From the Departments of Microbiology and
Chemistry, Faculty of Medicine, Kagawa
Medical University, Miki-cho, Kita-gun, Kagawa 761-0793, Japan,
¶ Core Research for Evolutional Science and Technology, Japan
Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan, and the
Department of Molecular and Cellular Biology,
Institute for Frontier Medical Sciences, Kyoto University, Kyoto
606-8397, Japan
Received for publication, April 24, 2000, and in revised form, December 7, 2000
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ABSTRACT |
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Clostridium histolyticum type I
collagenase (ColG) has a segmental structure, S1+S2+S3a+S3b. S3a and
S3b bound to insoluble collagen, but S2 did not, thus indicating that
S3 forms a collagen-binding domain (CBD). Because S3a+S3b showed the
most efficient binding to substrate, cooperative binding by both
domains was suggested for the enzyme. Monomeric (S3b) and tandem
(S3a+S3b) CBDs bound to atelocollagen, which contains only the
collagenous region. However, they did not bind to telopeptides
immobilized on Sepharose beads. These results suggested that the
binding site(s) for the CBD is(are) present in the collagenous region.
The CBD bound to immobilized collagenous peptides,
(Pro-Hyp-Gly)n and (Pro-Pro-Gly)n, only when
n is large enough to allow the peptides to have a
triple-helical conformation. They did not bind to various peptides with
similar amino acid sequences or to gelatin, which lacks a
triple-helical conformation. The CBD did not bind to immobilized
Glc-Gal disaccharide, which is attached to the side chains of
hydroxylysine residues in the collagenous region. These observations
suggested that the CBD specifically recognizes the triple-helical
conformation made by three polypeptide chains in the collagenous region.
Collagens are the major components of the extracellular matrix.
They are the most abundant proteins in mammals, constituting a quarter
of their total weight. Collagens are not only structural proteins with
a high tensile strength but also affect cell differentiation, migration, and attachment. Nineteen different types are known to date,
and type I collagen is the major species in higher vertebrates. In the
endoplasmic reticulum of fibroblasts, collagen precursor peptides are
hydroxylated by various dioxygenases (prolyl hydroxylases and a lysyl
hydroxylase), glycosylated at the hydroxylysine residues, and folded
into a triple helix. HSP47 associates with procollagen during this
modification and/or folding processes and is assumed to function as a
collagen-specific molecular chaperone (1). The precursor is secreted
into the extracellular space, and its N- and C-terminal propeptides are
removed by procollagen peptidases. Cleavage triggers the arrangement of
collagen monomers into a staggered array called fibrils, which are
insoluble macromolecular assemblies having lengths up to a few hundred
micrometers. One can observe 67-nm-wide cross striations (overlap and
gap zones) on the fibrils by electron microscopy, which are formed by
the regular arrangement of collagen monomers. Each collagen monomer consists of collagenous and noncollagenous regions. The former have a
regular amino acid sequence, which is a repeat (338 times in type I
collagen) of the Gly-X-Y triplet, forming a long
(300 nm in type I collagen) triple-helical domain. Proline and
hydroxyproline residues are most common in the X and
Y positions, respectively, and this combination stabilizes
the helix to the highest extent (2). The latter regions lack the
sequence and conformation characteristic of the former, and are named
telopeptides, because they are located at the N and C termini of the
collagenous region.
Clostridium histolyticum is a strictly anaerobic
Gram-positive bacterium that is one of the causative agents of gas
gangrene. The pathogen produces a variety of collagenases in large
quantity, which efficiently degrade collagen in connective tissue.
Although the enzyme has been widely used in biological experiments, its detailed biochemical characteristics have not been fully understood (3). Previously, we have shown that C. histolyticum has two divergent collagenase genes (colG and colH) in
its chromosome, which encode two distinct enzymes; class I collagenase
(ColG)1 and class II
collagenase (ColH) (4, 5). Their deduced amino acid sequences showed no
significant similarity to those of eukaryotic matrix
metalloproteases. Comparison of the ColG and ColH sequences revealed their segmental structure; S1+S2+S3a+S3b for ColG and S1+S2a+S2b+S3 for ColH as shown in Fig. 1A (see below). A
truncated ColH consisting of only S1 showed full hydrolytic activity on a peptide substrate (6). When a consensus motif for zinc proteases, HEXXH, present in S1 was altered, catalytic activity was
drastically reduced (7). Thus, S1 was assumed to contain the catalytic domain. However, an isolated C-terminal fragment, S2b+S3 from ColH,
bound to lyophilized insoluble collagen to the same extent as
full-length ColH (6). This observation lead us to conclude that this
C-terminal fragment contains the collagen-binding domain (CBD).
Considering the complex macromolecular structure of collagen fibrils,
however, the structure of the substrate molecule/moiety recognized by
CBD is not fully understood. It could be even a disparate molecule
closely associated with collagen fibrils.
For applications, we have shown that the CBD can be used to anchor
peptide-signaling molecules to collagen-containing tissues (8). Fusion
proteins between growth factors and S2b+S3 bound not only to insoluble
collagen in vitro but to connective tissue in
vivo. A fusion protein carrying basic fibroblast growth factor joined to the N terminus of S2b+S3 remained in type I collagen-rich tissue when injected subcutaneously into nude mice and exerted a
growth-promoting effect at the sites of injection much longer than
basic fibroblast growth factor alone. To apply the findings more
widely to drug delivery systems, it is essential to diminish the
antigenicity of the CBD. One straightforward approach is to synthesize
mimics of the CBD by rational drug design, based on its molecular
structure. To achieve this objective, detailed information is needed,
i.e. the minimal region for collagen binding, the minimal receptor moiety, and the structure of the bimolecular complex. Furthermore, elucidation of the molecular mechanism of collagen binding
provides insights to understand the biosynthesis and degradation of
extracellular matrix proteins. Thus, we carried out binding experiments
using various C-terminal fragments of class I collagenase (ColG) and
subcomponents or mimics of human type I collagen.
