Procollagen Peptidase: an Enzyme Excising the Coordination Peptides of Procollagen (Connective Tissue/Collagen/Calf/Acrylamide Electrophoresis/Dermatosparaxis)
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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 pep- tides 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 pro- collagen 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 electro- phoresis, 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 dermato- sparaxic 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 vol- ume 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 be- fore 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 mus- cle, 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.