Mutations in Cartilage Oligomeric Matrix Protein Causing
Pseudoachondroplasia and Multiple Epiphyseal Dysplasia Affect Binding
of Calcium and Collagen I, II, and IX*
Jochen
Thur
§,
Krisztina
Rosenberg¶
,
D. Patric
Nitsche
§,
Tero
Pihlajamaa**,
Leena
Ala-Kokko**,
Dick
Heinegård¶

,
Mats
Paulsson
§, and
Patrik
Maurer
§§§
From the
Institute for Biochemistry, Medical Faculty,
University of Cologne, D-50931 Köln, Germany,
¶ Department of Cell and Molecular Biology, Section for Connective
Tissue Biology, Lund University, S-22100 Lund, Sweden, and
** Collagen Research Unit, Biocenter and Department of Medical
Biochemistry, University of Oulu, FIN-90014 Oulu, Finland
Received for publication, October 18, 2000, and in revised form, November 13, 2000
 |
ABSTRACT |
Mutations in type 3 repeats of cartilage
oligomeric matrix protein (COMP) cause two skeletal dysplasias,
pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia
(MED). We expressed recombinant wild-type COMP that showed structural
and functional properties identical to COMP isolated from cartilage. A
fragment encompassing the eight type 3 repeats binds 14 calcium ions
with moderate affinity and high cooperativity and presumably forms one
large disulfide-bonded folding unit. A recombinant PSACH mutant COMP in
which Asp-469 was deleted (D469
) and a MED mutant COMP in which
Asp-361 was substituted by Tyr (D361Y) were both secreted into the cell
culture medium of human cells. Circular dichroism spectroscopy revealed
only small changes in the secondary structures of D469
and D361Y,
demonstrating that the mutations do not dramatically affect the folding
and stability of COMP. However, the local conformations of the type 3 repeats were disturbed, and the number of bound calcium ions was
reduced to 10 and 8, respectively. In addition to collagen I and II,
collagen IX also binds to COMP with high affinity. The PSACH and MED
mutations reduce the binding to collagens I, II, and IX and result in
an altered zinc dependence. These interactions may contribute to the
development of the patient phenotypes and may explain why MED can also
be caused by mutations in collagen IX genes.
 |
INTRODUCTION |
Pseudoachondroplasia
(PSACH)1 and multiple
epiphyseal dysplasia (MED) constitute two autosomal dominantly
inherited forms of osteochondrodysplasias characterized by severe to
moderate short limb dwarfism with normal skull. Their clinical features
include pronounced joint laxity and limitation of joint movement. The major clinical complication is caused by premature osteoarthritis of
the weight-bearing joints (1). Both syndromes exhibit considerable clinical heterogeneity and show a broad phenotypical overlap with PSACH
at the severe end of the disease spectrum and MED at the mild end of
the disease spectrum (2, 3). Genetic linkage to chromosome 19 was
recently demonstrated for both mild and severe forms and was soon
followed by the identification of mutations in cartilage oligomeric
matrix protein (COMP) that cause both PSACH and MED (4, 5).
COMP is a secreted pentameric glycoprotein built from modular units and
belongs to the thrombospondin protein family (6-8). A coiled-coil
domain at the amino terminus mediates the pentamerization resulting in
a bouquet-shaped subunit arrangement (7, 9). It is followed by four
epidermal growth factor (EGF)-like domains, a region encompassing eight
so-called thrombospondin type 3 (T3) repeats and a carboxyl-terminal
globular domain. COMP is found in cartilage, ligaments, and tendons (6,
10). Cells from synovial tissue express COMP in culture (11-13).
Although COMP release to synovial fluid and blood plasma can serve as a
diagnostic marker in early rheumatoid arthritis and osteoarthritis (14, 15) and increases after traumatic injury (16-19), its precise function
is not established. The binding of COMP to collagens I and II with high
affinity provides indications for structural roles (20).
All mutations in the COMP gene, which are known to cause PSACH
or MED, result in either a single amino acid substitution, a small
deletion, or an insertion of one or two amino acids (4, 5, 21-32).
Thus far, no premature stop codon has been found. Most (44 of 48) known
mutations in COMP affect residues in the T3 repeats, whereas 4 mutations are located in the carboxyl-terminal domain. 72% of the
mutations affect acidic residues. For thrombospondin-1, it was
postulated that the region encompassing the T3 repeats is responsible
for the binding of 13 calcium ions (33). By analogy, it was speculated
that the mutations of acidic residues in the T3 repeats may disturb the
calcium binding, folding, and stability of COMP. Large inclusions in
the endoplasmic reticulum of PSACH and MED patient chondrocytes contain
COMP, collagen IX, and aggrecan and point to a destabilizing effect of
the mutations. However, the inclusions were found only in chondrocytes
and not in tenocytes and ligament cells (24, 34-36).
