From the ¶ Shriners Hospital for Children and the
Department of Biochemistry and Molecular Biology, Oregon
Health Sciences University, Portland, Oregon 97201
Received for publication, March 20, 2001, and in revised form, April 27, 2001
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ABSTRACT |
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HSP47, a collagen-specific molecular chaperone,
interacts with unfolded and folded procollagens. Binding of
chicken HSP47 to native bovine type I collagen was studied by
fluorescence quenching and cooperative binding with a collagen
concentration at half saturation (Khalf) of
1.4 × 10 HSP47 (47-kDa heat shock protein) is a rough endoplasmic reticulum
protein believed to function as a collagen-specific molecular chaperone
(1-3). It was shown that HSP47 (also known as colligin, J6, gp46, and
CB48) is co-expressed with collagens in cells such as fibroblasts and
chondrocytes (4, 5). In mouse embryo development, HSP47 is expressed
mainly in mesoderm and mesoderm-derived tissues, and the expression
correlates both temporally and spatially with that of type I and II
collagen (6). HSP47 knockout mice show an embryonic lethal phenotype
(7). HSP47 is isolated easily by affinity chromatography on
gelatin-Sepharose (8, 9) and was later shown to interact also with
triple helical collagen types I-V (10). It was found that all these
collagens have a similar affinity for HSP47 with dissociation constants
of about 10 A number of potential functions for HSP47 during procollagen
biosynthesis have been described. HSP47 binds to nascent procollagen chains and therefore may help prevent the premature association of
procollagen chains and/or assist with the translocation of procollagen
chains into the rough endoplasmic reticulum (11). HSP47 also interacts
with procollagen chains in the rough endoplasmic reticulum when triple
helix formation is inhibited by Conflicting results were obtained with co-expression of HSP47 and
procollagens. In human embryonic kidney 293 cells, the secretion of
type III procollagen was delayed when HSP47 was co-expressed (15),
whereas an enhancement of the secretion of human type I procollagen was
found in mouse HSP47-expressing insect cells (16). The exact function
of HSP47 during procollagen biosynthesis has not been established.
The structural requirements for HSP47 binding to procollagens have been
studied recently using synthetic collagen-like peptides (17) and also
in a yeast two-hybrid system (18). It was found that the collagen-like
peptide (Gly-Pro-Pro)n possesses sufficient information to
bind to HSP47 and that hydroxylation of the peptide (Gly-Pro-Hyp)n prevents this interaction (17). By using the two-hybrid system in yeast it was shown that the (Gly-Pro-Pro) tripeptide unit is essential to binding and that HSP47 preferentially recognized the triple helical conformation in these peptides (18). Another study suggested that HSP47 interacts with and stabilizes correctly folded procollagen (19).
In this report we show that HSP47 binds triple helical type I collagen
cooperatively and with a much higher affinity than synthetic peptides.
We also explore the effect of HSP47 on the thermal stability and
refolding of type I and pN type III
collagen.1
Purification of Chicken HSP47--
The purification method was
based upon previously published protocols for the purification of HSP47
(9, 20) but has been modified to improve both the yield and purity of HSP47.