DNA Manipulations and Sequencing--
Restriction endonucleases
were purchased from Takara Shuzo Co. (Kyoto, Japan), Toyobo (Osaka,
Japan), and New England BioLabs (Beverly, MA). The DNA ligation kit was
a product of Takara Shuzo. All recombinant DNA procedures were carried
out as described by Sambrook et al. (9). All constructs were
sequenced to confirm the reading frame on an automated nucleotide
sequencer (model ABI PRISM 377, PerkinElmer Life Sciences, Foster City,
CA). A Thermo Sequenase fluorescence-labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech, Buckinghamshire, UK)
and Purification of GST Fusion Proteins--
The segmental
boundaries of C. histolyticum class I collagenase (ColG)
were determined from an alignment of the amino acid sequences of three
clostridial collagenases (4). Nucleotide fragments encoding the
C-terminal segments were obtained by PCR using either pCHG5 or pCHG51
(4) plasmid DNA as the template and the following pairs of
synthetic primers; 5'-CCCGGGACAGATAATGCGGATATTAG-3' and
5'-CTCGAGTTAATCTTCGTTCTTTATTTCTA-3' for segment 2 (S2,
Thr677-Asp772);
5'-CCCGGGACAACAACACCTATAACTAA-3' (S3a-5' primer) and
5'-CTCGAGCTATTTCTCGTTACCTAATCCTT-3' for segment 3a (S3a,
Thr773-Lys896);
5'-CCCGGGAACGAGAAATTGAAGGAAAA-3' and 5'-CTCGAGTTATTTATTTACCCTTAACT-3' (S3b-3' primer) for segment 3b (S3b,
Asn894-Lys1008); and S3a-5' primer and S3b-3'
primer for segments 3a and 3b (S3a3b,
Thr773-Lys1008). These fragments were cloned
into the pT7Blue T-vector. After their correct nucleotide sequences
were confirmed, they were inserted into pGEX-4T-2 vector DNA between
the SmaI and XhoI sites to express the C-terminal
fragments as GST fusion proteins. Escherichia coli DH5
To confirm that each fusion protein was produced as designed, the
proteins were cleaved by incubation with thrombin (Amersham Pharmacia
Biotech, 10 units/mg of fusion protein) for 10 h at room
temperature. Two hundred picomoles of the cleavage products was
separated on a 12.5% SDS gel and transferred onto a sheet of
polyvinylidene diflouride membrane (Bio-Rad Laboratories, Hercules, CA)
by electroblotting (10). The membrane was briefly washed with distilled
water, and stained by Coomassie Brilliant Blue R as described by the
manufacture. Bands corresponding to the C-terminal fragments were cut
out and subjected to the amino acid sequencing using a protein
sequencer (model 492, PerkinElmer Life Sciences). The sequence was
determined up to the 20th residue for each fragment.
Purification of C-terminal Fragments--
The thrombin-cleaved
protein fractions were dialyzed three times against 1 liter of
phosphate-buffered saline at 4 °C to remove glutathione. The
N-terminal GST fragment was removed by applying the fraction to a
glutathione-Sepharose 4B column (bed volume, 0.5 ml). To measure the
molecular mass of the purified C-terminal fragment, the sample was
diluted with distilled water, mixed with 3,5-dimethoxy-4-hydroxycinnamic acid solution (10 mg/ml 50% ethanol), and analyzed by matrix-assisted laser desorption time-of-flight mass
spectrometry (MALDI-TOF MS, model Kompact MALDI IV, Kratos, Manchester,
UK) in the positive linear mode with time-delayed extraction and sperm
whale myoglobin as an internal standard.
Fluorescence and Circular Dichroism--
S2, S3a, S3b, and S3a3b
were dissolved at concentrations of 50 µg/ml in 50 mM Tris-HCl, 100 mM NaCl, 1 mM
CaCl2 (pH 7.5, CB buffer) containing varying concentration
(0-9 M) of urea. Fluorescence was measured by using an
excitation wavelength of 290 nm with emission at 350 nm on a
fluorescence spectrophotometer (model F-2000, Hitachi, Tokyo, Japan).
Circular dichroism (CD) spectra were measured from 300 µg of protein
in 300 µl of CB buffer in a 1-mm cell in a spectropolarimeter (model
J600, Jasco, Tokyo, Japan). The spectra for denatured proteins were
obtained by dissolving the same amount of lyophilized protein in the
same volume of CB buffer containing 9 M urea.
Purification of Full-length Type I Collagenase--
A nucleotide
fragment corresponding to the reading frame encoding mature ColG
(Ile1-Lys1008) was prepared by PCR using
C. histolyticum genomic DNA and a pair of oligonucleotide
primers: 5'-CCCCAAGCTTGCTAGCATAGCGAATACTAATTCTGAG-3' and
5'-GGATCCTTATTTATTTACCCTTAACT-3'. The amplified fragment was inserted
into pT7Blue T-vector DNA. A recombinant plasmid carrying an insert
with the correct nucleotide sequence was chosen. To express the
collagenase, a pET-11a vector (Novagene, Madison, WI) was employed. A
unique EcoRI site in the vector was deleted by the fill-in
reaction using Klenow enzyme prior to use (pET-11a
A glutamate residue in the putative catalytic center,
413HEYTH417, was mutated to a glutamine by
site-directed mutagenesis. PCR was carried using pCHG4 (4) plasmid DNA,
and two mutagenic primers;
5'-GAAGAATTGTTTAGACATCAATATACTCACTATTTACAA-3' and
5'-TTGTAAATAGTGAGTATATTGATGTCTAAACAATTCTTC-3'. After the mutation
was confirmed by nucleotide sequencing, the insert EcoRI
fragment (0.80 kilobase) was used to replace the corresponding fragment
in pEG101. The mutant collagenase, rColG(E414Q), was produced and
purified in the same manner as the wild type.
Specific activity against insoluble collagen was measured as described
previously (7). One unit of enzyme activity equals 1 µmol of
L-leucine equivalents liberated from collagen in 5 h at 37 °C under the specified conditions. The N-terminal amino acid
sequence up to the 20th residue was determined as described above. The
molecular mass was determined by MALDI-TOF MS.
Collagen Binding Assay--
Binding of the CBD to type I
collagen was determined as follows; 10 mg of collagen (acid-soluble
collagen, Sigma type I) was incubated in 100 µl of protein mixture at
room temperature for 30 min. The basal mixture (100 µl) contained 5 µg of bovine serum albumin (Sigma), 5 µg of chicken ovalbumin
(Sigma), 5 µg of horse myoglobin (Sigma), 5.42 µg (0.2 nmol) of
S3a3b (ColG), and 2.76 µg (0.2 nmol) of S3b (ColG) in CB buffer. The
water phase was examined by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). When necessary, the salt concentration in
CB buffer was modified (0, 0.1, 0.5, or 1.0 M). To alter
the pH of the buffer, Tris-HCl was replaced with 0.1 M
BisTris-HCl (pH 6.0 or 7.0) or Tris-HCl (pH 7.0, 8.0, or 9.0). The
effect of divalent cations was examined by replacing CaCl2
with 1 mM EGTA or 1 mM EGTA plus 2 mM CaCl2.
Semi-quantitative measurements of binding were carried out as described
previously (6), where the amount of the unbound fusion protein was
quantitated by GST assay using 1-chloro-2,4-dinitrobenzene as the substrate.