MED also shows a genetical heterogeneity. This clinically defined
entity is caused not only by mutations in COMP but also by mutations in
the COL9A2 and the COL9A3 genes that encode the
2 and
3 chains of
collagen IX, respectively (37-41). The mutations cause skipping of
exon 3 for both chains and lead to collagenous domains with in-frame
deletions of 12 amino acids. Thus far, no mutations have been reported
for the
1 chain of collagen IX that complements the
2 and
3
chains in the heterotrimeric nonfibrillar collagen IX (42, 43).
Collagen IX is a structural component of cartilage and was suggested to
play a stabilizing role in collagen fibrils (44). The findings that
mice expressing
1(IX) collagen chains with a central deletion
develop a mild dwarfism and osteoarthritis (45) and that mice lacking
collagen IX develop late-onset osteoarthritis (46, 47) supported this role.
To understand the consequences of mutations in COMP that cause PSACH
and MED, we used recombinant COMP and several fragments thereof to
demonstrate that the T3 repeats are responsible for calcium binding and
that calcium binding is affected by the mutations. We also show that
mutated COMP is still able to bind to collagens, but with altered zinc
dependence. The fact that COMP also binds to collagen IX and that this
binding is interfered with by the mutations points to a common
mechanism for the development of phenotypes in patients affected
in either the COMP or collagen IX gene.
 |
MATERIALS AND METHODS |
Recombinant Expression of COMP in 293 Cells--
The cDNA
clone coding for full-length COMP (8) was a kind gift of Å. Oldberg
(Lund University, Lund, Sweden) and served as the template for
PCR amplification of the different cDNA fragments. The cDNA
coding for COMP lacking the signal peptide (rCOMP) was amplified
according to standard protocols with Expand Polymerase (Roche
Diagnostics) with primer 1 (5'-GCCCGCTAGCCCAGGGCCAGATCCCGC-3') and primer M1 (5'-CAATGACTGCGGCCGCCTAGGCCCTCCGCAG-3'). The EGF-T3 cDNA encoding residues 227-524 was generated with primer 3 (5'-GCCCGCTAGCCCACTTCTGCCCCGACG-3') and primer M2
(5'-CAATGACTGCGGCCGCTTAGGCGTTCTCGGGGCAC-3'), and the T3 cDNA
encoding residues 268-524 was amplified with primer 4 (5'-GCCCGCTAGCCCGCGACACAGACCTGG-3') and primer M2. The PCR
products were cloned in pUC18 linearized with EcoRV.
Excision of the NheI/NotI fragments and ligation
into the pCEP-Pu expression vector (48) linearized with
NheI/NotI joined the 5' end of the cDNA to
the 3' end of the coding sequence for the BM40 signal peptide. T3-D361Y cDNA was generated by PCR using primer 4 and mutagenesis primer 361m (5'-GGTCTGTATACTTCTGGTCATCATTC-3'). A separate reaction was performed with primer M2 and primer 361c
(5'-GACCAGAAGTATACAGACCGGGATG-3'). These PCR products served as the
template for a final PCR with primer 4 and primer M2 to amplify
T3-D361Y cDNA that was ligated into the pCEP-Pu. T3-D469
cDNA was generated using the combination of mutagenesis primer 469m
(5'-GTCGTCGTCATCGCATGCATCACCCTT-3') with primer 4 and primer 469c
(5'-GGTGATGCATGCGATGACGACGAC-3') with primer M2. Additional silent
mutations created a novel Bst1107I in T3-D361Y and a new
SphI site in T3-D469
to identify mutated clones. D361Y
and D469
cDNA clones were constructed by digesting T3-D361Y and
T3-D469
, respectively, with ClaI/BstBI and
ligating these fragments into the
ClaI/BstBI-digested rCOMP cDNA. Correct sequences of all clones were confirmed by sequencing (ABI Prism 377 DNA
Sequencer; Applied Biosystems).
For stable episomal expression, 5 × 105 human
embryonic kidney cells (293-EBNA; Invitrogen) were
electroporated with 3 µg of purified plasmid. The transfected cells
were selected with 1 µg/ml puromycin and grown to confluence in
Dulbecco's modified Eagle's medium:Ham's F-12 with 10% fetal calf
serum (Life Technologies, Inc.). Secretion of recombinant proteins into
the medium was confirmed by SDS-PAGE of samples from transfected and
nontransfected cells. Serum-free culture medium was harvested,
supplemented with 0.5 mM phenylmethylsulfonyl fluoride and
25 mM Tris-HCl to give a pH of 8.6, and filtered through
Sephadex G-25 (Amersham Pharmacia Biotech). This medium was applied to
a Q-Sepharose FastFlow ion-exchange column (Amersham Pharmacia
Biotech) equilibrated in 50 mM NaCl, 0.5 mM
phenylmethylsulfonyl fluoride, 50 mM Tris-HCl, pH 8.6, and
eluted with a gradient of 50-1000 mM NaCl in the same
buffer. COMP-containing fractions were subjected to gel filtration on Sepharose CL-6B (Amersham Pharmacia Biotech) equilibrated in 50 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride,
and 50 mM Tris-HCl, pH 8.6. Final purification and
concentration were achieved by ion-exchange chromatography on a
Resource Q or a Source Q column (Amersham Pharmacia Biotech) using
buffers as described in the first ion-exchange chromatography. The
buffer of the purified sample was changed to 5 mM Tris-HCl,
pH 7.4, on a PD10 column (Amersham Pharmacia Biotech), and aliquots
were stored at
80 °C. The purity and size of all proteins were
assessed by SDS-PAGE with or without prior reduction of disulfide
bonds. Amino-terminal sequencing on an ABI473A/476A Sequencer
(PerkinElmer Life Sciences) confirmed the identity of the proteins.
