From the
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
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)
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
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.
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-
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
All procedures were performed at 4 °C or in an ice bath
unless otherwise stated.
The enzymatic preparation after the heparin-Sepharose
chromatography (step IV) was used for immunization of F1 mice (Balb/c
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 K
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).
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
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 NH
Determination of the cleavage site was assayed by sequencing
the carboxyl end of the released pN-
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).
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.
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) .
The extracted Lys-C peptides were separated on a
reversed-phase HPLC column (C
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
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.
To determine the peptide bond cleaved by PCI-NP,
pN-collagen type I was digested with the enzyme, and the isolated
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
The sequences for K1 and K3 were used to
screen protein and nucleic acid data banks. No significant homology
with known sequences was observed.
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-
The kinetic properties of
the enzyme such as the activation energy (17,000 cal
mol
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,
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
(
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.
Activity was determined under standard reaction
conditions and expressed in cpm of cleaved substrate. ND, not
determined.
Kinetic studies were performed as described under
``Experimental Procedures.'' K
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.
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).
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.
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)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) .
-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) .
Preparation of Radiolabeled Substrates
type I chains were labeled.
Preparations at 1300 cpm/µg of amino procollagen type I (1 mg/ml)
were usually obtained.
Enzyme Assay
, 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
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 NH
Ac.
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 NH
Ac
and stored at -80 °C.
Production of Monoclonal Antibody
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
and 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
Estimation of PCI-NP Molecular Size by SDS-PAGE
, 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
Ac. 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
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 NH
Ac, 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
Sequence Analysis
Enzymatic Cleavage of Membrane-bound PCI-NP
Separation of the Generated Peptides
/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
-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
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.
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
).
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.
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.
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 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).
(
)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.
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.
Table: Purification of PCI-NP from fetal calf
skin (250 g)
Table: K and V
of the enzymatic reaction determined at increasing
temperatures
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
Table: Amino acid composition of PCI-NP
Table: Amino acid sequencing of PCI-NP
indicates that the sequence
analysis was deliberately stopped.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.