Procollagen Peptidase: an Enzyme Excising the Coordination Peptides of Procollagen (Connective Tissue/Collagen/Calf/Acrylamide Electrophoresis/Dermatosparaxis)

Proc. Nat. Acad. Sci. USA Vol. 68, No. 12, pp. 3054-3058, December 1971

Procollagen Peptidase: An Enzyme Excising the Coordination Peptides of Procollagen (connective tissue/collagen/calf/acrylamide electrophoresis/dermatosparaxis)

CHARLES M. LAPIERE, ALBERT LENAERS, AND LEONARD D. KOHN Service de Dermatologie, H6pital de Baviere, University of Liege, 4000 Liege, Belgium; and Laboratory of Biochemical Pharmacology, The National Institute of Arthritis and Metabolic Diseases, Bethesda, Maryland 20014 Communicated by C. B. Anfinsen, September 27, 1971

ABSTRACT A heritable connective tissue disorder of creased length being the result of an additional peptide at the cattle, dermatosparaxis, is characterized by an extreme N-terminal end of the molecules, i.e., the abnormal a-chains, fragility of the skin and the presence of additional peptides at the N-terminal extremities of the collagen a p-al amd p-a2, appeared to be the normal a, and a2 chains chains, p-al and p-a2. extended at their N-termini by an additional peptide of 5,000- The existence of an enzyme activity is demonstrated in 10,000 molecular weight. The suggestion was made that these normal connective tissues that is capable of cleaving these peptides were involved in the registration of the a chains after additional N-terminal peptides from dermatosparaxic collagen. The activity is demonstratable with dermato- synthesis, and the hypothesis was presented that the resultant sparaxic collagen in solution, as well as with reconstituted association product was a precursor of the collagen normally dermatosparaxic collagen fibrils polymerized in vitro. found in the extracellular space, a normal collagen precursor It has a pH optimum of about 7.0 and is inhibited by or "procollagen" from which coordination peptides were not EDTA and mercaptoethanol. Differences in Km and Vmaz removed. values exist depending on the substrate utilized, i.e., p-al or p-a2; and the presence of additional amounts of one Collagen a chains similar to the p-al chains of dermato- substrate, p-al, alters the concentration requirement for sparaxic calves have been demonstrated in tissue cultures of the second substrate, p-a2- The product of the excision human fibroblasts (6) and in both rat (7) and chick (8) bone reaction with p-al as substrate is an equiniolar amount of cultures. Pulse-chase experiments demonstrated that these normal al monomer; the product when p-a2 is substrate is an equimolar amount of normal a2 monomer. pre-a chains were procollagen forms and a nonspecific protease, The enzyme is present in normal calf skin, tendon, pepsin, was used to convert them to normal al chains. The aorta, cartilage, and lung; it can be demonstrated in the proteolytic conversion of a soluble collagen precursor [pro- skin of rats and humans. The enzyme activity is absent in collagen] into the polymerizable type of collagen normally dermatosparaxic connective tissues, thus suggesting that found in connective tissues was a hypothesis presented by dermatosparaxis is caused by the absence of a normal enzyme function rather than by the production of an ab- Schmitt (9) in 1960, and recently extended by Speakman (10) normal collagen. on theoretical grounds. Coordination peptides would be re- quired to initiate the formation of the collagen triple helix. Inbreeding and selection in the cattle population of Central An enzyme would cleave the additional peptides during or and High Belgium has resulted in the disclosure of an un- after the formation of the stable, triple-helical, collagen struc- wanted recessive anomaly of the connective tissue. This ture. A defective or absent excision enzyme might result in a heritable disorder, characterized by an extreme fragility of disorder of the connective tissue. the skin, was described in 1967 as dermatosparaxis (1, 2). The In the present report, we demonstrate the existence of such dermis of dermatosparaxic animals contains disorganized an enzyme in normal connective tissues and its absence in collagen bundles, the parallel packing of individual collagen cases of dermatosparaxis. filaments within the collagen fibers appear to be impaired, and the individual filaments display a weakly staining cross- MATERIALS AND METHODS striation (3, 4). The abnormal collagen was shown (5) to contain a signifi- Preparation of the Collagen Substrate. Dermatosparaxic and cant proportion of two additional types of monomers hitherto normal calves were obtained from the Department of Animal undescribed. The first, p-al, resembles in its aminoacid com- Genetics of the University of Libge in Cureghem. All animals position the a, chain of collagen extracted from the skin of a were killed and their tissues were collected as described (5). normal calf, but its molecular weight is 10% greater. The Collagen was extracted from homogenized tissues by sequen- second, p-a2, was similar to the normal a2 chain in its amino- tial treatment with 0.15 M NaCl, 1.0 NaCl, and 0.1 M acetic acid composition, but its molecular weight was 5% greater. acid (5, 11). The substrate for all experiments described here The abnormal a chains contained 6-8 half-cystine residues in was the collagen extracted from dermatosparaxic skin by the p-al and one in p-a2. Microscopically, the dermatosparaxic cold 1.0 M NaCl solution; it was purified by a standard procollagen was 25 nm larger than the normal collagen, the in- cedure (11). Before inclusion in the assay mixture, aliquots of the stored, lyophilized substrate preparation were solubilized Abbreviations: TCA, trichloroacetic acid; PCMB, p-chloro- in cold potassium phosphate at pH 7.6 (r/2 = 0.4) and di- mercuribenzoate. alyzed against a 100-fold excess of 0.4 M NaCl for 24 hr at 0°C. 3054 Downloaded by guest on September 27, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Procollagen Peptidase 3055