Gel-filtration Studies--
Size exclusion chromatography
experiments were performed at room temperature on a high performance
liquid chromatography (HPLC) system equipped with a Superdex 75 column
(1 × 30 cm, Amersham Pharmacia Biotech) at a flow rate of 0.5 ml/min. S3b (20 µg) was dissolved in 50 µl of CB buffer or the same
buffer containing 1 mM EGTA instead of Ca2+,
and chromatographed. The following proteins were used as molecular mass
standards: Measurement of Hydrodynamic Radius--
The hydrodynamic radius
of S3b was measured at 20 °C using a dynamic laser light-scattering
instrument (model DynaPro 99, Protein Solutions, Charlottesville, VA).
S3b was dissolved in CB buffer or CB buffer containing 1 mM
EGTA instead of Ca2+ at a concentration of 5.66 mg/ml. The
samples were filtrated through a 0.02-µm filter before analysis.
Binding to Atelocollagen--
Bovine skin atelocollagen was
purchased from Koken (Tokyo, Japan). To remove the water-soluble
fraction, atelocollagen was treated with water-soluble carbodiimide
prior to use. Ten milligrams of atelocollagen powder were washed in 1 ml of ice-cold 0.1 M potassium phosphate (pH 5.5) three
times. It was suspended in 1 ml of the same buffer, and solid
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC,
Pierce, Rockford, IL) was added to the suspension to a final
concentration of 0.1 M. The suspension was incubated at
4 °C for 24 h with gentle shaking. Atelocollagen was collected by centrifugation at 15,000 × g for 5 min and washed
with 1 ml of CB buffer three times. The pretreated atelocollagen was
transferred into an Ultrafree microcentrifugal device with a 0.22-µm
low binding Durapore membrane (Millipore, Bedford, MA), which was
placed in a microcentrifuge tube. The buffer was removed by
centrifugation at 15,000 × g for 5 min. The
atelocollagen precipitate was incubated in 100 µl of the basal
protein mixture for 30 min at room temperature. The water phase was
collected by centrifugation and examined by SDS-PAGE.
Peptide Synthesis--
Telopeptides were synthesized on an
Applied Biosystems 432A peptide synthesizer using the standard
fluorenylmethyloxycarbonyl method on 4-hydroxymethyl
phenoxyacetamidomethyl resin. Their amino acid sequences, based on
those of human type I collagen (GenBankTM accession numbers,
P02452 and P08123) were as follows; QLSYGYDEKSTGGISVP (17 aa residues)
for Coupling of the Peptides to Sepharose Beads--
Peptides were
dried under vacuum before weighing to prepare solutions in 0.2 M NaHCO3, 1 M NaCl (pH 8.3). The
peptide solutions (6 mg/ml) were incubated at 4 °C for 48 h
prior to use. To prepare 150 µl of the peptide-coupled gel beads, 43 mg of CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) powder
was suspended in 1 ml of 1 mM HCl and incubated for 5 min.
The suspension was centrifuged at 500 × g for 1 min to
remove the water phase. This washing was repeated a total of three
times. The gel was washed once with 1 ml of distilled water in the same
way, and was suspended in 500 µl of the peptide solution. The
suspension was incubated on a rotary shaker for 2 h. The peptide
solution was removed by centrifugation, and the gel was incubated in 1 ml of 0.1 M Tris-HCl (pH 8.0) over night on a rotary shaker
to block any remaining active groups. The gel was sequentially washed
with 1 ml each of 0.1 M NaHCO3, 0.5 M NaCl (pH 8.3, Coupling buffer); 0.1 M sodium acetate, 0.5 M NaCl (pH 4.5); and Coupling buffer. CB
buffer was added to the gel to obtain the final suspension (1 ml). All
the above procedures were carried out at room temperature (25 ± 1 °C), and the suspension was stored at 4 °C. Gelatin-Sepharose 4B was purchased from Amersham Pharmacia Biotech.
Coupling of a Disaccharide to Sepharose
Beads--
Epoxy-activated Sepharose 6B powder (Amersham Pharmacia
Biotech) was rehydrated and washed with distilled water as described by
the supplier.
2-O- Examination of the Binding to Substrate-coupled Sepharose--
A
suspension containing 50 µl of Sepharose beads coupled with peptides,
gelatin, or disaccharide was added to an 0.5-ml microcentrifuge tube. The gel was centrifuged at 500 × g for 1 min and
washed once with 0.4 ml of CB buffer. Then it was suspended in 100 µl of the above protein mixture. The suspension was incubated at 25 ± 1 °C for 30 min on a rotary shaker, and the water phase was collected by centrifugation at 500 × g for 1 min. The
sample (30 µl) was mixed with 4× SDS reducing dye (10 µl), boiled
for 3 min, and analyzed by SDS-PAGE using 15% SDS-polyacrylamide gels
(13). To examine the bound fraction, the gel beads were washed twice with 0.4 ml of CB buffer and suspended in 50 µl of 2× SDS reducing dye. The suspension was boiled for 3 min to solubilize the bound protein(s). After centrifugation, the water phase (40 µl) was analyzed by SDS-PAGE.
Quantitative Analysis of the Binding to a Peptide
Substrate--
Binding of S3b, S3a3b, and rColG(E414Q) to a
collagenous peptide was measured by surface plasmon resonance using a
BIACORE apparatus (Biacore, Uppsala, Sweden). A peptide,
G(POG)8, was dissolved in 10 mM sodium acetate
(pH 6.0), and the solution was incubated at 4 °C for 48 h prior
to use. The peptide was covalently immobilized to a biosensor chip CM5
through the Purification of the C-terminal Fragments--
Various C-terminal
segments (S2, S3a, S3b, and S3a3b) of C. histolyticum class
I collagenase (ColG) were expressed as GST fusion proteins (Fig.
1B). The C-terminal fragments
were cleaved off from the N-terminal carrier by thrombin, and their
N-terminal amino acid sequences were determined. For each fragment, the
initial 20 amino acid residues coincided with the expected sequence.
The carrier was removed by affinity chromatography, and each C-terminal fragment was purified to homogeneity. Although the molecular masses calculated for S2, S3a, S3b, and S3a3b were 10,769, 14,223, 13,787, and
27,055 Da, the values estimated by SDS-PAGE were 16.5, 23.0, 16.5, and
38.0 kDa, respectively. To determine whether these discrepancies were
due to abnormal mobility, the molecular masses were measured by
time-of-flight mass spectrometry. The observed values were 10,758, 14,208, 13,776, and 27,070, for the respective fragments, coinciding
well with the calculated values.
Binding to Insoluble Collagen--
Binding of GST fusion proteins
to collagen was examined semi-quantitatively using lyophilized type I
collagen, which is insoluble at neutral pH. As shown in Table
I, the fusion protein containing S3 bound
while that containing S2 did not. The fusion proteins were cleaved at
the linker by thrombin, and the binding of the C-terminal fragments was
measured together with N-terminal GST as a control (Fig.
