©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization and Partial Amino Acid Sequencing of a 107-kDa Procollagen I N-Proteinase Purified by Affinity Chromatography on Immobilized Type XIV Collagen (*)

Alain Colige (§) , Alain Beschin (1), Bart Samyn (2), Yvette Goebels , Jozef Van Beeumen (2), Betty V. Nusgens , Charles M. Lapière (¶)

From the (1)Laboratory of Experimental Dermatology, B23/3, University of Liège, CHU Sart Tilman, B-4000 Belgium, the Laboratory of Cellular Immunology, Institute of Molecular Biology, VUB, Pardenstraat 65, 1640 Sint Genesius Rode, and (2)Vakgroep Biochemie, Fysiologie en Microbiologie, Ledeganckstraat 25,9000 Gent, Belgium

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Procollagen I N-proteinase (EC 3.4.24.14), the enzyme that specifically processes type I and type II procollagens to collagen, was isolated from extracts of fetal calf skin. After two chromatographic steps on concanavalin A-Sepharose and heparin-Sepharose, the semi-purified preparation was used to produce monoclonal antibodies. One reacting antibody was found to recognize not the enzyme itself but type XIV collagen on which the enzyme was bound. This binding, highly sensitive to ionic conditions (pH, salt concentrations) but not affected by non-ionic detergents, was used for affinity chromatography that strongly improved the purification procedure. The enzyme is extensively characterized: 1) it has a molecular mass of 107 kDa as determined by polyacrylamide gel electrophoresis in presence of SDS and of about 130 kDa when estimated by gel filtration on a Sephacryl-S300; 2) in standard assay (pH 7.5, 0.2 M NaCl, 35 °C), the activation energy for reaction with amino procollagen type I was 17,000 calories per mole. In the same conditions, K and V values were, respectively, 435 and 39 nM per hour but varied strongly with pH and salt concentration; 3) the enzyme cleaved the NH-terminal propeptide of type I procollagen at the specific site, the Pro-Gln bond in the 1 type I procollagen chain; 4) the enzyme contained a high proportion of Gly, Asx, and Glx residues but no Hyp or Hyl; 5) partial amino acid sequences obtained from internal peptides of the enzyme displayed no significant homology with known sequences. The association of procollagen I N-proteinase with a FACIT (fibril-associated collagens with interrupted triple helices) collagen as found here might be of physiological significance.


INTRODUCTION

Fibrillar collagens (types I, II, III) are synthesized as precursors (procollagens) formed by a central triple-helical collagen domain extended by propeptides both at the carboxyl and amino extremities. During the processing of procollagen to collagen, the carboxyl and amino propeptides are cleaved by specific proteinases. The procollagen C-proteinase removes the carboxyl propeptide of types I, II, and III procollagens(1) , the type III procollagen N-proteinase excises the amino propeptide of type III procollagen only(2, 3) , while the procollagen I N-proteinase (PCI-NP)()is specific of type I and type II procollagens. Dermatosparaxis is a heritable disease of the connective tissues that was first described in cattle (4) and more recently in human(5, 6) . It is characterized by a severe skin fragility caused by the absence or a reduced activity of PCI-NP(7) , resulting in the accumulation of amino procollagen type I and abnormal collagen polymers in skin and other tissues(8) .

PCI-NP activity was first detected in extracts of normal calf tissues by Lapière et al.(7) in 1971. The enzyme is a neutral, Ca-dependent proteinase that cleaves a Pro-Gln bond in the pro-1 type I chain and an Ala-Gln bond in the pro-2 type I chain(9, 10) . An enzyme complex of 500 kDa containing at least four distinct subunits was isolated from chicken tendon by Hojima et al.(11) in 1989. It is, however, not established if the multimeric association is required for a full catalytic activity or if only one of the proteins carries most of the enzymatic properties. The association of the enzyme with other proteins in vivo is suggested by experiments showing that it is immobilized on extracellular matrix macromolecules rather than being free in the extracellular fluids or attached to cell membranes(12) .

In the present study, we have identified and characterized a 107-kDa form of PCI-NP from bovine skin, which displays a full enzymatic activity. A specific binding of PCI-NP to type XIV collagen was also demonstrated.


EXPERIMENTAL PROCEDURES

Preparation of Radiolabeled Substrates

Amino procollagen type I was prepared from the skin of dermatosparactic calves and labeled according to Nusgens and Lapière(13) . With this procedure, only the amino propeptide of pN- type I chains were labeled. Preparations at 1300 cpm/µg of amino procollagen type I (1 mg/ml) were usually obtained.