Preparation of a Crude Microsomal Extract--
After removal of
the heads, 6 dozen 15-17-day-old chick embryos were homogenized in an
equal volume of homogenization buffer (10 mM Tris/HCl, pH
7.5, containing 0.25 M sucrose, 5 mM EDTA, 2 mM each phenylmethylsulfonylfluoride and
N-ethylmaleimide, and 1 µg/ml each leupeptin and pepstatin
A) in an ice-cold Waring blender at full speed for 2 min in a 4 °C
cold room. All further steps were carried out at 4 °C. Large tissue
debris was removed by centrifugation of the homogenate at 5,000 × g in an H-6000A rotor (Sorval) for 15 min. The supernatant
from this step was then centrifuged at 150,000 × g in
a 45 TI rotor (Beckman) for 1-2 h. The pelleted material from this
step was transferred to a plastic specimen container and frozen at
Gelatin Affinity Chromatography of Crude Microsomal
Extract--
Using a peristaltic pump, the crude microsomal extract
(~400 ml) was loaded at 0.5-1.0 ml/min onto a 200-ml column (XK50, 5.0 × 10.1 cm) of gelatin-Sepharose 4B (Amersham Pharmacia
Biotech) equilibrated with gelatin-Sepharose buffer A (50 mM Tris/HCl, pH 7.5, containing 0.2 M NaCl,
0.1% Tween 20, and 0.2 mM phenylmethylsulfonylfluoride). After loading the extract, the column was washed with 200 ml
gelatin-Sepharose buffer B (50 mM Tris/HCl, pH 7.5, containing 1.0 M NaCl, 0.1% Tween 20, 0.2 mM
phenylmethylsulfonylfluoride) and re-equilibrated with 400 ml of
gelatin-Sepharose buffer A. Gelatin-binding proteins were eluted with a
200-ml pH gradient from 7.5 to 5.0 of gelatin-Sepharose Buffer A
followed by 200 ml of gelatin-Sepharose buffer A, pH 7.5, and collected
in 10-ml fractions. The protein-containing fractions (identified by
SDS-polyacrylamide gel electrophoresis) were pooled, neutralized with
Tris base to pH 7.5, concentrated to 45 ml, and dialyzed overnight
against 1 liter of monoQ buffer A (20 mM
triethanolamine/HCl, pH 7.0, containing 80 mM NaCl and 0.1 mM 4-(2-aminoethyl)-benzolsulfonylfluoride.)
MonoQ Anion Exchange Chromatography--
The dialyzed
gelatin-Sepharose pH eluate was loaded into a 50-ml fast protein liquid
chromatography Superloop (Amersham Pharmacia Biotech), run over a monoQ
HR 5/5 column, washed with 25 ml of monoQ buffer A, and collected in
5-ml fractions. The flow-through fractions were highly enriched with
the protein HSP47 but also contained some of the endoplasmic
reticulum-resident cyclophilin B. Bound proteins were eluted
with a 40-ml gradient of 0-100% monoQ buffer B (20 mM
triethanolamine/HCl, pH 7.0, containing 0.8 M NaCl),
followed by 5 ml of 100% monoQ buffer B, and collected in 1-ml fractions.
Molecular Sieve Chromatography of Gelatin-binding
Proteins--
The monoQ flow-through fractions were pooled and
concentrated to 6-8 ml using Centricon-10 concentrators (Amicon)
according to manufacturer recommendations. The HSP47 and cyclophilin B
were separated in runs of 2 ml each by chromatography in
gelatin-Sepharose buffer A over two tandemly arranged Superose 12 HR
16/50 columns (Amersham Pharmacia Biotech) and collected in 2-ml
fractions. After SDS-polyacrylamide gel electrophoresis, the peak
fractions containing HSP47 were pooled, concentrated to about 1 mg/ml,
and stored on ice for later analysis.
Other Materials--
Type I and pN type III collagens were
extracted from fetal bovine skin. Type II collagen was extracted from
fetal bovine cartilage. (Pro-Pro-Gly)10 and
(Pro-Hyp-Gly)10 was purchased from the Peptide Institute,
Inc. (Osaka, Japan), and (Pro-Gly-Pro)n was from
Sigma-Aldrich. (Pro-Gly-Pro)n (average molecular
mass of about 10 kDa, which corresponds to an average chain
length of n = 40) was fractionated on a Sephadex 75 column and divided into two pools with a Kav of
0.41 and 0.66, respectively.
Fluorescence-based Collagen Binding Assays--
The measurement
of fluorescence spectra was performed on an SLM8000C instrument (SLM
Instruments, Inc., Urbana, IL) using the software provided by the
manufacturer. For most experiments the excitation wavelength was 275 nm
(4-nm bandwidth), but in some cases excitation was at 295 nm. Emission
scans were obtained over the wavelength range of 300-400 nm (2-nm
bandwidth) for excitation at 275 nm or 310-410 nm for excitation at
295 nm. In most of the binding assays, stock solutions of bovine
collagens (0.5-2.0 mg/ml) were added to a solution of tissue-purified
chicken HSP47 (0.05-0.2 mg/ml) in 50 mM Tris/HCl, pH 7.5, containing 0.2 M NaCl in a 1 × 1-cm cell in a
thermostatted block at 25 °C with constant stirring. A constant
temperature was maintained by a circulating water bath (RCS, Lauda
Division, Brinkmann Instruments). After each addition of substrate, the
samples were equilibrated for 2 min before emission spectra were
acquired. In some experiments recombinant mouse HSP47 was used instead
of chicken HSP47. Analysis of the binding data was done by subtracting
the emission of the collagens (this is a very small correction) and
correcting for dilution.