1C). Folding of the purified C-terminal fragments was
analyzed by measurement of fluorescence and CD in the presence and
absence of urea. The fluorescence intensity of S2 at 350 nm started to
increase at 7 M urea, whereas that of S3a, S3b, and S3a3b
increased at 5 M urea. The latter proteins all gave similar curves (Fig. 2A). Because all
the proteins seemed to be unfolded in the presence of 9 M
urea, CD spectra were also recorded in this condition (Fig. 2,
B-E). In the absence of urea, the spectrum was
characteristic for the respective protein, but the uniqueness was lost
in the presence of urea. The molar ellipticity profile of S3a3b (Fig.
2E) correlated well to the averaged pattern for S3a (Fig.
2C) and S3b (Fig. 2D). Binding of monomeric (S3b)
and tandem (S3a3b) S3 to type I collagen were measured in various buffers. Neither addition of sodium chloride up to 1 M
(Fig. 3A) or alteration of the
pH within a range between 6.0 and 9.0 (Fig. 3B) affected
binding significantly. However, addition of 1 mM EGTA
instead of Ca2+ inhibited binding significantly (Fig.
3C). The effect of the chelator was reversed by the addition
of a 1 mM excess of Ca2+. The apparent
molecular mass of S3b was measured by size exclusion chromatography.
The value was 11.4 ± 0.12 kDa (mean ± S.D. of three
experiments) in the presence of 1 mM Ca2+ and
16.0 ± 0.08 kDa in the presence of 1 mM EGTA. The
hydrodynamic radius (RH) of S3b was 1.93 nm in
the presence of 1 mM Ca2+ as measured by
dynamic laser light scattering. This size corresponds to about 14.2 kDa, based on the standard size/mass relationship for globular
proteins. When Ca2+ was replaced with 1 mM
EGTA, the RH value and estimated molecular mass
were 2.16 nm and 18.7 kDa, respectively.
Binding to Tropocollagen Segments--
When collagen is treated
with pepsin, the nonhelical regions (telopeptides) are digested, giving
the triple-helical region named atelocollagen. Binding to commercially
available atelocollagen was measured to determine which region is
recognized by S3. The binding assay was carried out in the same
condition as with collagen using the basal protein mixture. Although
binding of S3b and S3a3b was observed, significant amounts of
atelocollagen
Various subcomponents of human type I collagen were coupled to
Sepharose beads, and the binding of S3b and S3a3b to these gels was
examined (Fig. 4, B and C). When the proteins
were incubated with the gel beads coupled with a synthetic peptide
corresponding to one of the four telopeptides present in human type I
collagen, S3b and S3a3b remained in the water phase (Fig.
4B, lanes 1F-4F). When the proteins were
incubated with beads coupled with a collagenous peptide,
(prolyl-hydroxyprolyl-glycine)10 [(POG)10],
S3b and S3a3b disappeared from the water phase (lane 6F).
The bound fraction was recovered by boiling the affinity gel in SDS
sample buffer (lane 6B). Although a small amount of BSA was
present in this fraction, a similar amount of the protein was also
visible in the fraction bound to Tris-blocked Sepharose beads used as a
control (lane 5B). A disaccharide,
2-O-
To examine if S3 specifically binds to the collagenous peptide, cleared
lysates were prepared from uninduced E. coli cells, which
express GST-S3a3b or GST-S3b. Each lysate containing 1 mg of protein in
CB buffer was incubated at 25 ± 1 °C for 30 min with 50 µl
of (POG)10-Sepharose beads, and the bound fractions were
examined by SDS-PAGE. In each fraction, one distinct band was present
that seemed to correspond to the respective GST fusion protein (Fig.
4D). Because each band showed a strong signal by Western
blotting and immunodetection using affinity-purified anti-S3b rabbit
antibody (data not shown), it was concluded that the bands were
GST-S3a3b and GST-S3b, respectively.
Finally, the binding of S3a3b and S3b to the collagenous peptide,
(POG)10, were examined in the same set of buffers as used for the collagen binding assay shown in Fig. 3, which was carried out
in the buffers with varying salt concentrations, varying pH values, and
varying Ca2+ concentrations. Their binding to this peptide
was similar to those against type I collagen (Fig.
5).
Binding to Various Peptides--
Various substrates related to
(POG)10 were immobilized on Sepharose beads, and their
binding activity to S3a3b and S3b was examined. First, the number of
repeats of the POG triplets was altered (Fig.
6A). The proteins bound to
(POG)8-Sepharose to a similar extent as to
(POG)10-Sepharose, but they bound poorly to
(POG)5-Sepharose. The better binding to the longer peptides was also true for prolyl-prolyl-glycine (PPG). Binding reached a
maximum level when the number of repeats was 8 (Fig. 6B).
The proteins did not bind to Sepharose beads coupled with any of the following 24-mer peptides; (PG)12, (PPPG)6,
all-D-(PPG)8, (APG)8, or
(PAG)8 (Fig. 6C, lanes 1-5) or to
gelatin-Sepharose beads (Fig. 6C, lane 6).
Estimation of Dissociation Constants--
The time course of
binding between S3b and a collagenous peptide was determined by surface
plasmon resonance (Fig. 7). The reaction
was fast enough to reach equilibrium within about 30 s. The
dissociation constant (KD) and corrected maximum response (cRUmax) were calculated by plotting the
equilibrium resonance values. KD and
cRUmax values for S3b were 5.47 × 10 Binding of the Recombinant ColG to the Peptide
Substrate--
Full-length ColG was purified from a recombinant
E. coli cell lysate (Fig.
8A, lane 1).
Because the synthetic peptides recognized by the CBD were expected to
be hydrolyzed by this full-length enzyme, we also prepared an enzyme
with a conservative mutation in the putative catalytic center located
in the catalytic domain (S1) (Fig. 8A, lane 2).
N-terminal amino acid sequences of the wild type and the mutated enzyme
were identical and matched the deduced sequence with the exception of
the removal of the initial methionine residue. Two extra residues,
Ala-Ser, were present at their N termini, compared with mature ColG.
The molecular masses measured by MALDI-TOF MS were 114,208 and 114,181 Da for the wild type and the mutated enzyme, respectively. The observed
values coincided well with the respective calculated values, 114,120.8 and 114,119.8 Da. The specific activity of recombinant collagenase against insoluble collagen was 881 ± 145 units/mg of protein, comparable to the value obtained for the authentic enzyme (826 ± 42 unit/mg of protein). The mutated enzyme, rColG(E414Q), had a low
level of hydrolytic activity (34.7 ± 9.6 unit/mg of protein) against insoluble collagen. The latter enzyme bound to
(POG)10-Sepharose beads but not to Tris-blocked beads (Fig.