Enzyme Assay

The substrate solution (25 µl, 32500 cpm) and the enzyme preparation were diluted to 250 µl in the assay buffer to obtain a final concentration of 50 mM sodium cacodylate, pH 7.5, 200 mM KCl, 2 mM CaCl, 2.5 mM NEM, 0.5 mM PMSF, and 0.02% Brij (standard assay buffer). After 16 h at 26 °C, the reaction was stopped by adding 50 µl of EDTA solution (0.2 M EDTA, pH 8, 0.5% SDS, 0.5 M DTT) and 300 µl of 99% ethanol. The samples were then kept for 30 min at 4 °C and centrifuged for 30 min at 9500 g. Collagen and uncleaved radioactive pN-collagen substrate were pelleted, whereas the freed amino propeptides remained in solution. An aliquot of the supernatant (200 µl) was assayed by liquid scintillation spectrometry. Enzyme activities were calculated after correction for background values obtained from samples in which 50 mM EDTA was added before the incubation as an inhibitor of PCI-NP activity and which never exceeded 2.5% of the total radioactivity in the assay.

Purification of PCI-NP

All procedures were performed at 4 °C or in an ice bath unless otherwise stated.

Step I: Preparation of Bovine Skin Extracts

Skin was collected from fetal calves at the third trimester stage. In our standard procedure, 250 g of material was ground at liquid nitrogen temperature and homogenized with an Ultra Turrax (8000 rpm) in 500 ml of washing buffer (50 mM sodium cacodylate, pH 7.5, 0.25 M sucrose, 2 mM CaCl, 2.5 mM NEM, 0.5 mM PMSF, and 0.02% NaN). After centrifugation (20,000 g for 10 min), the pellet was collected, and the washing procedure was repeated once. Pellets were then suspended in 950 ml of extraction buffer (50 mM sodium cacodylate, pH 7.5, 1 M KCl, 2 mM CaCl, 0.02% Brij) supplemented with 1.25 mM NEM and 0.25 mM PMSF. After shaking for 18 h at 4 °C, the samples were centrifuged for 10 min at 15,000 g. The supernatants were collected, and extraction of the pellets was repeated once.

Step II: Ammonium Sulfate Precipitation

The proteins in the pooled supernatants were precipitated by adding ammonium sulfate at 40% saturation. The solution was stirred 18 h at 4 °C and centrifuged at 15,000 g for 30 min. The precipitate was dissolved in extraction buffer and dialyzed.

Step III: Affinity Chromatography on Concanavalin A-Sepharose

The sample was loaded on a 300-ml concanavalin A-Sepharose (Pharmacia LKB Biotechnology, Uppsala, Sweden) column and extensively washed with the extraction buffer. Elution was carried out in the same buffer containing 0.5 M -methyl-D-mannoside. Active fractions were pooled and dialyzed against buffer H (50 mM sodium cacodylate, pH 7.5, 0.2 M NaCl, 2 mM CaCl, 0.02% Brij).

Step IV: Chromatography on Heparin-Sepharose

The enzyme preparation from step III was applied to a 75-ml heparin-Sepharose (Pharmacia) column equilibrated in buffer H. After washing, elution was performed with a linear gradient prepared from 250 ml of buffer H and 250 ml of buffer H containing 0.95 M KCl. The most active fractions, eluting between 0.6 and 0.8 M KCl, were pooled and dialyzed against TCNa buffer (50 mM Tris, pH 7.5, 0.2 M NaCl, 2 mM CaCl).

Step V: Affinity Chromatography on Immobilized 37D9 Monoclonal Antibody

A maximum of 50 ml of the preparation at step IV was applied to an affinity column prepared as described below. After two successive washings in TCNa buffer and in 0.2 M ammonium acetate (NHAc), the enzyme was eluted with 0.6 M NHAc.

Step VI: Second Chromatography on Heparin-Sepharose

The fractions collected in step V containing the enzymatic activity were pooled and loaded on a 0.5-ml heparin-Sepharose column. After washing in 0.8 M NHAc, PCI-NP was eluted at 1.2 M NHAc and stored at -80 °C.

Production of Monoclonal Antibody

The enzymatic preparation after the heparin-Sepharose chromatography (step IV) was used for immunization of F1 mice (Balb/c C57 Black/6, Studie Centrum voor Kernenergie, Mol, Belgium). Mice were intraperitoneally inoculated twice at 3-week intervals with 20 µg of antigen emulsified in Freund's adjuvant. 10 days after the second injection, the animals were boosted with 20 µg of antigen in saline and sacrificed 3 days later. The hybridoma clones were screened for their ability to produce a monoclonal antibody able to immunoprecipitate PCI-NP activity in the presence of goat anti-mouse IgG coupled to agarose beads (Sigma). Only one hybridoma supernatant (clone 37D9) out of 3000 promoted significant and reproducible immunoprecipitation. The secreted monoclonal antibody was subclassed as an IgG1. After purification on a protein G column, 20 mg of antibody was coupled to 15 ml of Affi-Gel Hz hydrazide following instructions of the manufacturer (Bio-Rad) with an efficiency of 80% and used for step V of the PCI-NP purification (see above).