Collagen Thermal Stability and Refolding Monitored by Optical
Rotary Dispersion--
The thermal stability and refolding of bovine
type I and pN type III collagen was monitored at 365 nm using a 241MC
polarimeter (Perkin-Elmer) with a 10-cm path-length thermostatted cell.
The temperature was controlled by two circulating water baths (RCS, Lauda Division, Brinkmann Instruments) and measured with a digital thermometer (Omega Engineering, Inc., Stamford, CT) and a thermistor inserted into the cell. Both the temperature and the ORD signals were
recorded and digitized on an HP9070A measurement and plotting system
(Hewlett-Packard) connected to an IBM-compatible computer. Protein
concentrations were determined by amino acid analysis.
Chicken HSP47 was extracted and purified from chick embryos, and
mouse HSP47 was expressed in Escherichia coli. Fig.
1A shows a 5-20%
SDS-polyacrylamide gel of purified chicken HSP47. The final yield of
highly purified HSP47 from 6 dozen chick embryos was 4-8 mg. Fig.
1B shows a 10% SDS-polyacrylamide gel of purified mouse
HSP47 expressed in E. coli. The yield of purified and
refolded mouse HSP47 was about 30 mg from a 500-ml culture. Binding of HSP47 to type I collagen was measured by observing the tryptophane fluorescence of HSP47 that is quenched by the addition of type I
collagen. These measurements are facilitated by the fact that type I
collagen and collagen-like peptides do not contain tryptophane. Fig.
2 shows the change in fluorescence as a
function of added type I collagen. A sigmoidal curve is observed,
indicating cooperative binding. The data were analyzed using the Hill
equation. For the binding of chicken HSP47 to type I collagen, a
Khalf of 1.4 × 107 M, and a Hill coefficient of
4.3 was observed. Similar results are observed for the binding of mouse
HSP47 recombinantly expressed in Escherichia coli. Chicken
HSP47 binds equally well to native type II and type III procollagen
without the carboxyl-terminal propeptide (pN type III collagen),
but binding to triple helical collagen-like peptides is much weaker.
Weak binding occurred to both hydroxylated and nonhydroxylated
collagen-like peptides, and a significant chain length dependence was
observed. Binding of HSP47 to native type I collagen had no effect on
the thermal stability of the triple helix. Refolding of type I collagen
in the presence of HSP47 showed minor changes, but these are probably not biologically significant. Binding of HSP47 to bovine pN type III
collagen has only minor effects on the thermal stability of the triple
helix and does not influence the refolding kinetics of the triple helix.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7 M (10). The interaction of HSP47
with both unfolded and folded procollagens is unusual and distinguishes
HSP47 from other molecular chaperones.
,
'-dipyridyl, an inhibitor of
prolyl-4-hydroxylase (9). A potential role in vesicular trafficking was
explored with Brefeldin A and monensin (9, 12, 13). In Brefeldin
A-treated cells HSP47 remained associated with procollagen, whereas in
cells treated with monensin no HSP47 binding was detected. Together
this shows that HSP47 seems to release procollagen on entry to the
Golgi. Procollagens are not secreted as individual molecules, but
rather are secreted in aggregates by a cisternal maturation pathway
(14), and HSP47 could potentially be involved in this process.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C for storage until later purification steps could be carried
out. After four such microsomal pellets had been prepared and stored,
they were thawed together in an equal volume of extraction buffer (50 mM Tris/HCl, pH 7.5, containing 0.2 M NaCl, 1%
Triton X-100, and all the previously used protease inhibitors). After
the pellets had completely thawed, 0.5-1.0 µl of diisopropyl
fluorophosphate/ml of extract was added, and this solution was stirred
vigorously for 4 h on ice. The final microsomal extract was
obtained by centrifugation of this solution at 150,000 × g in a 45 TI rotor (Beckman) for 1-2 h.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7
M and a Hill coefficient P of 4.3 were found (Fig.