8B). Positive binding of the mutated enzyme to
G(POG)8 peptide was also observed by surface plasmon
resonance. In this binding profile (Fig. 8C), however, a
slow binding reaction was present in addition to the fast binding
reaction. Due to this complexity, we could not estimate the
KD value for binding between full-length enzyme and the collagenous peptide.
We previously reported that a C-terminal fragment (S2b+S3) of
C. histolyticum class II collagenase (ColH) binds to
insoluble type I collagen (6). Recently, we cloned the structural gene for the class I enzyme (ColG) from the same strain, and found that S3
was tandemly repeated at its C terminus (4). Comparison of the
predicted amino acid sequences of these divergent collagenases allowed
us to precisely predict the boundary between each segment. Based on
these results, we produced various C-terminal segments of ColG as GST
fusion proteins. The results of semi-quantitative binding experiments
indicated that the S3 monomer is a functional collagen-binding domain
(CBD). Tandem S3 (S3a3b) bound to insoluble collagen more efficiently
than S3b, suggesting cooperative binding of the two functional domains
to this macromolecular substrate.
The fusion proteins were cleaved by thrombin, and the C-terminal
fragments were purified by affinity chromatography. Collagen-binding domain peptides, S3a, S3b, and S3a3b, all gave a Neither addition of salt up to 1 M or alteration of the pH
between 6 and 9 affected binding significantly, suggesting that ionic
interactions do not play a major role in the interaction between the
CBD and collagen. A plasma glycoprotein, von Willebrand factor,
mediates platelet adhesion by binding both to collagen in a damaged
blood vessel and to a glycoprotein on the platelet membrane. The A3
domain interacts with collagen in the perivascular tissue of damaged
cell walls. Based on a crystal structure analysis, Huizinga et
al. (17) suggested that binding is achieved primarily through
ionic interactions between negative charged residues on the domain and
positively charged residues in collagen. Elucidation of the precise
mode of CBD binding by structural methods is essential to understand
its mechanism.
The molecular mass of S3b estimated by size exclusion chromatography
and the value estimated by laser light scattering were the same in the
presence and absence of Ca2+. It was suggested that S3b is
a monomer in both conditions. The reduction of the Stokes radius and
hydrodynamic radius in the presence of Ca2+ was consistent
with observations for various Ca2+-binding proteins,
e.g. troponin C or calmodulin (18-20). Because ionic
interactions are not likely to play a major role in the binding, the
enhanced binding in the presence of Ca2+ may be due to a
conformational change through exposure of a hydrophobic site as occurs
in calmodulin (21, 22). The efficient binding of the CBD to collagen in
the presence of Ca2+ seems to be one of the reasons why
collagenase requires the cation for full activity (23).
C-terminal fragments of class I collagenase (S3b and S3a3b) bound to
various types of collagen.2
Because their amino acid sequences are more diverse in the
noncollagenous region (telopeptides) than in the collagenous region, it
was hypothesized that the binding site(s) is(are) present in the
collagenous region. This working hypothesis was supported by the facts
that CBD binds to atelocollagen but not to synthetic telopeptides. In
type I collagen, the collagenous region consists of three polypeptide chains each of which contains 338 Gly-X-Y peptide
units. Although various residues are present at the X and
Y positions, we initially used the representative
oligopeptide (POG)10 to mimic collagen, because this
triplet is the most frequent in type I collagen. Alternatively, the CBD
might recognize a disaccharide,
2-O- Among various triplets, Gly-Pro-Hyp is known to contribute the most to
the formation of a triple helix. The thermal stability of triple
helices has been reported for (POG)n peptides of various
length. The melting temperatures (Tm) are
<2 °C when n = 5, 44.5 °C when n = 8 (24), and 61 °C when n = 10 (25). Because we
carried out all the binding experiments at 25 ± 1 °C, it was
assumed that most of the (POG)5 molecules are in a random conformation, whereas most of the (POG)8 and
(POG)10 molecules are triple-helical. Binding was observed
when the peptides are supposed to be triple-helical. To see if these
results are due to steric hindrance, we immobilized the
(POG)5 and (POG)10 peptides on NHS-Sepharose
beads, which have a 9-atom spacer arm, and examined binding of CBD and
obtained essentially the same results (data not shown). The length of
the collagenous peptides estimated from x-ray crystallography are
longer than the measured diameter of S3b (3.86 nm), also suggesting
that CBD recognizes the conformation generated only when the number of
triplet reiteration is large enough (26). Another series of
oligopeptides, (PPG)n, were known to have a triple-helical
conformation when n is sufficiently large (27). When
n increased, the binding of S3 increased, and it seemed to
reach a maximum when n = 8. To exclude the possibility that binding is due to nonspecific hydrophobic interactions, binding to
various peptides that have similar sequences of the same length (24 residues) without the proper triple-helical conformation was examined.
Two peptides, (Pro-Gly)12 and
(Pro-Pro-Pro-Gly)6, lack triplets and, hence, the
triple-helical conformation despite their similar hydrophobicity. The
enantiomer of the original peptide, (D-Pro-D-Pro-Gly)8, should have a
triple-helical conformation but in the reverse orientation. Two similar
peptides, (APG)8, and (PAG)8 were assumed to be
in a random conformation at room temperature for the following reasons.
When only one triplet within a peptide containing eight POG triplets
(Tm = 44.5 °C) was replaced with PAG or APG, the
melting temperature decreased to 38.3 °C and 39.9 °C,
respectively (24). An extrapolation from these results suggests that
the melting temperature is well below room temperature when all eight
triplets are replaced with these less stable peptides (28). CBD bound
to none of these peptides, clearly indicating that binding is not due
to nonspecific hydrophobic interactions but rather depends on
the triple-helical conformation. This conclusion was also supported by
the lack of binding of CBD to gelatin, where the triple-helical
conformation is destroyed by heat treatment, but the primary structure
of collagen is preserved. The result is consistent with the previous
observation that collagen binding of a C-terminal fragment of class II
collagenase was not inhibited by gelatin (6). We carried out
quantitative analysis of the binding between the CBD and a collagenous
peptide with eight POG triplets by surface plasmon resonance. Monomeric
S3 (S3b) bound to this substrate with low but significant affinity. The
reaction was too fast to estimate association/dissociation rate
constants (kas/kdis) by
this method. This fast reaction might contribute to efficient recycling
of the enzyme. Tandem S3 (S3a3b) did not show cooperative binding when
the immobilized ligand density was low. "Bridging" might be
necessary for cooperative binding by two independent domains.
Alternatively, two domains might form a single continuous binding
surface. Analysis using a sensor chip on which a longer (>25 aa)
peptide is immobilized should give more insight.