Kinetic Studies

For kinetic studies, 1.5 ng of purified PCI-NP (amount determined from amino acids analysis) and 5-60 µg of radiolabeled substrate (50-600 nM) were incubated for 1 h in the standard assay buffer and processed as described above. When the amount of cleaved substrate was less than 15% of the total amount of substrate, the enzyme activity was proportional to the amount of enzyme added and to the time of incubation. Values of Kand V were determined according to a Lineweaver-Burk plot and the least squares test method. Results were also confirmed by the direct Eisenthal and Cornish-Bowden (14) plot. Different pH (7.2 and 8.3) and NaCl concentrations (0.2 and 0.4 M) were also tested.

Activation Energy

Purified PCI-NP (1.5 ng) and 20 µg of substrate were incubated in 250 µl of standard assay buffer for 1 h at 15, 20, 25, 30, and 35 °C. Activation energy was calculated from the slope of the Arrhenius plot (log reaction velocity versus 1/temperature in degrees Kelvin).

Estimation of PCI-NP Molecular Size by SDS-PAGE

1 ml of enzymatic preparation after heparin-Sepharose chromatography (step IV) was dialyzed (50 mM sodium cacodylate, pH 7.5, 0.2 M NaCl, 2.5 mM NEM, 2 mM CaCl, 0.5 mM PMSF) and rotated for 2 h at room temperature with 0.2 ml of 37D9 monoclonal antibody coupled to Affi-Gel (1 mg/ml of gel) pre-equilibrated in the same buffer. After washing, sequential elution of the bound material was performed at increasing NaCl molarity in the same buffer (0.3, 0.4, 0.5 M; 2 400 µl of buffer at each NaCl concentration). For all collected fractions, the enzyme activity was assayed, and protein labeling with biotin was performed according to a standard protocol (Biotin labeling kit, Boehringer). Adequate volumes of labeled samples (depending upon the estimated protein concentration) were then mixed with the same volume of Laemmli buffer with or without 100 mM DTT and denatured for 5 min at 100 °C. After electrophoresis on 7.5 or 12.5% polyacrylamide gels, proteins were transferred to Hybond C extra (Amersham Corp.). Free reactive sites on membranes were then saturated in 1% BSA. After incubation with streptavidin coupled to horseradish peroxidase (Sigma), labeled proteins were visualized by use of ECL (Amersham) followed by exposure to x-ray film (Kodak). Relative intensities of the different proteins bands were determined using a LASER scanning densitometer (Ultroscan XL, Pharmacia). Apparent molecular weights under reducing or non-reducing conditions were estimated by comparison to biotinylated molecular weight standards (Bio-Rad).

Estimation of PCI-NP Molecular Size by Gel Filtration

The enzymatic preparation (step V) was applied to the second heparin-Sepharose (step VI) and eluted with the extraction buffer (see above) instead of 1.2 M NHAc. An aliquot of the active fraction was then chromatographed on a 2.5 100-cm column of Sephacryl S-300 (Pharmacia) previously equilibrated in the extraction buffer. PCI-NP molecular size was estimated by comparing the enzyme activity profile with the elution volume of molecular weight markers.

Cleavage Specificity by PCI-NP

Determination of the cleavage site was assayed by sequencing the carboxyl end of the released pN-1 propeptide. 2 mg of pN-collagen I were incubated at 35 °C with PCI-NP (0.8 ml of enzyme preparation after step IV). After 18 h, high molecular weight proteins were precipitated by addition of the same volume of 99% ethanol followed by centrifugation (10,000 g, 30 min). The supernatant containing the cleaved amino-terminal propeptides was then lyophilized, resolubilized in 500 µl of water, dialyzed against 0.2 M NHAc, and lyophilized again. The sample was denatured for 5 min at 100 °C in Laemmli buffer and migrated in 13% acrylamide:piperazine diacrylamide (100:1) gel (Bio-Rad). Electrophoresis was carried out in 50 mM Tris, 0.1% SDS adjusted to pH 8.4 with boric acid instead of glycine. After transfer on PVDF membrane in 50 mM Tris/boric acid buffer (pH 9.5), proteins were stained with Coomassie Brilliant Blue. The band corresponding to pN-1 propeptide and fragments of membrane without protein as control were cut out, destained in 100% methanol, incubated for 30 min in methanol containing 0.2% polyvinylpyrrolidone, and extensively washed in distilled water. Peptide samples and control membranes were then incubated at 25 °C with 1 µg of carboxypeptidase Y (Boehringer) in 40 mM sodium citrate, pH 6, 7% acetonitrile or with 3 µg of carboxypeptidase P (Boehringer) in 40 mM sodium citrate, pH 4, 1% acetonitrile. Aliquots of the solutions were taken after 10 min and 1, 2, 4, and 20 h of incubation and adjusted to pH 9 with 0.1 N NaOH. Released amino acids were then derivatized by incubation for 15` at 70 °C with DABS-Cl (Fluka) and resolved on a reversed-phase C (250 4.6 mm) HPLC column (Gold Systems, Beckman, San Ramon, CA). For each time point, the measured amount of amino acids was corrected for background values calculated from protein-free PVDF membrane fragments handled in parallel.

