the journal of vol. no. inc printed in u.s.a. the ... · this report describes the influence of...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 hy The American Society for Biochemistry and Molecular Biology, Inc

Vol. 262, No. 35, Issue of December 15, pp. 17144-17148,1987 Printed in U.S.A.

The Influence of Peptidyl-Prolyl Cis-Trans Isomerase on the in Vitro Folding of Type I11 Collagen*

(Received for publication, May 5, 1987)

Hans Peter BachingerS From the Research Unit, The Shriners Hospital for Crippled ChiMren and the Department of Biochemistry, Oregon Health Sciences University, Portland, Oregon 97201

Peptidyl-prolyl cis-trans isomerase was extracted from pig kidney cortex and partially purified. Enzyme activity was monitored against the cis-trans isomerization of succinyl-Ala-Ala-Pro-Phe-methylcoumaryl amide by means of a two-step process using chymotrypsin as the trans cleaving activity. The in vitro refolding of denatured type I11 collagen, which is rate- limited by the cis-trans isomerization of peptide bonds, was studied in the presence of peptidyl-prolyl cis-trans isomerase by optical rotatory dispersion and by resistance to tryptic digestion. A %fold increase in the initial rate of folding was observed compared to the uncata- lyzed refolding. This rate increase is comparable to the rate increase found for the CT-phase in the refolding of urea-denatured ribonuclease A, but it is smaller than the increase in the rate of isomerization of succinyl- Ala-Ala-Pro-Phe-methylcoumarylamide.

The mechanism for the in vitro folding of the collagen triple helix has been studied (1-4). For type I11 collagen, pN type 111,’ and the amino-terminal propeptide of type I11 procollagen, folding starts at a set of disulfide bonds at the carboxyl- terminal end of the molecules and proceeds in a zipper-like fashion toward the amino-terminal end. Two phases are observed kinetically. An initial fast phase represents the addition of hydrogen bonds between tripeptide units with the peptide bonds in trans conformation. Because the triple helix can only accommodate trans peptide bonds, any tripeptide units with peptide bonds in the cis conformation in the coiled state have to isomerize. The percentage of peptide bonds, to which proline contributes the nitrogen, in the cis conformation in unfolded type I11 collagen was estimated from kinetic experiments to be about 3-6% (1, 2), and 12% was measured by nuclear magnetic resonance for type I collagen (6). This isomerization is a much slower process and for type I11 collagen gives rise to zero-order kinetics over a large fraction of the conversion.

Fischer et al. (6) recently described an enzyme which catalyzes the cis-trans isomerization of peptide bonds. Peptidyl-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed Shriners Hospital for Crippled Children, 3101 S. W. Sam Jackson Park Rd., Portland, OR 97201.

‘The abbreviations used are: pN type I11 collagen, partially processed type I11 procollagen which retained the amino-terminal propeptide; SDS, sodium dodecyl sulfate: Tricine, N-tris(hydrox- ymethy1)methylglycine; Hepes, N-2-hydroxyethylpiperazine-N’-2- ethanesulfonic acid; MCA, methylcoumaryl amide.

prolyl cis-trans isomerase was extracted from pig kidneys and assayed by the trans specificity of bovine chymotrypsin for substrates with a proline residue in the Pa-position of the peptide substrate. These authors also showed that one of the slow phases in refolding of ribonuclease A is catalyzed by peptidyl-prolyl cis-trans isomerase, even though ribonuclease A is a much poorer substrate than the shorter peptides (7). Studies of the biosynthesis of collagen indicate that the carboxyl-terminal propeptides are responsible for chain association, which has to precede folding (8-11). Because the carboxyl-terminal propeptides are synthesized last, there is enough time for the unfolded chains to establish an equilibrium between cis and trans peptide bonds before folding can start from the carboxyl-terminal end. Collagen is therefore a potential substrate for peptidyl-prolyl cis-trans isomerase. This report describes the influence of peptidyl-prolyl cis-trans isomerase on the in vitro refolding of type I11 collagen.