A recombinant wild type collagenase, rColG, showed full activity
against insoluble collagen despite the two extra residues at its N
terminus. This suggested that the structures necessary for substrate
binding and hydrolysis are conserved in the recombinant enzyme.
However, it is likely that the collagenous peptide ligands are
hydrolyzed by its catalytic activity during the binding assay. Previously, we have reported a mutational analysis on the catalytic center of the C. histolyticum class II collagenase (ColH)
(7). As for this enzyme, conservative mutations in the putative
catalytic center, HEXXH, significantly affected its
catalytic activity without affecting its binding activity (6, 7). Thus,
it seems unlikely that the conservative mutation, E414Q, alters the
global conformation of the homologous enzyme. As expected, the mutated
full-length enzyme bound to a collagenous peptide immobilized on the
beads. However, surface plasmon resonance analysis revealed that the enzyme has a slow binding mechanism in addition to the fast one governed by CBD. Because S2 did not bind significantly to collagen, gelatin, (POG)5, or (POG)10 (data not shown),
it was suspected that the slow interaction occurs between the catalytic
domain (S1) and the substrate.
A simple model peptide, (POG)n has an amino acid sequence like
the collagenous region of various collagen molecules. We showed that
the peptide possesses the structural information that allows specific
binding of the CBD of a bacterial collagenase. Because actual collagen
molecules have diversity in the amino acid residues at the X
and Y positions, the CBD has a binding spectrum wide enough
to allow this diversity. This binding spectrum would contribute to the
wide substrate specificity of the bacterial enzymes, which is different
from the cleavage of specific collagens by eukaryotic matrix
metalloproteases (29). In the collagen biosynthetic process, a possible
chaperone protein, HSP47, transiently interacts with procollagen
molecules in the endoplasmic reticulum to regulate their folding and/or
modification (30, 31). Koide et al. (12) showed that HSP47
interacts with (PPG)n peptides but not with (POG)n
peptides. They also showed that prolyl 4-hydroxylase has the same
substrate preference based on prolyl 4-hydroxylation at the
Y position. Our results showed that the CBD of C. histolyticum class I collagenase lacks such selectivity.
Elucidation of the underlying molecular mechanism would give insights
into the evolution and structure-function relationships of various
collagen-binding proteins. From a practical view point, the binding of
CBD to atelocollagen is beneficial; i.e. it extends the
clinical applications of the drug delivery system we proposed
previously (8), because atelocollogen is one of the more widely used
biomaterials in clinical fields. To apply the system more generally,
however, it is necessary to replace the CBD with low molecular mimics
to reduce or eliminate its antigenicity. To achieve these
objectives we have begun attempts to make cocrystals between the CBD
and collagenous peptides.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
21M13 or M13 reverse dye primer (PerkinElmer Life Sciences) was
used for sequencing the constructs in the pT7Blue T-vector (Novagen,
Madison, WI). An ABI PRISM dye terminator cycle sequencing ready kit
with AmpliTaq DNA polymerase, FS (PerkinElmer Life Sciences), and pGEX
primers (Amersham Pharmacia Biotech, Uppsala, Sweden) were used for
sequencing the GST fusion constructs.
-competent cells were transformed with each ligation mixture by
electroporation. After the nucleotide sequence of each fusion gene was
confirmed, E. coli BL21 was transformed with the recombinant plasmids. Each clone was grown in 100 ml of 2YT-G medium to an optical
density at 600 nm of 2.5. Isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 0.1 mM, and cells were grown for
further 2 h. Purification of the fusion protein was performed with
a glutathione-Sepharose 4B column (bed volume, 0.5-ml; Amersham Pharmacia Biotech) as described by the manufacturer.
RI). The
colG fragment was inserted into the vector between
NheI and BamHI sites, and E. coli
BL21-CodonPlus(DE3)-RIL (Stratagene, La Jolla, CA) was transformed with
the resultant plasmid (pEG101). The cells were grown in 2 liter of LB
broth supplemented with 100 µg/ml ampicillin and 30 µg/ml
chloramphenicol to an optical density at 600 nm of 0.7. Isopropyl-
-D-thiogalactopyranoside was added to a final
concentration of 1 mM, and the cells were grown for 2 more
hours. Cells were harvested by centrifugation at 7000 × g for 15 min at 4 °C, suspended in 40 ml of CB buffer, and disrupted two times in a French pressure cell at 10,000 p.s.i. Ammonium sulfate was added to the cleared lysate to 60% saturation, and the precipitate was dissolved in 7 ml of 50 mM Tris-HCl
buffer (pH 7.5). The sample (approximately 8.5 ml) was applied to a
size exclusion column (Sephacryl S-200, 2.2 × 85 cm). Enzyme
activity in the eluate was monitored by the Azocoll assay as described previously (11). The pooled fraction was then applied to an ion
exchange column (Q-Sepharose, 2.5 × 5.5 cm) pre-equilibrated with
50 mM Tris-HCl (pH 7.5), and proteins were eluted with a 400-ml gradient of 0-0.5 M NaCl in 50 mM
Tris-HCl (pH 7.5). Activity eluted around 0.2 M NaCl. The
pooled fraction was dialyzed two times against 1 liter of 50 mM Tris-HCl (pH 7.5). It was divided into six aliquots, and
each was applied to an ion-exchange fast protein liquid chromatography
column (MonoQ, bed volume 1 ml, Amersham Pharmacia Biotech). The column
was pre-equilibrated with 50 mM Tris-HCl (pH 7.5) eluted
with a 20-ml linear gradient of the same buffer as described above. The
combined active fractions were divided into nine aliquots, and each was
applied to a size exclusion fast protein liquid chromatography column
(Superose 12 10/30, Amersham Pharmacia Biotech) pre-equilibrated with
10 mM Na-HEPES (pH 7.4), 150 mM NaCl, 1 mM CaCl2.
-globulins, 158 kDa; bovine serum albumin (BSA), 68 kDa;
chicken ovalbumin, 44 kDa; and cytochrome c, 12.5 kDa. The
measurements were carried out in triplicate.
1(I) N-telopeptide; SAGFDFSFLPQPPQEKAHDGGRYYRA (26 aa residues)
for
1(I) C-telopeptide; QYDGKGVGLGP (11 aa residues) for
2(I)
N-telopeptide; and GGGYDFGYDGDFYRA (15 aa residues) for
2(I)
C-telopeptide. These peptides were cleaved off from the resin,
deprotected by the supplier's standard procedure, and purified by
reversed phase chromatography on a Shimadzu HPLC system using a Tosoh
ODS-80TM column. The purity of the final peptide fractions
was greater than 95% estimated by the same HPLC system. Identity of
the peptides was confirmed by amino acid sequencing using an Applied
Biosystems 492 protein sequencer. Collagenous peptides,
(PPG)5, (PPG)10, (POG)5, and (POG)10, were purchased from Peptide Institute, Inc.