Amino Acid Analysis of PCI-NP

A PVDF membrane fragment containing about 0.3 µg of PCI-NP was hydrolyzed with 6 N HCl in the gas phase for 24 h at 106 °C under an inert argon atmosphere. Precolumn derivatization of the free amino acids was performed with phenylisothiocyanate on a 420-amino acid analyzer (ABI, Foster City, CA). The phenylthiocarbamyl derivatives were analyzed on-line on a C18 reverse phase liquid chromatography column (Brownlee, ABI, Foster City, CA).

Sequence Analysis

Amino-terminal sequence analysis of the intact protein and the peptides was performed on the model 476A protein sequencer (ABI, Foster City, CA) operating in the pulsed liquid mode with on-line phenylthiohydantoin analysis. The amino-terminal sequence analysis of the blotted PCI-NP was performed in a cross-flow reaction cartridge using modified run cycles. For sequence analysis of the peptides, a trifluoroacetic acid-treated glass fiber disk was covered with polybrene before application of the sample.

Enzymatic Cleavage of Membrane-bound PCI-NP

The in situ digest was performed as described by Fernandez et al.(15) . Briefly, the PVDF fragments, containing approximately 30 µg of PCI-NP, were cut in small parts, destained with 0.5 ml of 200 µM NaOH, 20% acetonitrile for 1 min, followed by one wash with 0.5 ml of Milli-Q water for 30 min at 37 °C. 100 µl of digest buffer (1% RTX-100, 10% acetonitrile, 100 mM Tris-HCl, pH 8.0) were added to the sample together with 3 µg of endoproteinase Lys-C (Wako, Osaka, Japan) (E/S = 1/10 (w/w)). The digestion was carried out at 37 °C for 24 h, and cleaved peptides were recovered as previously described(15) .

Separation of the Generated Peptides

The extracted Lys-C peptides were separated on a reversed-phase HPLC column (C/C, 2.1 100 mm, 5 µm) installed on the SMART system (Pharmacia). A linear gradient was formed with two solvents: solvent A = 0.05% trifluoroacetic acid/MQ-water and solvent B = 0.04% trifluoroacetic acid, 70% acetonitrile. The applied gradient was as follows: 0-60 min, 1-60% B; 60-70 min, 60-100% B; 70-75 min, 100% B; 75-80 min, 100-1% B.

Mass Analysis

The correctness of the sequences determined was verified by comparing the calculated masses (based on the average residual masses of the individual amino acids) with the experimentally determined value using a VG Tofspec matrix-assisted laser desorption mass spectrometer (VG Analytical, Cheshire, UK). One-fourth (1-5 pmol) of the peptide fraction was mixed with a 50 mM solution of -cyano-hydroxy-cinnamic acid (Aldrich) and applied to the multisample stage. Calibration of the mass scale was performed by preliminary analysis of a mixture of gramicidin S and bovine insulin (Sigma). Samples were ionized using a N laser (337 nm), while the laser energy was pulled out at a level that yielded the best signal performance.

Amino Acid Analysis and Sequencing of Type XIV Collagen

The amino acid composition was determined after derivatization of amino acids with DABS-Cl by reversed-phase chromatography. 50 µg of type XIV collagen present in the 0.4-0.5 M NaCl fractions of the heparin-Sepharose chromatography (step IV) were sequenced as described for PCI-NP.


RESULTS

Purification of PCI-NP

The crude skin homogenate (250 g, wet weight) was first washed with a low ionic strength buffer. This preliminary step allowed removal of proteins in solution (10 mg/g of skin) without significant extraction of PCI-NP activity (60 5%). summarizes the purification of the enzyme from the washed skin homogenate. Through steps I to IV, PCI-NP was purified 90-fold with 45% recovery. However, many proteins were still present in the active fraction as judged from SDS-PAGE analysis. This semi-purified preparation of enzyme was injected in mice to produce monoclonal antibodies that could be used to improve the purification of PCI-NP. Only one antibody (37D9) showed a significant and reproducible immunoprecipitating activity and was used for affinity chromatography. This additional step (step V) strongly improved the purification of the enzyme, and its specific activity could not be determined since the protein concentration was too low to be measured with accuracy. A final low volume heparin-Sepharose column was used to concentrate and further purify the enzyme. At this step, more than 50% of the proteins present at step V were removed as judged from the silver-stained electrophoresis patterns of the different fractions. A concentration of PCI-NP of about 1 µg per ml was deduced by comparing the intensity of the specific band (see below) with the intensity of standard BSA bands, suggesting that about 25 µg of enzyme were recovered from 250 g of fetal skin.