EXPERIMENTAL PROCEDURES

Type ZZZ Collagen-Type 111 collagen was extracted from human amnion by pepsin digestion according to the procedure for the isola- tion of type VI1 collagen (12). The 1.8 M NaCl precipitate was purified by DEAE-cellulose chromatography in 50 mM Tris/HCl buffer, pH 7.5, containing 2 M urea and 0.2 M NaC1. Proteases were inhibited by the addition of 0.001 M diisopropyl fluorophosphate.

Peptidyl-prolyl Cis-Trans Isomerase-Peptidyl-prolyl cis-trans isomerase was extracted from pig kidneys according to the procedure of Fischer et al. (6) and partially purified. All procedures were carried out at 4 “C. Pig kidney cortex was homogenized in 0.05 hi Tris/HCl buffer, pH 7.4, containing 0.2 M sucrose, and the extract was spun for 60 min at 10,000 X g in a Sorvall RC5B centrifuge. The supernatant was adjusted to pH 5.5 with l M sodium acetate, pH 4.0. After centrifugation for 60 min at 13,000 X g, the supernatant was adjusted to pH 7.0 with 1 M Tris solution. Ammonium sulfate was added to a final concentration of 40%, and the precipitate was discarded. The supernatant was adjusted to 60% ammonium sulfate, and the precipitate containing peptidyl-prolyl cis-trans isomerase was dissolved and dialyzed against 10 mM Tris/HCl buffer, pH 7.6. The sample was chromatographed on a Bio-Gel DEAE A-50 column (Bio-Rad) in the same buffer. The unbound material was dialyzed against 0.05 M Tricine buffer, pH 8.0, and the enzyme was applied to a Sephadex CM-50 column (Pharmacia Biotechnology, Inc.) where it could be eluted with the same buffer containing 1.2 M KCI. Remaining proteases were inhibited with 0.001 M diisopropyl fluorophosphate. In a modification of the original procedure, the Sephadex CM-50 fraction containing isomerase activity was separated on a Superose 12 column (1.6 x 120 cm) in 0.05 M Tris/HCl buffer, pH 7.8, containing 1.2 M KC1 and 0.02 M EDTA using a Pharmacia FPLC system. Active fractions from the Superose column were further purified using a reverse phase column (Pharmacia ProRPC) in 50 mM sodium phosphate buffer, pH 7.0. Elution was performed with a linear gradient from 0 to 70% (v/v) acetonitrile in 50 mM phosphate buffer, pH 7.0.

pH 7.8. Active fractions were pooled and dialyzed against 35 mM Hepes buffer,

Enzyme Assay-Enzyme activity was assayed qualitatively according to the method of Fischer et al. (6) with succinyl-Ala-Ala-Pro-Phe- methylcoumarylamide (Peninsula Laboratories, Belmont, CA) as substrate. The following reactions take place.

17144

Enzyme-catalyzed in Vitro Folding of Type 111 Collagen

chymotrypsin S-Ala-Ala-Pro-Phe-MCA (trans) > S-Ala-Ala-Pro-PheO- + MCNHs+

S-Ala-Ala-Pro-Phe-MCA (cis)

The reaction was monitored by the increase in fluorescence of methylcoumaryl amine (MCNHZ) above 430 nm in a stopped-flow spectrophotometer (Dionex Corp., Sunnyvale, CA). The excitation wave- length was 380 nm, and fluorescence was observed with a filter (3-72, Corning C.S.) with a cutoff point of 430 nm. The analog output signal was recorded with a digitizer (390AD, Sony-Tektronix) and traces evaluated on a IBM PC/XT (IBM Corp.) computer. The experimental curves were fitted with two exponentials by a nonlinear least squares method. The fast phase corresponds to the chymotryptic cleavage of the trans peptide substrate, whereas the slow phase corresponds to the isomerization of the cis peptide substrate into the trans conformation followed by fast chymotryptic cleavage. The ratio of cis to trans isomers was calculated from the ratio of the amplitudes of the slow phase and the fast phase.