(Osaka, Japan). The synthesis and purification of the other collagenous
peptides were described elsewhere (12).
-D-Glucopyranosyl-D-galactose
(5 mg, Glc-Gal) was purchased from Toronto Research Chemicals (North
York, ON, Canada) and dissolved in 150 µl of 0.1 M NaOH
prior to use. The swollen gel beads (75 µl) were mixed with all the
carbohydrate solution. After the suspension was incubated at 37 °C
for 16 h with gentle mixing, the beads were washed with 300 µl
of 0.1 M NaOH and subsequently with 400 µl of 1 M ethanolamine-HCl (pH 8.0). The beads were suspended in
400 µl of 1 M ethanolamine-HCl (pH 8.0) at 45 °C for
10 h to block any remaining active groups. The gel was
sequentially washed alternatively with 0.4 ml each of 0.1 M
sodium acetate, 0.5 M NaCl (pH 4.5) and 0.1 M
Tris-HCl, 0.5 M NaCl (pH 8.0) three times. The beads were
washed with 0.4 ml of CB buffer three times and used for the binding assay.
-amino group following the supplier's protocol. To
minimize interaction between multiple ligands and a single protein
molecule ("bridging"), the peptide was used at a low concentration
(0.1 mg/ml) to achieve a low ligand density (432.5 RU). Resonance was
measured in 10 mM Na-HEPES (pH 7.4), 150 mM
NaCl, 1 mM CaCl2, 0.005% Tween 20 with a flow
rate of 20 µl/min at 25 °C. After each cycle the chip was
regenerated with a 180-s pulse of 0.1 M HCl. Values for
apparent dissociation constants, KD(app), were
calculated from equilibrium binding data at eight protein
concentrations (100 nM to 300 µM) for S3b,
and at eight protein concentrations (30 nM to 100 µM) for S3a3b. Data were directly fit using the following equation by least square method,
where cRU is the response at equilibrium corrected for bulk
refractive index errors using a sham-coupled flow cell blocked with
ethanolamine, [protein] is the analyte concentration, and KA is the association constant.
KD(app) is the reciprocal of
KA.
(Eq. 1)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Collagen binding of various C-terminal
fragments. A, segmental structure of the two C. histolyticum collagenases. HEXXH, putative catalytic
center. B, purified GST fusion proteins (2 µg each).
M, molecular mass markers; lane 1, GST-S2;
lane 2, GST-S3a; lane 3, GST-S3b; lane
4, GST-S3a3b. The samples were subjected to SDS-PAGE on a 12.5%
polyacrylamide gel. Numbers on the left are molecular masses (in kDa)
of the markers. C, collagen binding of the cleaved products
of various GST fusion proteins. The cleaved product (0.1 nmol) were
incubated at 25 ± 1 °C for 30 min in the absence ( ) or
presence (+) of 5 mg of insoluble type I collagen. The supernatants
were subjected to SDS-PAGE on a 15% polyacrylamide gel. M,
molecular mass markers; lanes 1, GST and S2; lanes
2, GST and S3a; lanes 3, GST and S3b; lanes
4, GST and S3a3b. Numbers on the left are
molecular masses (in kDa) of the markers. The position of GST is
indicated on the right.
Collagen-binding activities of fusion proteins carrying various
C-terminal segments of class I collagenase
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Fig. 2.
Fluorescence and CD. A,
proteins were incubated in CB buffer with increasing concentration of
urea, and the change in fluorescence intensity at 350 nm
(FI350) was measured to monitor denaturation. S2
(triangle), S3a (circle), S3b (closed
square), and S3a3b (open square). B-E, the
CD spectra were measured for native (thick lines) and 9 M urea treated (thin lines) proteins; S2
(B), S3a (C), S3b (D), and S3a3b
(E).
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Fig. 3.
Collagen binding of S3 in various
buffer. Two hundred picomoles each of S3a3b and S3b, and 5 µg
each of BSA, ovalbumin, and myoglobin in 100 µl were incubated at
25 ± 1 °C in the absence ( ) or presence (+) of 10 mg of
insoluble type I collagen in various buffers. A, varying
salt concentration. M, molecular mass markers; lanes
1, without NaCl; lanes 2, 0.1 M NaCl;
lanes 3, 0.5 M NaCl; lanes 4, 1 M NaCl. B, varying pH. M, molecular
mass markers; lanes 1, 0.1 M BisTris-HCl (pH
6.0); lanes 2, 0.1 M BisTris-HCl (pH 7.0);
lanes 3, 0.1 M Tris-HCl (pH 7.0); lanes
4, 0.1 M Tris-HCl (pH 8.0); lanes 5, 0.1 M Tris-HCl (pH 9.0). C, varying Ca2+
ion concentrations. M, molecular mass markers; lanes
1, with 1 mM CaCl2; lanes 2,
with 1 mM EGTA; lanes 3, with 1 mM
EGTA and 2 mM CaCl2. Numbers on the
left are molecular masses (in kDa) of the markers.
-chains were observed in the water phase (data not
shown). Thus, we pretreated atelocollagen with water-soluble
carbodiimide to cross-link the
-chains to prevent their release into
the water phase. This substrate showed binding to S3b and S3a3b, and
negligible amounts of
-chains were observed in the water phase (Fig.
4A).
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Fig. 4.
Binding of S3 to various type I collagen
segments. Two hundred picomoles each of S3a3b and S3b, and 5 µg
each of BSA, ovalbumin, and myoglobin in 100 µl were incubated at
25 ± 1 °C with various collagen segments (A-C).
A, atelocollagen. M, molecular mass markers; ,
in the absence; +, in the presence of atelocollagen. B,
various oligopeptide-coupled Sepharose beads. M, molecular
mass markers; C, control incubated in the absence of beads;
F, free fraction remained in the water phase; B,
fraction bound to the beads. Lane 1, N-telopeptide of
1(I); lane 2, C-telopeptide of
1(I); lane
3, N-telopeptide of
2(I); lane 4, C-telopeptide of
2(I); lanes 5, Tris (mock-coupled beads); lanes
6, (POG)10. C, Glc-Gal-Sepharose. The
samples in panels A-C were subjected to SDS-PAGE on a 15%
polyacrylamide gel. D, affinity protein binding from cleared
lysates using (POG)10-Sepharose. L, cleared lysate (10 µg
of protein); B, fraction bound to the beads. Lanes
1, GST-S3a3b; lanes 2, GST-S3b. Numbers on
the left are molecular masses (in kDa) of the markers.