Determination of PCI-NP Molecular Size

Enzyme at step IV of purification was loaded on a 37D9 affinity column and progressively eluted by increasing the NaCl concentration. Aliquots of each of the collected fractions were used to measure the PCI-NP activity and to determine in parallel the protein pattern after SDS-PAGE. To visualize even the minor protein bands, a fluorimetric technique (see ``Experimental Procedures'') was used that allowed the detection of subnanogram amounts of protein. As illustrated in Fig. 1, only one band with an apparent molecular mass of 107 kDa in the presence of DTT varied proportionally to the PCI-NP activity. No other protein of higher or lower molecular weight (detected, respectively, on 7.5 or 12.5% polyacrylamide gel) showed a similar pattern, providing strong evidence that the 107-kDa band was the PCI-NP. In non-reducing conditions, this protein migrated faster (84 kDa), suggesting the presence of at least one intramolecular disulfide bond. A completely similar analysis was also carried out on the active fractions collected from the second heparin-Sepharose column (step VI of purification) with similar results (not shown). In addition, PCI-NP molecular size was estimated by gel filtration on Sephacryl-S300. A single peak of enzyme activity representing more than 95% of the loaded activity was observed at elution volumes corresponding to an apparent molecular size of about 130 kDa. SDS-PAGE analysis of the collected fractions confirmed the presence of the 107-kDa band (reduced) varying proportionally to PCI-NP activity, while contaminant polypeptides displayed a different pattern of elution (not shown).


Figure 1: Estimation of PCI-NP molecular weight by SDS-PAGE. 1 ml of enzymatic preparation after step IV of purification was loaded on a 37D9 affinity column and progressively eluted by increasing NaCl concentration (lane1, 0.2 M; lanes2 and 3, 0.3 M; lanes4 and 5, 0.4 M; lanes6 and 7, 0.5 M). Collected fractions were assayed for their enzyme activity and labeled with biotin. Adequate volumes of biotinylated samples were then migrated on a 7.5% (A) or on a 12.5% (B) SDS-PAGE and transferred on Hybond C extra membrane. After incubation with streptavidin coupled to horseradish peroxidase, labeled proteins were visualized by use of ECL followed by exposure to x-ray film (A, 2 min; B, 1 min).



Western blots of enzyme preparations at different steps of purification incubated with the 37D9 monoclonal antibody revealed three major bands of about 200, 220, and 290 kDa but no 107-kDa protein. This observation strongly suggested that the antibody did not recognize PCI-NP itself but was specific of another protein present in the preparation and on which the enzyme could bind (see below). In a complementary experiment, the serum of a mouse immunized with the 107-kDa band was shown to reproducibly and significantly inhibit, by 15-20%, the PCI-NP activity in a standard assay. No such inhibition was observed with the pre-immune serum of the mouse or with other control sera, confirming the specificity of the inhibition and providing an additional evidence that the 107-kDa protein is PCI-NP. No monoclonal antibody could be derived from that mouse.

Cleavage Specificity by PCI-NP

Enzyme preparations at different steps of purification were incubated at 26 °C with labeled pN-collagen type I for 15 min to 1 day, and aliquots of the digested products were examined by SDS-PAGE. As expected, pN- chains were progressively converted to chains. This conversion was blocked by EDTA. There was no internal cleavage of either chains or amino propeptides even after prolonged incubation. In other experiments, denatured pN-collagen type I, native pN-collagen type III, or type XIV collagen was incubated for 3 days with the purified enzyme (step V) without any apparent degradation or processing.

To determine the peptide bond cleaved by PCI-NP, pN-collagen type I was digested with the enzyme, and the isolated chains were analyzed by Edman degradation. No sequence was obtained, confirming previous reports (9, 11) indicating that the amino-terminal residues of type I collagen chains were blocked by a modified glutamine residue. Amino acids at the carboxyl end of the released pN-1 propeptide were then analyzed by digestion with either carboxypeptidase Y or P. Analysis of the time course-released products by reverse-phase HPLC revealed an Ala-Pro-COOH sequence with carboxypeptidase Y and a Phe-Ala-Pro-COOH sequence with carboxypeptidase P (Fig. 2). In the pN-1 propeptide, this sequence is unique and situated immediately upstream of the published cleavage site by PCI-NP(9) , indicating the specificity of the cleavage performed by our enzyme.