Refolding of Type IIZ Colhgen-Refolding of type 111 collagen was measured by optical rotatory dispersion at 365 nm in a 241 MC polarimeter (Perkin-Elmer) equipped with a 100-mm thermostatted quartz cell. Two thermostats (RCS, Lauda Division, Brinkmann Instruments) were used to change the temperature quickly, and the temperature was monitored inside the cell with a thermistor and digital thermometer (Omega Engineering, Inc., Stamford, CT). The analog output was recorded on a XY plotter (HP 7090A. Hewlett- Packard Co., Palo Alto, CA).

Other Methods-SDS-polyacrylamide gel electrophoresis was performed on 8-25% gradient gels on a Phastsystem (Pharmacia Bio- technology, Inc.), and proteins were visualized by silver staining according to the manufacturer's instructions (Pharmacia Biotechnol- ogy, Inc.). Refolding kinetics were also monitored by the resistance of the triple helix to tryptic digestions according to published procedures (2, 13). Intermediate fragments were analyzed on 10% SDS- polyacrylamide gels (2). Protein concentrations were determined by amino acid analysis on a Waters Pic0 Tag System (14).

RESULTS

Peptidyl-prolyl cis-trans isomerase was partially purified by chromatographic procedures. Fig. 1 shows an SDS-polyacrylamide gel of enzyme preparations (treated with or without dithiothreitol) together with marker proteins. Enzyme purity was assessed by gel electrophoresis following Superose chromatography (lanes 2 and 4 ) and after final reverse phase chromatography (lanes 1 and 3 ) . The unreduced enzyme fraction after reverse phase chromatography showed two protein bands (lane 1 ) with apparent molecular weights of 24,000 and 12,000. At present, it is unknown which band represents the active enzyme or whether the slower migra'ing band represents a disulfide-linked dimeric form. The reauced fraction showed only one band (lane 3 ) with an apparent molecular weight of 11,500. No amino-terminal amino acid sequenc- ing was performed to show that this band represents a ho- mogeneous polypeptide chain. Therefore enzyme concentrations were not known, and the amount of total protein in the active fractions was determined by amino acid analysis. En- zyme fractions after reverse phase chromatography as well as fractions after Superose chromatography showed no proteo- lytic activity against the peptide substrate, and both were used for various experiments. The slow phase of the chymotryptic cleavage of succinyl-Ala-Ala-Pro-Phe-MCA as a func- tion of temperature was measured in a stopped-flow spectrophotometer equipped with a fluorescence detector. Measure-

17145

ments were performed in 0.035 M Hepes buffer, pH 7.8, with a final peptide concentration of 7.3 X M and a chymotrypsin concentration of 2.5 X M. The results are sum- marized in Table I. The equilibrium constants, rate constants, activation energy, and reaction enthalpy are in good agreement with the values reported for cis-trans isomerization of peptide bonds (1, 15-19) and the slow phase of glutaryl-Ala- Ala-Pro-Phe-p-nitroanilide (20). Fig. 2 shows a comparison of the kinetics of the chymotryptic cleavage of succinyl-Ala- Ala-Pro-Phe-MCA in absence and presence of partially purified enzyme (30 pg/ml protein, Superose fraction) at 10 "C.

95,500

55,000 43,000

36,000 29,000

18,400

12,400

- F 1 2 3 4 5

FIG. 1. SDS-polyacrylamide gel electrophoresis of partially purified peptidyl-prolyl cis-trans isomerase. Proteins were separated on a 8-25% gradient gel. Lane 1, nonreduced enzyme fraction after reverse phase chromatography; lane 2, nonreduced enzyme fraction after Superose column; lane 3, reduced enzyme fraction after reverse phase chromatography; lane 4, reduced enzyme fraction after Superose column; lane 5, marker proteins with the corresponding molecular weights indicated to the right. F indicates the dye front of the gel.