2A). For recombinantly expressed mouse HSP47 binding to type
I collagen, a Khalf of 1.1 × 10
7 M and a Hill coefficient P of 3.2 were
determined (Fig. 2B).
View larger version (23K):
[in a new window]
Fig. 1.
SDS-polyacrylamide gels of purified
HSP47. The final purity of HSP47 was assessed by
SDS-polyacrylamide gel electrophoresis. A, the purity of
chicken HSP47 is shown by electrophoresis on a 5-20%
SDS-polyacrylamide gel in lane 1. Lane 2 shows
molecular mass markers, the molecular masses of which (in kDa) are
indicated on the right. B, the purity of
recombinant mouse HSP47 is shown by electrophoresis on a 10%
SDS-polyacrylamide gel in lane 1. Lane 2 shows
molecular mass markers, the molecular masses of which (in kDa) are
indicated on the right
View larger version (14K):
[in a new window]
Fig. 2.
Binding of HSP47 to native type I
collagen. The fluorescence of HSP47 was monitored at 340 nm, and
the quenching observed by binding to type I collagen is plotted against
the type I collagen concentration. A, binding of chicken
HSP47; B, binding of recombinant mouse HSP47. The
curves represent best fits to the equation F = Fmax
CP/(KhalfP + CP), where F is the fluorescence of
HSP47 at concentration C of type I collagen,
Fmax is the fluorescence of HSP47 at maximum
quenching, Khalf is the collagen concentration
at half saturation, and P is the Hill cooperativity parameter.
To determine the structural determinants of the HSP47 binding to the
triple helix, binding studies using different collagens and
collagen-like peptides were done using the same technique. Fig.
3 shows the fluorescence quenching of
chicken HSP47 by type I collagen, type II collagen, and pN type III
collagen. All three collagens seem to bind about equally to HSP47.
However, binding of collagen-like peptides is much weaker. Again, a
cooperative effect is observed when the high and low molecular weight
fractions of (Pro-Gly-Pro)n are compared; the higher
molecular weight fraction shows better binding to HSP47. Binding of the
classical collagen-like peptides (Gly-Pro-Pro)10 and
(Gly-Pro-Hyp)10 to HSP47 is very weak, and these peptides
seem to be a poor choice for determining a change in binding between
hydroxylated and nonhydroxylated triple helices.
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Binding of HSP47 to collagen could influence the properties of the
triple helix. The effect of HSP47 on the thermal stability of type I
and pN type III collagen was studied. Because the interaction of HSP47
with collagen is abolished at pH 5, and collagens tend to form fibrils
at neutral pH and elevated temperatures, measurements were done in 50 mM Tris/HCl buffer, pH 7.5, containing 0.4 M
NaCl. Fig. 4A shows that the
thermal stability of type I collagen is not affected by the presence of
HSP47. There is a small effect on the melting behavior of pN type III
collagen. We have observed previously that pN type III collagen has a
tendency to form aggregates even in the high salt buffer. This is
evident from a fraction of higher melting material. In the presence of
HSP47, this aggregation is not observed, and the melting curve is
slightly shifted to lower temperatures (Fig. 4B).
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Because HSP47 interacts with both folded and unfolded triple helices,
the refolding of type I collagen in Tris/HCl buffer, pH 7.5, containing
0.15 M NaCl was measured. The type I collagen was denatured
for 15 min at 45 °C, and refolding was observed at 25 °C. Type I
has been shown previously to refold very poorly, and also in these
experiments we only get less than 30% of the initial signal back after
90 min of refolding. In the presence of HSP47, the recovered signal is
slightly higher (Fig. 5A).
More remarkable is the change from an exponential kinetic to a
zero-order kinetic typically observed for the refolding of type III
collagen (21). However, these changes are quite small. Refolding of pN type III collagen proceeds with an initial zero-order reaction and
reaches at least 90% of the initial signal. The initial rate was
independent of the presence or absence of HSP47 (Fig.
5B).