Figure 2: Time course digestion of the 1 amino propeptide by carboxypeptidases Y (A) or P (B). 1 amino propeptide immobilized on PVDF membrane was incubated with carboxypeptidases Y or P during a time course experiment. At the indicated times, aliquots of cleaved products in solution were derivatized with DABS-Cl and analyzed on a reverse phase C HPLC column. For each time point, the measured amounts of amino acids were corrected for background values (see ``Experimental Procedures'').



Kinetics Properties

The K and V values of the reaction were determined at various temperatures in the standard assay buffer (pH 7.5, 0.2 M NaCl) using 1.5 ng (0.056 nM) of PCI-NP and 50-600 nM of labeled pN-collagen type I. As seen in , a significant enzymatic activity was already measured at 15 °C, but incubation at higher temperatures strongly increased the rate of cleavage. At 35 °C in presence of 600 nM of substrate, we determined that one molecule of PCI-NP cleaved about 430 molecules of pN-collagen type I within 1 h. These data obtained at various temperatures were used to determine, from an Arrhenius plot, the activation energy of the enzyme (17,000 cal mol).

In other experiments, the effects of pH and NaCl concentration were assayed at 35 °C (I). Increasing the NaCl molarity from 0.2 to 0.4 or the pH from 7.2 to 8.3 markedly increased the K and the V values. However, assay buffer adjusted to both pH 8.3 and 0.4 M NaCl did not further modify the kinetic properties.

Amino Acid Composition of PCI-NP

The amino acid analysis was performed on about 250 ng of PCI-NP, as determined from protein staining of the blot. As shown in , the proportion of Gly, Glx, and Asx is slightly higher than usually observed in other proteins. No Hyp or Hyl residues, the specific markers of collagen, were found. In addition, the analysis indicated that about 2.6 pmol of PCI-NP were hydrolyzed. This corresponds to about 260 ng of a 100-kDa protein, confirming our estimation by protein staining after electrophoresis.

Sequencing Analysis

Since a first amino-terminal sequence analysis on about 10 pmol (1 µg) of the blotted PCI-NP protein indicated that the protein was amino-terminally blocked, we cleaved the membrane-bound protein (30 µg) enzymatically to obtain sequence information for some internal peptide fragments. Endoproteinase Lys-C, which cleaves specifically at the carboxyl-terminal end of every lysyl residue, was chosen because the lysine content in PCI-NP (4%) seemed appropriate to obtain peptides of various lengths that could be easily resolved by reverse phase liquid chromatography analysis. A control digest was performed on a blank piece of PVDF (Coomassie stained but containing no protein) to identify peaks originating from background or enzyme autoproteolysis. After extraction, the peptides were separated on a reverse phase liquid chromatography column containing a mix of C/C chains, and different fractions were collected. Several peptides were subjected to amino-terminal sequence analysis (3/4 of the material), but only two fractions, K1 and K3, contained a pure peptide that could be unambiguously sequenced up to the final lysyl residue. Three other fractions contained a mix of two or more fragments or had a very low initial sequence yield (<1 pmol) (). As a final control, the remainder (1/4) of the sequenced fractions was subjected to matrix-assisted laser desorption/ionization mass analysis to verify the obtained sequences. For fraction K3, the calculated mass, 1633.7 Da, is in perfect agreement with the experimentally determined one, 1633.2 Da. Fraction K1 yielded no mass probably because there was too little material left.

The sequences for K1 and K3 were used to screen protein and nucleic acid data banks. No significant homology with known sequences was observed.

Type XIV Collagen Is the Protein Recognized by 37D9 Monoclonal Antibody

The electrophoretic mobility of the protein recognized by 37D9 monoclonal antibody was analyzed in different conditions. This protein barely entered the gel without reduction but migrated as one minor and two major bands of about 200, 220, and 290 kDa in presence of DTT. Digestion with purified bacterial collagenase yielded a single band of 190 kDa. Immunofluorescence studies using the 37D9 monoclonal antibody demonstrated that this protein colocalized with type I collagen fibers in calf skin and tendon. Altogether, these results suggested that it could be either type XII or type XIV collagen(16) . As additional evidence, the CY 15B8 monoclonal antibody(16) , which is specific of bovine type XIV collagen, was shown to coprecipitate PCI-NP and type XIV collagen with the same efficiency as the 37D9 monoclonal antibody. Finally, amino acid sequencing of four internal Lys-C peptides revealed that on a total of 48 residues, 44 residues were identical to the sequence of chicken type XIV collagen (data not shown).