TABLE I Influence of the temperature on the rate constant and on the

fractionul concentration, [cis/, of the cis isomer in 0.035 M Hepes buffer, pH 7.8

Reaction enthalpy A H o = -4.8 kJ.mol". Activation energy A H $ = 79 kJ. mol".

t 102 k; ..,.... lcisl

"C S"

10.0 1.13 f 0.05 0.100 f 0.002 15.0 1.95 f 0.03 0.106 * 0.002 20.0 3.18 f 0.06 25.0

0.108 f 0.002 6.31 f 0.04 0.110 f 0.002

17146 Enzyme-catalyzed in Vitro Folding of Type 111 Collagen

I

I f B _.LIIc-L

0 100 200

Tlme (sed FIG. 2. Kinetics of the chymotryptic cleavage of succinyl-

Ala-Ala-Pro-Phe-methylcoumarylamide in presence and absence of peptidyl-prolyl cis-trans isomerase. The reaction was measured at 10 "C in 0.035 M Hepes buffer, pH 7.8. The peptide concentration was 7.3 X M, and the chymotrypsin concentration was 2.5 X lo6 M. Velocity of the cis-trans isomerization in absence of peptidyl-prolyl cis-trans isomerase ( A ) and in presence of peptidyl- prolyl cis-trans isomerase (30 pg of protein/ml, Superose fraction) ( B ) were measured in the stopped-flow spectrophotometer with fluorescence detection.

The rate of the cis-trans isomerization in the presence of peptidyl-prolyl cis-trans isomerase is increased about 100- fold.

The a-l(II1) chain of collagen contains a large number of sites which can be cleaved by trypsin when the protein is unfolded but which are not accessible when the chains are in the triple helical conformation. In in vitro folding experiments, the growing triple helix protects an increasing number of sites which gives rise to fragments of increasing length (2). Type I11 collagen was denatured for 20 min at 45 "C and refolded at 25 "C for various lengths of time. Trypsin was added at various times of refolding and the fragments were analyzed on an SDS-polyacrylamide gel. Gel A in Fig. 3 shows the pattern of fragments obtained after refolding at 25 "C. In gel B, partially purified enzyme was added (10 pg/ml protein, ProRPC fraction) before refolding. The fragments and complete a-l(II1) chains appeared earlier in the presence of peptidyl-prolyl cis-trans isomerase, indicating an increase in the rate of folding. The time required to achieve complete folding was about 45 min in the control experiment, whereas it took only 25 min in the presence of peptidyl-prolyl cis-trans isomerase. This rate increase was abolished in the presence of 0.001 M p-hydroxymercuribenzoate, which was shown to in- hibit peptidyl-prolyl cis-trans isomerase (6), and the rate of folding was the same as in the control experiment.

Refolding of type I11 collagen by measurement of the time- dependent change in optical rotation is shown in Fig. 4. The overall shape of the refolding curve was the same in the presence of a low concentration (10 pg/ml protein, Superose fraction, curue B ) or absence (curue C ) of peptidyl-prolyl cis- trans isomerase, indicating that refolding occurs by the same mechanism. The difference was in the steepness of the linear increase, which is proportional to the rate of cis-trans isomerization (2). With higher concentrations of peptidyl-prolyl cis-trans isomerase (25 pg/ml protein, Superose fraction, curue A ) , a deviation from the linear increase is observed at

0 2.5 5 7.5 10 15 20 25 30 45 90 C

Renaturation Time (min) FIG. 3. Folding kinetics of type I11 collagen monitored by

trypsin digestion. Type I11 collagen was denatured for 20 min at 45 "C in 0.05 M Tris/HCl buffer, pH 7.5, containing 0.2 M NaCl and refolded for various times at 25 "C. After trypsin digestion for 2 min at 20 "C triple helical fragments were visualized on a 10% SDS- polyacrylamide gel. Gel A shows the appearance and disappearance of intermediate fragments and formation of (Y chains without peptidyl-prolyl cis-trans isomerase. For gel B, peptidyl-prolyl cis-trans isomerase was added (10 pg/ml protein, ProRPC fraction).

about 0.25" conversion, followed by another linear, flatter increase. The initial rates of the linear phase increased with increasing concentration of peptidyl-prolyl cis-trans isomerase (Fig. 5 ) . No indication for saturation of the initial rate with increasing enzyme concentration is observed in the range of enzyme concentration obtainable so far. This is in agreement with the results obtained for the enzyme-catalyzed isomerization of the peptide substrate (6). An approximately %fold increase in the initial rate of folding was observed by the addition of peptidyl-prolyl cis-trans isomerase at 30 pg/ ml protein (Superose fraction).