The substrate solution was clarified by centrifugation (150,000 X g for 60 min) and adjusted to a protein concentration of about 1 mg/ml. The composition of this substrate, in terms of its various monomeric and polymeric components after denaturation at A CT pH 4.0 and 600C, as determined by analytical gel electrophoresis, is illustrated in Fig. 1. By comparison with an analo- gous collagen extract prepared from the skin of a normal calf, P-a2 P-ai'l 21 S the abnormal collagen monomers, p-al and p-a2, of dermatosparaxic skin are readily identified and distinguished from the - normal monomers a, and a2 (5). Recognition of the conversion II I' of p-al to a, and of p-a2 to a2, as measured by gel electro- phoretic analyses, served as the basis of the enzyme assay. Enzyme Assay. 0.25-ml aliquots of the substrate solution (2 aI p 4 that contained 250 ,g of protein were added to an equal volume of the enzyme solution to be tested. The tubes were incubated in a water bath at 260C and pH 7.2 for 6 hr unless otherwise noted. After incubation, samples were cooled to 00C, and 0.5 ml of potassium phosphate at pH 7.6 (r/2 = 0.4), adjusted to pH 3.5 by a fresh solution of 25% trichloroacetic acid, was added. FIG. 2. Densitometer recordings of acrylamide gel electro- After 20 min, the samples were centrifuged for 15 min at phoresis of dermatosparaxic collagen after incubation with 40,000 X g. To an 0.8-ml aliquot of the supernatant fluid was various enzyme preparations. The band pattern of a standard gel added 0.3 ml of cold absolute ethanol. After 20 min, the re- is schematically illustrated between the two left diagrams; the top sultant precipitate was collected by centrifugation at 40,000 of the gels is on the right. Besides the quantitative changes in the X g for 15 min. The precipitated collagen was washed once normal (al and a2) and abnormal monomers (p-al and p-C2) with 30% ethanol and heated to 600C for 10 min in 0.2 ml of between (A) and (B), note also the changss in the patterns of the 0.15 MI glycine adjusted to pH 4.0 with acetic acid. The de- dimers (,B). As compared to A, the nonspecific proteolysis illus- natured collagen samples were layered on acrylamide gels in trated in C is characterized by a reduction in p-al, with no propor- the presence of sucrose and electrophoresed for 3 hr tional increase in a,. In D the presence of collagen fragments 10% (5, migrating faster than a2 indicates the occurrence of 12). Band patterns of gels stained with amidoschwarz were collagenolysis. recorded with a Densicord Photovolt densitometer connected to an integrator. Typical densitometer traces of the denatured substrate before and after enyzmatic digestion are presented in Fig. 2A and B. The conversion of p-al to a, or of p-a2 to a2 was quanti- tated by averaging the decrease in p-a, with the increase in al and by averaging the decrease in p-a2 with the increase in a2.