-glucopyranosyl-D-galactopyranose, is
present in the triple-helical region at the hydroxylysine residues. We
coupled this disaccharide to the Sepharose beads, and S3 did not bind
(Fig. 4C).
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Fig. 5.
Binding of S3 to
(POG)10-Sepharose in various buffers. Two hundred
picomoles each of S3a3b and S3b, and 5 µg each of BSA, ovalbumin, and
myoglobin in 100 µl were incubated at 25 ± 1 °C with 50 µl
of (POG)10-Sepharose beads. For each binding experiment,
the following three samples are shown: C, control incubated
in the absence of the beads; F, free fraction in the water
phase; B, fraction bound to the beads. The buffers are the
same as in Fig. 3. A, varying salt concentration.
M, molecular mass markers; lanes 1, without NaCl;
lanes 2, 0.1 M NaCl; lanes 3, 0.5 M NaCl; lanes 4, 1 M NaCl.
B, varying pH. M, molecular mass markers;
lanes 1, 0.1 M BisTris-HCl (pH 6.0); lanes
2, 0.1 M BisTris-HCl (pH 7.0); lanes 3, 0.1 M Tris-HCl (pH 7.0); lanes 4, 0.1 M
Tris-HCl (pH 8.0); lanes 5, 0.1 M Tris-HCl (pH
9.0). C, varying Ca2+ ion concentration.
M, molecular mass markers; lanes 1, with 1 mM CaCl2; lanes 2, with 1 mM EGTA; lanes 3, with 1 mM EGTA and
2 mM CaCl2. Numbers on the
left are molecular masses (in kDa) of the markers.
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Fig. 6.
Binding of S3 to various peptide-coupled
beads. Two hundred picomoles each of S3a3b and S3b, and 5 µg
each of BSA, ovalbumin, and myoglobin in 100 µl were incubated at
25 ± 1 °C with 50 µl of various peptide-coupled Sepharose
beads. For each binding experiment, the following three samples are
shown: C, negative control incubated in the absence of the
beads; F, free fraction in the water phase; B,
fraction bound to the beads. A, (POG)n.
B, (PPG)n. C, lanes 1,
(PG)12; lanes 2, (PPPG)6;
lanes 3, all-D-(PPG)8; lanes
4, (APG)8; lanes 5, (PAG)8;
lanes 6, gelatin-Sepharose.
5
M and 2.34 × 103 RU, respectively.
Binding of S3a3b resulted in similar sensorgram profiles,
although the response was larger than that of S3b at the same
concentration (data not shown). KD and
cRUmax values for S3a3b were 6.30 × 10
5
M and 3.41 × 103 RU, respectively.
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Fig. 7.
Estimation of KD
value by surface plasmon resonance. A collagenous peptide,
G(POG)8, was immobilized on the surface of thin gold
membrane at a low density. S3b solution with varying concentration (100 nM to 300 µM) was injected at a time of
100 s, and the time course of the binding was observed.
Inset, equilibrium response values were plotted against
protein concentration, and fitted to the equation described under
"Experimental Procedures" by the least square method to calculate
KD and cRUmax values. Dissociation
constant values measured by surface plasmon resonance do not correspond
to values measured in solution, being affected by chemical modification
and/or immobilization by one of the reactants.
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Fig. 8.
Binding of a recombinant ColG to collagenous
peptides. A, purified recombinant ColG proteins (2 µg
each). M, molecular mass markers; lane 1, wild
type; lane 2, mutated collagenase, rColG(E414Q), in which a
glutamate residue in the putative catalytic center (Glu414)
was replaced with a glutamine residue. The samples were subjected to
SDS-PAGE on a 12.5% polyacrylamide gel. Numbers on the
left are molecular masses (in kDa) of the markers.
B, binding of rColG(E414Q) to ligand-coupled Sepharose
beads. M, molecular mass markers; C, control
incubated in the absence of the beads; F, free fraction
remained in the water phase; B, fraction bound to the beads.
Lanes 1, Tris-blocked; lanes 2,
(POG)10. C, sensorgram of the binding of
rColG(E414Q) to a collagenous peptide, G(POG)8. The
concentration of the analyte ranges from 1 nM to 30 µM. Slow binding reaction overlapped to the fast binding
reaction.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet CD spectrum. By using a k2d server available on the Web (14, 15), the
percentages of secondary structure were estimated as follows:
-helix
5%,
-sheet 47%, random 48% for S3a;
-helix 9%,
-sheet
44%, random 48% for S3b;
-helix 5%,
-sheet 47%, random 48%
for S3a3b. These percentages agree with a secondary structure
prediction carried out by using the PredictProtein (available on the
Web) (16). The CD spectrum of the tandem domain (S3a3b) resembles the
average of the S3a and S3b spectra, which suggests that the secondary structure of each monomer is conserved in the tandem domain. When treated with 9 M urea, all of these spectra became
characteristic of non-native proteins, suggesting that they are
unfolded in this solvent.
-glucopyranosyl-D-galactopyranosyl, group
attached to hydroxylysine residues in the polypeptides. Thus, we also
examined binding to immobilized Glc-Gal. S3 bound only to the
peptide-coupled beads, indicating that the domain recognizes the
polypeptides but not the disaccharide. In various buffers, CBD
interacted with the peptide similarly to insoluble type I collagen,
supporting the idea that the collagenous peptide chains are the major
or only binding site in collagen.
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ACKNOWLEDGEMENTS |
---|
We thank Kayoko Yamashita and Yuki Taniguchi for their technical assistance. We also thank David B. Wilson (Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY) for invaluable discussion and assistance in preparing the manuscript.
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FOOTNOTES |
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* This work was supported by a Grant-in Aid for Scientific Research (C) from Japan Society for the Promotion of Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) .
** Present address: Dept. of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima City 770-8506, Japan.
§ To whom correspondence should be addressed: Dept. of Microbiology, Kagawa Medical University, 1750-1 Ikenobe, Miki-cho, Kagawa 761-0793, Japan. Tel./Fax: 81-87-891-2129; E-mail: microbio@kms. ac.jp.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M003450200
2 T. Toyoshima, O. Matsushita, J. Minami, N. Nishi, A. Okabe, and F. Itano, manuscript submitted.
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ABBREVIATIONS |
---|
The abbreviations used are: ColG, C. histolyticum type I collagenase; ColH, C. histolyticum type II collagenase; CBD, collagen-binding domain; MALDI-TOF MS, matrix-assisted laser desorption time of flight mass spectrometry; CD, circular dichroism; HPLC, high performance liquid chromatography; BSA, bovine serum albumin; EDC, 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; aa, amino acid(s); GST, glutathione S-transferase; PCR, polymerase chain reaction; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; CB buffer, collagen binding buffer; Hyp and O, three-letter code and one-letter code of 4-hydroxy-L-proline residue.
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REFERENCES |
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