DISCUSSION

Peptidyl-prolyl cis-trans isomerase catalyzes the rotation of a peptide bond by 180". The peptide bond contains delo-

Enzyme-catalyzed in Vitro Folding of Type 111 Collagen 17147

I 1 I 1 I 10 20 30 40 50

I 1 I 1 10 20 30 40 50

Tim (ndnl

FIG. 4. Folding kinetics of type I11 collagen monitored by optical rotatory dispersion. The optical rotation was measured at 365 nm to follow the renaturation of type I11 collagen at 25 "C in 0.05 M Tris/HCl buffer, pH 7.5, containing 0.2 M NaC1. Curve C shows refolding in the absence of enzyme, curues B and A were measured in the presence of peptidyl-prolyl cis-trans isomerase (10 and 25 pg/ml protein, respectively, Superose fraction).

0.09

.- z

I C 0.07

a -

0.05

0.03 10 20 30

[Enzyme] (pg protein ml" 1

FIG. 5. Dependence of the initial rate of folding of type I11 collagen on the concentration of partially purified peptidyl- prolyl cis-trans isomerase. The initial rate of folding of type I11 collagen was calculated from experiments performed under the same conditions as in Fig. 4. A linear increase in the initial rate upon increasing enzyme concentration was observed.

calized electrons which leads to a partial double bond char- acter and makes the peptide bond planar. Formally, a local- ization of the electrons on the carboxyl group would allow free rotation around the single C-N bond. This mechanism is the cause for the acid catalysis of cis-trans isomerization of peptide bonds in ribonuclease (21) and polyproline (16). In a random polypeptide, peptide bonds in which an imino acid contributes the nitrogen were found to have significant amounts of cis peptide bonds. Equilibrium constants K,rana,eis in the range of 1-40 were reported (15,22,23), whereas values around 1000 were estimated for other amino acids (24). Due to the relatively slow rate of cis-trans isomerization, proline residues have an important influence on protein folding (15,

The experiments described here using succinyl-Ala-Ala- Pro-Phe-MCA as a substrate confirm the results obtained by Fischer et al. (6) using glutaryl-Ala-Ala-Pro-Phe-p-nitroanilide. The in uitro folding of type I11 collagen involves the

25-27).

isomerization of peptide bonds, because only peptide bonds in the trans conformation can be accommodated in the triple helix. Peptidyl-prolyl cis-trans isomerase recognizes unfolded type 111 collagen chains as a substrate and catalyzes the isomerization of peptide bonds which leads to an increase in the rate of folding. A comparison of the intermediate fragments produced by the tryptic digestion shows that the same size fragments appear at earlier time points. The folding mechanism, therefore, remains unchanged while the rate is increased. This is also shown by the presence of the linear increase of the optical signal over a large fraction of the conversion at enzyme concentrations below 20 pg/ml (Supe- rose fraction). Above 20 pg/ml partially purified peptidyl- prolyl cis-trans isomerase, a deviation from the linear increase is observed at about 0.25 degree of conversion, where the molecules are folded to about one-quarter of the length. In this region, the cleavage site for vertebrate collagenase is located, which can also be cleaved by prolonged incubation with trypsin, indicating a less stable region of the type I11 collagen triple helix. The deviation from linearity could be due to a nucleation needed to overcome this less stable region which is faster than the cis-trans isomerization in the uncat- alyzed folding and therefore not observed. The 3-fold increase in the initial rate at 30 pg/ml partially purified peptidyl-prolyl cis-trans isomerase is similar to the rate increase observed for the folding of urea-denatured ribonuclease A in presence of peptidyl-prolyl cis-trans isomerase (7). However, both unfolded proteins are apparently much poorer substrates than the tetrapeptide.