f The assumption implicit in this assay is that the staining in- I/ po W.O.IW tensity of the different molecular species is proportional to their protein concentration. Over the range of 10-80 ,ug of Is~*-o monomer protein applied to the gels, this assumption is valid. - Accordingly, gel assays were performed at least in duplicate ~P-ai,,, and at two protein concentrations within this range. The specificity of the procollagen peptidase resulted in the decrease I...a a in p-al being equal to the increase in ail and similarly for p-a2 '' mW8' m/- _ and a2. Nonspecific protease activity, i.e., proteolytic activity of normal and dermatosparaxic liver, spleen, kidney, or muscle, resulted in destruction of p-al, but without the concomi- tant or proportional increase in a, monomers; to a lesser de- gree, p-a2 was destroyed without a proportional increase in a2 (Fig. 2C). Collagenolytic activity of the enzyme extracts N D could be detected by the recognition of the formation of FIG. 1. Gel electropherograms of normal (N, left) and der- smaller fragments in association with destruction of both matosparaxic (D, right) calf-skin collagen extracted with 1.0 M normal and abnormal monomer forms (Fig. 2D). NaCI. The technique used for the acrylamide gel electrophoresis In order to eliminate the possibility that treatment with was derived from that of Sakai and Gross (12); it has been simpli- tissue extracts or that collagen purification after the assay fied by the omission of the spacer gel. Before application, collagen could induce a preferential collection of the normal samples (250 jg in 200 ,ul) were denatured, the density of the polypep- solution was increased by addition of 10% sucrose, and 50,I of the tides, the amount of collagen (hydroxyproline) recovered at denatured samples were applied to the top of the gel. Electro- the various steps of the enzyme assay was measured. Significant phoresis was performed at 100 V and 2.5-3 mA per tube for 3 hr. differences were not detected between active and control Collagen monomers are labeled as a, dimers as f3; note also the samples, and the final recovery of collagen was similar in all presence of higher polymers. assays (85 + 5%). Downloaded by guest on September 27, 2021 3056 Biochemistry: Lapiere et al. Proc. Nat. Acad. Sci. USA 68 (1971) TABLE 1. The conversion of tendon extract p-a1 into al and of p-a2 into a2 by the enzyme preparation derivedfrom normal and dermatosparaxic calf

p-al converted to a, P-a2 converted to a2 Dermatosparaxic collagen substrate Decrease Increase Average Decrease Increase Averge in the presence of in p-al in ac change in P-a2 in a2 change 250 Ml normal-tendon extract -23 +20 21.5 -14 + 13 13.5 250,gl dermatosparaxic-tendon extract 0 0 0 0 0 0 250,g1 boiled normal-tendon extract 0 0 0 0 0 0 250 IA normal-tendon extract and 250 jul dermatosparaxic-tendon extract -22 +21 21.5 -14 +14 13 250 dul dermatosparaxic-tendon extract and 250 Ml boiled normal-tendon extract 0 0 0 0 0 0

250 Ml of the dermatosparaxic collagen substrate and 250 M1l of tissue extract were incubated at 260C for 6 hr and analyzed by gel electrophoresis. The initial contents of p-ai, a,, p-a2, and a2 in the substrate preparation were 32, 25, 25, and 13 lg, respectively; the protein content of all enzyme extracts was about the same. Changes after incubation are quoted in micrograms.

