29. the Release of Collagenase Inhibitors from Procollagen Additional Peptides by Pepsin Treatment

Home , Collagenase, Procollagen peptidase

140 Proc. Japan Acad., 54, Ser. B (1978) [Vol. 54(B),

29. The Release o f Collagenase Inhibitors from Procollagen Additional Peptides by Pepsin Treatment*'

By Yutaka NAGAI,* * ) Hiroshi SHINKAI,* * * and Yoshif umi NINOMIYA* *

(Communicated by Yasuj i KATSUx1, M. J. A., March 13, 1978)

During the course of studies on the regulation of collagenase activity in vivo and in vitro, an inactive complex of the enzyme with inhibitors, accumulating in cultured media of embryonic chick' and human skin2~ explants, was observed. This type of inactive enzyme was also detected in synovial fluids from patients with osteo- and rheumatoid arthritis.3~,4) Analysis of the inactive enzyme have revealed that the molecular sizes of inhibitors were about 12,000 and 6,000, respectively, regardless of the sources of enzymes.5~ Further- more, the presence of excess amount of free inhibitors of the same molecular sizes was detected in the embryonic chick skin culture system,5> suggesting that these inhibitors are derived from connective tissue macromolecule metabolites, but not from procollagenase, a precursor of the enzyme. A question arised from these observations is whether the inhibitors are from preexisting macromolecules in tissues or not. Therefore, attempts to use matrix-free cell culture systems were undertaken to see if release of collagenase inhibitors is related with synthesis of matrix components by the cells. Recent observation with human skin fibroblast culture system has shown that collagenase inhibitors with molecular weights of about 15,000 and 8,000-6,000 are released from procollagen preparation by limited proteolysis with pepsin (to be published elsewhere). This paper describes evidence that collagenase inhibitors are derived from the N- and/or C-terminal additional peptide portion(s) of procollagen, using chick tendon cell culture system. A novel function of procollagen additional peptide(s) is proposed with special reference to selective protection of newly formed collagen against proteolysis by collagenase. Matrix-free cells were prepared by enzyme (bacterial collagenase *' This work was supported in part by Scientific Research Grants from the Ministry of Education, Science and Culture of Japan (211203, 244075 and 257278). * *' Department of Tissue Physiology , Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101. ***' Department of Dermatology , Osaka University School of Medicine, Fukushima-ku, Osaka 553. No. 3] Collagenase Inhibitors from Procollagen 141 plus trypsin) digestion of tendons from 16-day-old chick embryos according to the method of Dehm and Prockop.6) About 3 x 106 cells were incubated with 500 ~tCi of [3H] proline (G) (Daiichi Pure Chemicals Co. Tokyo) at 37°C for 5 hr in 44 ml of a modified Krebs medium supplemented with D-glucose (final 0.1%), R-aminopropioni tril f umarate (100 pg/ml), L-ascorbate (50 pg/ml), L-glutamine (final 3 mM) and ferrous sulfate (final 3 ,tM). At the end of incubation, 4.4 ml of a mixture of protease inhibitors consisting of 0.25 M EDTA, 0.1 M N-ethylmaleimide and 10 mM phenylmethanesulfonyl fluoride was added. The medium was centrifuged and procollagen in the supernatant was purified by ammonium sulfate precipitation (30% saturation) followed by DEAE-cellulose (Whatman DE-32) chromatography according to the method of Hoffmann et a1.7> Loca- tion of procollagen in the effluent fractions from DEAE-cellulose column was determined by measuring radioactivity and by SDS-poly-