Properties of the Binding between PCI-NP and Type XIV Collagen

Characterization of the binding between PCI-NP and type XIV collagen was performed using 37D9 immunoaffinity columns. The enzyme immobilized at 0.25 M NaCl (pH 7.4) could be eluted by slight modifications of the ionic conditions such as 0.35 M NaCl at pH 7.4, 0.25 M NaCl at pH 9.0, or 0.2 M NaCl at pH 7.4 in presence of traces of an ionic detergent (0.0001% SDS). Even high concentrations of non-ionic detergents (3% Triton X-100 or 3% Brij) or 0.4 M urea were ineffective to desorb the fixed enzyme. It was also observed that excess of soluble type XIV collagen could elute PCI-NP activity from immobilized type XIV collagen while BSA (1 mg/ml) or serum (1%) was not able to do it, confirming the specificity of the binding.


DISCUSSION

A type I procollagen N-proteinase (PCI-NP) presenting a 107-kDa apparent molecular mass was purified from fetal calf skin. After six major purification steps, about 25 µg of enzyme were isolated from 250 g of tissue with a recovery of 16% based on enzymatic activity. The purified enzyme was extensively characterized. It specifically cleaved native pN-collagen type I and did not process pN-collagen type III. It was also unable to cleave and to degrade denatured pN-collagen type I, pN- propeptide, type I collagen, or type XIV collagen. Amino acid sequencing of the carboxyl end of the amino propeptide of the chain demonstrated that the 107-kDa PCI-NP cleaved the Pro-Gln bond reported to be cleaved during the processing of pro- I polypeptide (9).

The kinetic properties of the enzyme such as the activation energy (17,000 cal mol), the K, and the turnover number of the enzyme (237 nM and 430 h, respectively, at 35 °C, 0.2 M NaCl, and pH 7.2) were similar to values reported by Hojima et al.(10) . However, a major difference exists between these two enzymatic preparations. Hojima et al.(10, 11) described the extensive purification from bovine or chicken leg tendons of a 500-kDa enzyme, as determined by gel filtration, formed by the association of 4-6 different subunits, some of them exhibiting catalytic activity. A minor 300-kDa partially degraded form was also observed. By contrast, we purified from fetal calf skin an enzyme with an apparent molecular mass estimated at about 130 kDa by gel filtration. In addition, SDS-PAGE analysis of active fractions collected from three different chromatographic columns revealed that one band with an apparent molecular mass of 107 kDa (reduced) varied always proportionally to PCI-NP activity, while other polypeptides present in the preparations had a different pattern of elution that did not coincide with enzyme activity. These data suggest that the fully active enzyme consists in a single protein of 107 kDa, a size close to what was observed for the 102-kDa procollagen C-proteinase (1) rather than in a complex of polypeptides as suggested by Hojima et al.(10, 11) . The reason for this difference is not clear, but the use of an affinity chromatography in our procedure could have improved purification by removing tissue proteins displaying affinity for PCI-NP. We hypothesize that the 90-kDa subunit (unreduced) from the 500-kDa complex prepared by Hojima et al.(10, 11) corresponds to our PCI-NP (84 kDa, unreduced), while the other polypeptides in this complex represent PCI-NP-bound proteins as we observed it with type XIV collagen (see below).

An amino acid sequence analysis was performed using electrophoretically purified enzyme. As for most of the secreted proteins, the amino-terminal amino acid of PCI-NP was blocked and could not be subjected to an amino-terminal sequence analysis by Edman degradation. To overcome this problem, internal peptides of membrane-bound enzyme were produced and analyzed. An unambiguous sequence of 14 amino acids in length was determined and used to screen proteins and nucleic acid data banks. No significant homology was found, suggesting that PCI-NP was not cloned or sequenced so far, even as an unidentified protein.

One crucial point in this work was to produce a monoclonal antibody that could be used for immunopurification of PCI-NP. About 3000 hybridomas were produced from mice immunized with a semi-purified enzymatic preparation, but only one of them secreted a monoclonal antibody able to immunoprecipitate PCI-NP activity. This antibody, however, was not directed toward the enzyme itself but recognized a protein on which the enzyme was bound. This binding protein was determined to be type XIV collagen by various techniques, including SDS-gel electrophoresis, immunoblotting, analysis of amino acid composition, and partial microsequencing. The nature of the interaction between PCI-NP and type XIV collagen was investigated. This binding is highly specific since excess amounts of irrelevant proteins (0.1% BSA or 1% fetal calf serum) did not alter the interaction. It was also strongly dependent upon ionic conditions. Small increases of pH or salt concentration, as well as traces of ionic detergent (0.0001% SDS), prevented the binding of PCI-NP on type XIV collagen. By contrast, non-ionic detergents at concentrations as high as 3% had essentially no effect. Since extraction of PCI-NP from calf skin was also strongly dependent upon ionic conditions and not affected by non-ionic detergents,()this suggests that type XIV collagen might be a physiological ligand for PCI-NP in vivo. The exact function of type XIV collagen is not completely elucidated. In vivo, it is associated with type I collagen fibers via direct or indirect interactions(17, 18, 19) , and the current hypothesis is that it could be involved in the regulation of extracellular matrix assembly. Here, we propose another role for type XIV collagen that would be to immobilize PCI-NP in a close vicinity of type I collagen fibers, allowing the processing and the subsequent polymerization of the newly synthesized molecules under a strict spatial control.