One unit of enzyme activity is defined as the conversion of TABLE 2. Procollagen peptidase activity and nonspecific 1 g of p-al to a, or of p-a2 to a2 under the standard assay condi- proteolytic activity in the extracts of various tissues collectedfrom tions with a 6-hr incubation. normal (N) or dermatosparaxic (D) calves Preparation of the Procollagen Peptidase Extracts. 2-g tissue Procollagen peptidase activity samples from normal or dermatosparaxic animals were finely p-al p-a2 Nonspecific minced with scissors and homogenized in 3 times their volume converted to converted to proteolysis (w/v) of 0.15 M NaCl. Homogenization was for 30 sec at top Cal a!2 Collagen Casein speed with an Ultra-Thurax homogenizer. The homogenate was centrifuged at 40,000 X g for 20 min and the supernatant Skin N +++ +++ 0 + calf 0 0 0 fluid was collected as the crude enzyme extract. Samples were D + N at these rat +++ +++ 0 + maintained 0°C during procedures. Dialysis against rat L* +++ +++ 0 + 0.1 M barbital-acetate (pH 7.2), or against 0.1 M potassium human +++ +++ 0 + phosphate (pH 7.4), as well as storage at - 15°C for as long as Aorta N +++ +++ 0 ++ 3 months did not affect the activity of the extracts. D 0 0 0 ++ RESULTS Tendon N ++++ ++++ 0 + D 0 0 0 + Procollagen peptidase in normal tissues and its Lung N +++ +++ 0 ++ absence in dematosparaxis tissues D 0 0 0 ++ Table 1 and Fig. 2A and B demonstrate the effects of incuba- Cartilage N ++ ++ 0 + tion of dermatosparaxic collagen with enzyme preparations Kidney N + + + +++ N derived from normal or dermatosparaxic tendons. The equi- Liver + 0 + +++ D + 0 + +++ molar conversion of abnormal p-al monomers to normal a, Spleen N + + 0 +++ chains is evident, as is the equimolar conversion of abnormal Thymus N + 0 0 +++ Muscle N 0 0 0 ++ D 0 0 0 ++ Heart N 0 0 0 + 4 0 Serum N 0 0 0 +

A a Brain N 0 0 0 ++

3IO a)

a) 0) Procollagen peptidase activity was measured as the extent of a) c 0 conversion of p-arl into and p-a2 into a2 after 16 hr of incuba- c 2!0 a, 0 tion and after gel electrophoresis. The number of pluses is AA 0~~~ ZU proportional to the observed activity, complete conversion in I 0Oraa 16 hr being 4 + and no conversion being 0. The protein content of all enzyme solutions was about the same. Collage- 0 50 100 150 200 250 nolysis represents the occurrence of collagen fragments migrating OF EXTRACT HOURS faster than a2, as shown in Fig. 2D. Casein proteolytic activity p1 was measured by the degradation of denatured casein to Cl1- FIG. 3. Procollagen peptidase activity as a function of enzyme CCOOH-soluble fragments when incubated with the same concentration (6-hr incubations) (left) and time (150 ,l of ex- amount of tissue extract for 3 hr at 370C, or for 16 hr under tract) (right). The activity is expressed as ,ug of converted the standard assay conditions for procollagen peptidase. * monomers in the reaction mixture. Lathyritic Downloaded by guest on September 27, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Procollagen Peptidase 3057