At present it is unclear if peptidyl-prolyl cis-trans isomerase is involved in collagen folding in uiuo. The proposed mechanism for the in vivo folding of interstitial procollagens is based on studies of the following system: the in vitro refolding of type I11 collagen, pN type 111 collagen, and type 111 procollagen (2, 4); the folding of the amino-terminal propeptide of type I11 procollagen (1); the biosynthesis of type I11 procollagen (8, 11); the biosynthesis of type I procollagen (10); the formation of the triple helix of type I procollagen in cellulo (3, 28) and the folding of the carboxyl-terminal propeptide and assembly of type I procollagen (9). Individual pro-a chains are synthesized, and the globular domain of the amino-terminal propeptide folds forming intrachain disulfide bonds during synthesis. After complete synthesis of the pro-a chains, the carboxyl- terminal propeptides fold and form intrachain disulfide bonds, associate, and create a nucleus for triple helix formation. The triple helix formation then starts proceeding rapidly toward the amino-terminal end of the chains, and the carboxyl- terminal propeptides form interchain disulfide bonds. Rapid propagation of the triple helix is interrupted when a peptide bond in the cis conformation is encountered. The cis-trans isomerization of this bond allows helix formation to proceed rapidly until the next cis peptide bond is encountered. These cis-trans isomerizations are the rate-limiting step in the triple helix formation. Folding of the main triple helix is then followed by an association of the amino-terminal propeptides and formation of the short stretch of triple helix within the amino-terminal propeptide.

Is peptidyl-prolyl cis-trans isomerase involved in the in vivo folding of collagens? The in vitro folding experiments with pN type I11 collagen did not show the expected temperature dependence of the rate of folding (1). At low temperatures (7 "C), no linear phase was observed and a complex mixture of products was obtained. Between 20 and 25 "C, almost no temperature dependence was measured. For folding of type I procollagen in cellulo, it was first reported that the rate of

17148 Enzyme-catalyzed in Vitro Folding of Type III Collagen

folding at 37 "C is consistent with the rate of the in vitro folding at 25 "C, when the latter was corrected with an activation energy of 83.5 kJ.mol-' for 37 "C (3). At the same time, the value of the equilibrium constant between trans and cis peptide bonds had to be doubled, indicating about 1.5% cis peptide bonds within the cells. This value is much smaller than the value derived from kinetic studies i n vitro or from measurements by nuclear magnetic resonance in solution. A second report studied the temperature dependence of the rate of folding in cellulo. An activation energy of about 90 kJ. mol" was found (28). This is in good agreement with the reported activation energy of cis-trans isomerization of peptide bonds found i n vitro and in model compounds which range from 82 to 88 kJ . mol-' (16-19). These findings can be seen as an argument against the involvement of peptidyl- prolyl cis-trans isomerase in the i n vivo formation of the triple helix. However, the i n vitro folding studies with the small temperature dependence of the rate constant could indicate an involvement of the enzyme. Additionally, the time required for the secretion of type I procollagen at 37 "C is about 18 min (29). This is comparable to the time needed for the i n vitro folding of type I11 collagen (1). Already a 2-fold increase in the rate of folding would be more compatible with the time required for posttranslational modifications and transport to the cell surface.

So far very little is known about the mechanism of this enzyme. Studies with various inhibitors show that the enzyme contains thiol groups which are important for the catalytic activity ( 6 ) . Future experiments will have to show whether peptidyl-prolyl cis-trans isomerase is indeed involved in the folding of collagens i n vivo. It is interesting to note that extracts of all collagen-producing cell lines that were so far tested demonstrate peptidyl-prolyl cis-trans isomerase activity against the peptide substrate.'

Acknowledgments-I acknowledge the support of the Shriners Hos- pital for Crippled Children and the expert technical assistance of Barbara Fischer Smoody. I thank Dm. Robert Burgeson, Nick Morris, Lynn Sakai, and Robert Glanville for helpful discussions and reading of the manuscript.

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