M. (1981) FEBS Lett. 124, 253-256. 26. Constantinou, C. D., Vogel, B. E., Jeffrey, J. J. & Prockop, D. J. (1987) Eur. J. Biochem. 163, 247-251. 27. Mann, K. G. & Fish, W. W. (1972) Methods Enzymol. 26, 28-42. 28. Liau, G., Yamada, Y. & de Crombrugghe, B. (1985) J. Biol. Chem. 260, 531-536. 29. Welgus, H. G., Jeffrey, J. J. & Eisen, A. Z. (1981) J. Biol. Chem. 256, 9511-9515. 30. Traub, W. & Piez, K. A. (1971) Adv. Protein Chem. 25, 243-352. 31. Gross, J., Highberger, J. H., Johnson-Wint, B. & Biswas, C. (1980) in Collagenase in Normal and Pathological Connective Tissues, eds. Woolley, D. E. & Evanson, J. M. (Wiley, Chichester, U.K.), pp. 11-35. 32. Rose, G. D., Geselowitz, A. R., Lesser, G. J., Lee, R. H. & Zehfus, M. H. (1985) Science 229, 834-838. 33. Miller, E. J., Finch, J. E., Jr., Chung, E. & Butler, W. T. (1976) Arch. Biochem. Biophys. 173, 631-637. 34. Kivirikko, K. I. & Myllyla, R. (1982) Methods Enzymol. 82, 245-304. 35. Herskowitz, I. (1987) Nature (London) 329, 219-222. 36. Jaenisch, R. (1988) Science 240, 1468-1474. 37. Monson, J. M., Friedman, J. & McCarthy, B. J. (1982) Mol. Cell. Biol. 2, 1362-1371.