5 a, -1 N "I 3 -C 2 3

3 -7 0 1 2 3 -2 -1 0 _; x I02 FIG. 4. Kinetics of enzyme activity as a function of substrate concentration (a), and its modification by the addition of an excess of p-as (b and c). Substrate is taken as the amount of abnormal dermatosparaxic collagen polypeptides, p-al or p-a,. In a, it is expressed in ug for a reaction mixture (550 IAI) containing 250 Al of the collagen solution, 200 Jl of a 0.15 Ml NaCl extract of tendon, and 100 ,.d of 0.2 M NaCl. The velocity of the reaction (V) is defined as the amount of abnormal monomer (p-a> or p-ta) converted into normal polypeptides (as or a2) after an incubation of 6 hr at 260C. In (b) and (c), the conditions were similar, except for the replacement of the 100 AI of 0.2 M NaCl by 100 ,Al of a 0.2 AM NaCl solution containing 105 pg of p-al. Pure p-as preparations were obtained by carboxymethyl-cellulose chromatography. p-a2 monomers to normal a2 chains. Of particular interest is Under the conditions of the standard assay, the main portion the total absence of the specific enzyme activity in the extract of the collagen a chains was not denatured and cleaved, as from dermatosparaxic animals. If the normal tendon enzyme demonstrated by the constant viscosity of the reaction mixture preparation was heated for 10 min at 1000C, its enzyme activ- over a period of 24 hr at 260C. Both conversion reactions, ity was completely abolished. Mixing experiments established p-as to a, and p-a2 to a2, were inhibited by 0.15 M EDTA and that the dermatosparaxic enzyme preparations were not in- 0.1 M 2-mercaptoethanol. Excess calcium reversed the EDTA active because of an inhibitor and that the normal extracts inhibition, and p-chloromercuribenzoate, a sulfhydryl-group did not contain a thermostable activator (Table 1). reagent, was not inhibitory at saturating concentrations. Enzyme activity was proportional to protein concentration Kinetic studies (Fig. 4a) demonstrated that the Km value when either the conversion of p-al or p-a2 was considered for p-a2 was lower than that for p-as, whereas the Vmaz (Fig. 3). In both cases, the conversion of p-al to a, or of p-a2 was greater for p-al than p-a2. Also demonstrated was an to a2, the reaction was linear with respect to time (Fig. 3) and effect of one substrate, p-as, on the catalytic conversion of the went to completion. other, p-a2, in that the presence of added p-al lowered the Enzyme activity was present in normal tendon, normal Km value of p-a2 (Fig. 4c), without changing the kinetics of its skin, normal cartilage, normal aorta, and normal lung, but own conversion (Fig. 4b), and without changing the maximal was absent in the extracts of the same tissues from dermato- velocity of the p-a2 conversion reaction (Fig. 4c). sparaxic calves (Table 2). In contrast, the nonspecific pro- The enzyme was active with the p-al and p-a2 monomers, tease activity of both normal and dermatosparaxic extracts with the dimers composed of the monomers linked by con- from these tissues was the same, i.e., all these extracts had valent bonds, i.e., the (3 forms observed after denaturation equal collagenolytic activity and equal proteolytic activity (Figs. 1 and 2), and with polymerized collagen in the form of against casein. This last point must be considered, since fibrils. The action towards the (3 form is evident in the change trypsin, chymotrypsin, pepsin, papain, and ficin can all con- between Fig. 2A and B. The action of the enzyme on fibrils was vert p-al to as or p-a2 to a2, but will also very rapidly degrade demonstrated by examination of its activity under conditions casein under the conditions of the assay. Enzyme activity was in which the collagen substrate was maintained in a fibrillar detected in skin extracts of normal rats, lathyritic rats, and form (5). The dermatosparaxic collagen substrate [1 mg/ml in normal humans (Table 2). Low activities of procollagen pep- 0.4 M NaCl containing potassium phosphate (r/2 = 0.004, tidase were observed in the kidney and spleen of normal calves, pH 7.6)] was heated to 370C for 6 hr, and the gel was dis- but this specific activity was accompanied by a significant rupted by mechanical agitation; enzyme extracts from normal amount of nonspecific proteolytic activity. No specific enzyme or dermatosparaxic tendons were added after being heated to activity was detected in muscle, heart, blood serum, or brain of 370C. Incubations were for 16 hr at 370C; fibrils were col- normal or dermatosparaxic calves, although a significant lected by centrifugation at 3000 X g for 10 min at 370C, and amount of proteolytic activity was present in these tissue the fibrils in the precipitate, as well as the unpolymerized extracts. collagen remaining in the supernatant, were prepared for acrylamide gel electrophoresis. Both the fibrils and the col- Characteristics of the procollagen peptidase of lagen remaining in solution showed transformation of p-as tendon or skin and p-a2 into normal collagen polypeptides when treated with the normal tendon extract, but not when treated with derma- Enzyme activity was maximal at pH 7.0, and about 70% of tosparaxic tendon extract or with normal tendon extract that the maximum was at pH values as low as 5.5 or as high as 8.9. had been boiled before incubation. Downloaded by guest on September 27, 2021 3058 Biochemistry: Lapibre et al. Proc. Nat. Acad. Sci. USA 68 (1971)