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DISCUSSION |
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Binding of HSP47 to native type I-V collagens was investigated
previously by surface plasmon resonance measurements. A dissociation constant of 1.1 × 106 M was determined
for the binding of recombinantly expressed mouse HSP47 to porcine type
I collagen (10). This dissociation constant results from a relatively
high association rate of 2.08 × 104
M
1 s
1 and a rapid dissociation
rate of 2.36 × 10
2 s
1. No
cooperativity in binding was reported. The Khalf
determined here by fluorescence quenching is about an order of
magnitude smaller (1.4 × 10
7), and binding is
cooperative with a Hill coefficient of 4.3. Cooperative binding is
consistent with the finding that a strong chain length dependence is
observed for (Pro-Gly-Pro)n. The fraction with a
Kav of 0.41, corresponding to a greater chain
length than the fraction with a Kav of 0.66, binds HSP47 better. On a per tripeptide unit basis, increased
fluorescence quenching is observed going from
(Pro-Pro-Gly)10 to larger (Pro-Gly-Pro)n
polypeptides. However, the native collagens type I and pN III bind
HSP47 much stronger. Type I collagen (Gly-Xaa-Yaa)336
quenches the HSP47 fluorescence much more than even the longest
collagen-like peptides measured. The reason for this can be that there
is still a chain length effect or that HSP47 prefers other tripeptide
units over (Gly-Pro-Pro). Binding of (Gly-Pro-Pro)10 and
(Gly-Pro-Hyp)10 to HSP47 is weak, and not much difference
in fluorescence quenching is observed. It was proposed recently that
HSP47 binds to (Pro-Pro-Gly)10 but not to
(Pro-Hyp-Gly)10 and that this is a possible mechanism for
the release of HSP47 from collagen (17). The collagens measured here
are extracted from tissues and believed to be fully hydroxylated. Because HSP47 binds these collagens well, the proposed mechanism seems unlikely.
Because HSP47 binds to folded collagens, there is the potential that HSP47 modifies the properties of the collagen triple helix. The stability of type I collagen in the presence and absence of HSP47 shows no differences. It was shown previously that the interaction of HSP47 with collagen is strongly pH-dependent, with binding being abolished below pH 6.3 (9). Measuring the stability of the collagen triple helix around a neutral pH is difficult, because collagens tend to form fibrils at that pH and elevated temperatures. Aggregation has been observed previously with pN type III collagen, but these aggregates can be minimized by the addition of 0.4 M NaCl. Interestingly, during the measurement of the stability of pN type III collagen in the presence of HSP47, no aggregation was observed, whereas in the absence of HSP47, some higher melting aggregates were found under the same conditions. Thus binding of HSP47 influences the aggregation behavior of collagens, and this was shown in a recent paper (22).
Because HSP47 binds both unfolded and folded collagens, we tested the effect on the rate of refolding of the triple helix of type I and pN type III collagen. The folding mechanism for pN type III collagen has been described (21), and HSP47 does not have an effect on the rate of folding. In refolding experiments with type I collagen, HSP47 binding seems to provide a nucleation site for refolding. However, the increase in refolding is probably not biologically relevant.
From these studies, we conclude that HSP47 preferentially binds to
folded triple helices but does not influence the stability and folding
of the collagen triple helix. Therefore, it seems likely that HSP47 is
involved in the segregation of procollagen to the cisternal maturation pathway.
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ACKNOWLEDGEMENTS |
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We thank Diane Mechling, Bruce Boswell, Jay Gambee, and Bruce Donaldson for expert technical assistance.
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FOOTNOTES |
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* This work was supported by a grant from Shriners Hospitals.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.
§ Present address: Dept. of Biochemistry, Oregon State University, Corvallis, OR 97331.
To whom correspondence should be addressed: Shriners
Hospital for Children, Research Unit, 3101 S.W. Sam Jackson Park Rd., Portland, OR 97201. Tel.: 503-221-3433; Fax: 503-221-3451; E-mail: hpb@shcc.org.
Published, JBC Papers in Press, May 1, 2001, DOI 10.1074/jbc.M102471200
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ABBREVIATIONS |
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The abbreviations used are: pN type III collagen, type III procollagen without the carboxyl-terminal propeptide; Hyp, 4-hydroxy-L-proline.
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