Fig. 1. SDS-gel electrophoresis of chick tendon [3H]-procollagen purified by DEAE-cellulose chromatography. Electrophoresis was carried out with a 3% upper gel and a 5% lower gel in the presence of 3M urea at pH 8.3. Each sample contained 8000 dpm and a carrier collagen isolated from embryonic chick skin by pepsin treatment. After electrophoresis, the positions of migration of collagen components were determined by staining with 0.25% Coomassie brilliant blue as indicated by arrows, then the gels were sliced transversally into 1-mm discs for assay of 3H-radioactivity to locate labeled proteins. a): [3H]-procollagen; not reduced. b): [3H]-procollagen; reduced with 0.01 M dithiothreitol. 142 Y. NAGAI, H. SHINKAI, and Y. NINOMIYA [Vol. 54(B), acrylamide gel electrophoresis.8> For this purpose, a Beckman liquid scintillation system model LS-9000 was employed. The effluent fractions containing procollagen were pooled, supplemented with 2.8 mg of pepsin-treated chick skin collagen as a carrier, dialyzed against 0.1 M Tris-HC1 buffer, pH 8.0, containing 0.4 M NaCI and procollagen was precipitated by adding ammonium sulfate (final 30% saturation). The precipitate was shown to contain only procollagen (Fig. 1), since no appreciable radioactivity except pro r was observed before reduction (Fig. 1-a) and pro a1 and pro a2 with the ratio of 2.1 to 1 were obtained after reduction (Fig. 1-b) . An aliquot (5.86x105 dpm, 1 ml) of the purified procollagen in 0.05 M acetic acid was subjected to limited proteolysis with pepsin (20 pg) at room temperature for 20 hr, then chilled alcohol was added to precipitate collagen released. The supernatant was concentrated to remove alcohol, neutralized with 1 M Tris-HC1 buffer, pH 8.0, and submitted to Sephadex G-50 superfine chromatography. Inhibitory effect of the effluent fractions on collagenase activity was determined with purified tadpole enzyme, using 14C-labeled reconstituted guinea pig skin collagen fibrils as substrate.9~ As shown in Fig. 2, four distinct collagenase inhibitory activities were observed in the break through fraction and the fractions corre- sponding to molecular weights of about 15,000, 8,000 and 6,000, respectively. The molecular sizes of the latter three were quite similar to those released from chick skin latent collagenase by NaI treatment5~ and from human skin procollagen preparation by limited proteolysis with pepsin (to be published elsewhere). As regards the inhibiting activity in the break through fraction, this may not be from the procollagen but from carrier collagen, which might be contaminated due to incomplete precipitation at 60% alcohol concentration, since no correspondence with radioactivity was observed and collagen substrate shows a high affinity with collagenase at neutral pH.1o> Collagen is known to be synthesized and secreted as procollagen, a precursor of collagen, by the collagen-forming cells, then procollagen is converted to collagen by limited proteolysis with neutral proteases so-called "procollagen peptidase", releasing additional peptides from the N- and C-terminal portions of the molecule." However, the fate of the peptides released remains obscure so far. Pepsin treatment also provides a similar conversion of procollagen to collagen,12~ which could be precipitated at 60% alcohol concentration. SDS-polyacryl- amide gel electrophoresis of the precipitate showed to be composed of only a1 and a2 with the ratio of 2:1, based on the determination of radioactivity in the gel (data not shown). No smaller collagen frag- ments were observed. These results indicate that collagenase inhibi- No. 3] Collagenase Inhibitors from Procollagen 143

. r/VY1~I I"1./r1 Fig. 2. Elution profile of collagenase inhibiting principles in procollagen additional peptide fraction by Sephadex G-50 superfine chromatography. The supernatant (9.l x 104 dpm) of pepsin-treated procollagen at 60% alcohol concentration was concentrated, neutralized and applied to a Sephadex G-50 superfine column (1.4X47 cm) equilibrated with 0.05 M Tris-HC1 buffer, pH 7.5, containing 0.15 M NaCI and 5 mM CaC12. Fractions of 1.2 ml each were collected at a flow rate of 7.2 ml/hr. Aliquots of 0.2 ml each of effluent fractions were preincubated at room temperature for 30 min with 0.075 ml of purified tadpole collagenase (6.54 U/ml) in the presence of 0.03% bovine serum albumin, then collagenase activity was assayed with 0.22 ml of the incubation mixture, using 14C-labeled guinea pig skin collagen fibrils as substrate. 0 : 3H-radioactivity. A : relative collagenase activity. BSA, bovine serum albumin; CHTG, chymotryp- sinogen; CYT C, cytochrome C; TRAS, trasylol; BACIT, bacitracin.

tors are derived from the additional peptide portions of procollagen. As regards the function of the additional peptides of procollagen, following possibilities have been suggested : 1) association and align- ment of pro a chains to facilitate triple helix formation within the cells, 2) prevention of intracellular fibrillogenesis, 3) transmembrane and transcellular movement and so on.13> However, nothing is known as to the fate and role of either N- terminal (molecular weight: about 16,000) or C-terminal (molecular weight : about 35,000) additional peptide after being released from procollagen by limited proteolysis. The finding that the peptide(s) possesses collagenase inhibitory capacity would provide an additional function of the peptide(s) in the extra-cellular matrix. Of most interest in this study is the fact that regulation of collagenase activity or collagen catabolism is closely coupled with bio- synthesis of collagen. By this way, a newly synthesized collagen could selectively survive from proteolytic cleavage by collagenase. This is

144 Y. NAGAI, H. SHINKAI, and Y. NINOMIYA [Vol. 54(B),

consistent with autoradiographic observations that newly synthesized collagen is deposited at the epidermal-lamellar junction, migrating down to the bottom of the lamella and resorbed by the fibroblast after periods of 2-4 weeks.l4) Summary. Procollagen, a precursor of collagen, was isolated from cultured medium of matrix-free cells from tendon of 16-day-old chick embryos. Tendon procollagen was shown, by limited proteolysis with pepsin followed by gel chromatography, to possess collagenase inhibiting capacity at its N- and/or C-terminal additional peptide portion(s). The results suggest that collagenase inhibiting activity is manifested upon conversion of procollagen to collagen to protect it from proteolysis with collagenase.

References

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