DISCUSSION lytic conversion of the other, p-a2, and that this results from a The data demonstrate the existence of an enzyme activity in change in the affinity of substrate and enzyme, rather normal connective tissues that is capable of cleaving the ad- than from a change in the maximal rate of the reaction. Since ditional N-terminal peptides of the abnormal a-chain mono- the presence of added abnormal p-a1 monomers might alter mers (p-al and p-a2) present in dermatosparaxic collagen. the monomer to polymer ratio of dermatosparaxic collagen Enzyme activity can be demonstrated with the p-al and p-a2 solutions, the absence of an effect of the added p-al monomer monomers of salt-extracted collagen in solution, as well with on its own conversion to normal al chains (Fig. 4b) suggests the monomers present in collagen fibrils reconstituted in that this influence is the result of enzyme modulation rather vitro. Enzyme activity is absent in the connective tissue ex- than substrate modification. tracts of dermatosparaxic animals, thus suggesting that This work has been supported in part by the Belgian Research dermatosparaxis is produced by the absence of an enzyme Foundation, F.R.S.M. The authors acknowledge the excellent function rather than by the production of an abnormal col- technical assistance of Mrs. Y. Goebels and Mr. A. Stassen. lagen. 1. Hanset, R. and M. Ansay, Ann. Med. Vet., 7, 451 (1967). The enzymatic nature of the reaction has been established 2. Hanset, R., Hoppe-Seyler's Z. Physiol. Chem., 352, 13 by its thermolability, by its proportionality to protein con- (1971). centration, and by its linearity with respect to time. Speci- 3. O'Hara, P. J., W. K. Read, W. M. Romane, and C. H. ficity of the reaction is indicated by the similar and low activity Bridges, Lab. Invest., 23, 307 (1970). in 4. Simar, L. J., and E. H. Betz, Hoppe-Seyler's Z. Physiol. of nonspecific proteases normal and dermatosparaxic ex- Chem., 352, 13 (1971). tracts, in spite of the marked differences in procollagen pepti- 5. Lenaers, A., B. V. Nusgens, M. Ansay, and Ch. M. dase action. Nonspecific enzymes, such as trypsin or chymo- Lapibre, Eur. J. Biochem., in press (1972). trypsin, have an equal or greater nonspecific protease activity 6. Layman, D. L., E. B. McGoodwin, and G. R. Martin, than procollagen peptidase action under our assay conditions. Proc. Nat. Acad. Sci. USA, 68, 454 (1971). 7. Bellamy, G., and P. Bornstein, Proc. Nat. Acad. Sci. USA, The equimolar conversion of abnormal to normal monomers 68, 1138 (1971). has been indicated: p-ai to ai, and p-a2 to a2. It is difficult to 8. Muller, P. K., E. McGoodwin, and G. R. Martin, Biochem. distinguish the possibility of one procollagen peptidase at- Biophys. Res. Commun., 44, 110 (1971). tacking the p-al and p-a2 monomers at different rates and 9. Schmitt, F. O., Bull. N.Y. Acad. Med., 36, 725 (1960). 10. Speakman, P. T., Nature, 229, 241 (1971). affinities from the possibility that two procollagen peptidases 11. Van Caneghem, P., and Ch. M. Lapibre, Int. J. Radiat. exist, each specific for one chain. However, it is clear that the Biol., 18, 401 (1970). presence of one abnormal monomer p-al, influences the cata- 12. Sakai, T., and J. Gross, Biochemistry, 6, 518 (1967). Downloaded by guest on September 27, 2021