Different modules present in the NC-3 domain of the XIV collagen are potential binding sites. Among them, the tsp1 module (or NC-4-like domain) was carefully studied(20) . This module, first described in thrombospondin, is also found in FACIT collagens (types IX, XII, XIV) and in some fibrillar collagens ( V, XI, XI). It is characterized by a -stranded structure also observed in proteins that are involved in molecular recognition and that exert adhesion or binding functions such as Ig-related molecules(20) . To determine which module is really involved in the binding of PCI-NP, we are currently developing monoclonal antibodies against different epitopes of type XIV collagen. The hypothesis to test will be that these collagens, expressed in different tissues at variable levels(21) , could have binding properties for PCI-NP and eventually for other enzymes involved in the extracellular processing of procollagen molecules such as, for example, lysyl oxidase or type III procollagen N-proteinase.

The results presented in this study clearly indicate that we have isolated a fully active procollagen I N-proteinase (PCI-NP) of 107 kDa. An amino acid sequence was determined that will be useful for the cloning of the cDNA. We also described binding properties of type XIV collagen for PCI-NP, suggesting a new role for this collagen and perhaps for other FACIT collagens in vivo.

  
Table: Purification of PCI-NP from fetal calf skin (250 g)

Activity was determined under standard reaction conditions and expressed in cpm of cleaved substrate. ND, not determined.


  
Table: K and V of the enzymatic reaction determined at increasing temperatures

Kinetic studies were performed as described under ``Experimental Procedures.'' K and V were calculated according to the Lineweaver-Burk plot and the least squares test methods and confirmed by the direct Eisenthal and Cornish-Bowden (14) plot. Values in the table represent the mean of values ± S.D. obtained in three separate experiments, except for the 15 °C condition performed only once.


  
Table: Effect of pH and NaCl concentration on the enzymatic reaction at 35 °C

The study was performed as described for Table II except that pH was adjusted to 7.2 or 8.3 and the NaCl concentration to 0.2 or 0.4 M.


  
Table: Amino acid composition of PCI-NP

About 250 ng of PCI-NP immobilized on PVDF membrane were hydrolyzed during 24 h at 106 °C in 6 N HCl. Analysis of the PTC-derivatives was performed on a 420A analyzer (ABI).


  
Table: Amino acid sequencing of PCI-NP

Seven individual peptides peaks specific of PCI-NP were sequenced. The main sequences are given in the one-letter code on the first line and in bold if unambiguously identified. Other sequences found during the analysis are given below. An * indicates that the background in this cycle was too high to make any assignment for this residue. A dash indicates that there was no major increase for any PTH derivative of the main sequence in this cycle. indicates that the sequence analysis was deliberately stopped.



FOOTNOTES

*
This work was supported in part by the Belgian FRSM Grant n°3.4510.90, the Fonds de Recherche de la Faculté de Médecine de l'Université de Liège, the Action de Recherche Concertée n° 90-94/139 of the French Community of Belgium (to C. M. L.), and the Concerted Research Action (program 12052293) (to J. V. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Chargé de Recherche supported by the Belgian Fonds National de la Recherche Scientifique.

To whom correspondence should be addressed. Tel.: 32-41-662455; Fax: 32-41-667234.

The abbreviations used are: PCI-NP, procollagen I N-proteinase; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; HPLC, high pressure liquid chromatography; DTT, dithiothreitol; BSA, bovine serum albumin; NEM, N-ethylmaleimide; PMSF, phenylmethylsulfonyl fluoride; FACIT, fibril-associated collagens with interrupted triple helices.

A. Colige, Y. Goebels, B. V. Nusgens, and C. M. Lapière, personal observation.


ACKNOWLEDGEMENTS

We thank Dr. Darwin J. Prockop (Dept. of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, PA) for valuable criticisms and suggestions, Dr. P. de Baetselier (Laboratory of Cellular Immunology, VUB, Belgium) for the collaboration of his laboratory in this work, Dr. J. F. Beckers and co-workers (Faculty of Veterinary Medicine, ULg, Belgium) for supplying fetal bovine skin, and Dr. C. Lethias and Dr. M. van der Rest (Institute of Biology and Chemistry of Proteins, Ecole Normale Supérieure de Lyon, Lyon, France) for kindly providing us with the CY 15B8 monoclonal antibody against type XIV collagen. The skillful assistance of H. Cuaz for helping in the presentation of the manuscript is also acknowledged.


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