Solid-phase Ligand Binding Assays--
Collagen I and II were
purchased from Sigma. Collagen I had been isolated after pepsin
digestion from bovine dermal skin, and collagen II had been isolated
from bovine nasal septum. Each collagen was dissolved in 0.5 M acetic acid. Recombinant collagen IX was produced as
described previously (49). Ligand binding studies were performed in
solid-phase assays with collagens (2 µg/ml in 0.18 M
acetic acid) coated to 96-well plates (Maxisorb; Nunc) overnight at
20 °C. After washing with 0.05% Tween 20 in 50 mM Tris,
pH 7.4, 150 mM NaCl (TBS), the wells were blocked for
1.5 h with 10 mg/ml bovine serum albumin in TBS at room
temperature. After washing the wells were incubated for 1 h at
room temperature with ligands tCOMP, rCOMP, D361Y, and D469
diluted
in TBS containing 0.5 mM ZnCl2. The ligands
were used at different concentrations for the determination of
affinity. Nonspecific binding was controlled for each ligand
concentration in wells lacking a collagen coat but otherwise treated
identically. In separate experiments, the zinc-dependent
binding was measured with ligands (5 nM) diluted in TBS
containing zinc at different concentrations. Also the washing buffers
were supplemented with ZnCl2. Bound tCOMP, rCOMP, D361Y, and D469D were detected using a polyclonal antiserum against COMP (50)
followed by peroxidase-coupled swine anti-rabbit IgG (DAKO) using
tetramethylbenzidine as chromogenic substrate, and absorbance was
recorded at 450 nm. Identical titers of the antibodies against wild-type and mutant COMP were confirmed in enzyme-linked immunosorbent assay titrations in which wild-type and mutant COMP were coated onto
plastic and detected with different dilutions of the polyclonal antiserum.
Surface Plasmon Resonance Assay--
The BIAcore 2000 system
(BIAcore, Uppsala, Sweden) was used to characterize the
interaction between wild-type and mutant COMP and collagens.
Immobilization of tCOMP, rCOMP, D361Y, and D469
to the
carboxymethylated matrix of the CM5 sensor chip was performed as
described previously (20). The immobilization resulted in 9,100 resonance units for tCOMP, 4,600 resonance units for rCOMP, 3,300-3,600 resonance units for D361Y, and 3,400-5,200 resonance units for D469
. A blank surface was used as control. Binding was
determined at different concentrations of collagen I (2-136 nM) and collagen IX (11-88 nM). The collagens
were added to 20 mM HEPES, pH 7.4, 150 mM NaCl,
0.005% Tween 20 containing 0.05 mM zinc chloride before
injection to avoid fiber formation. A flow rate of 80 µl/min was
used. BIAevaluation software version 3.0 was used to calculate
Kd of the binding. Only curves in which the
association resulted in a signal response of more than 10 resonance
units were used for calculations.
CD Analysis--
CD spectra in the far UV region were recorded
in a thermostated quartz cell of 1 mm optical path length in a JASCO
715 CD spectropolarimeter at 25 °C. Spectra were measured with the
proteins dissolved in TBS (less than 5 µM
Ca2+) and after the addition of 0.25 and 1.5 mM
CaCl2. Reversal of the conformational change was examined
after the addition of 4 mM EDTA to the
Ca2+-containing samples. Mean molar ellipticities [
]
(expressed in degrees cm2 dmol
1)
were calculated on the basis of a mean residue molecular mass of 110 Da. Five spectra were accumulated to improve the signal/noise ratio,
and the spectra of buffers were subtracted. The percentage change in CD
at 215 nm was calculated as
[
]215 = 100 × ([
]Ca
[
]EDTA)/ [
]Ca, with
[
]Ca representing the signal at Ca2+
saturation, and [
]EDTA representing the CD signal in
the presence of excess EDTA. Calcium titrations were performed by the
addition of small aliquots of CaCl2 stock solution followed
by incubation for 5 min and recording of the CD signal at 215 nm or
recording of spectra. Secondary structure analysis was performed using
the variable selection method SelCon (51).
Fluorimetry and Ca2+ Titrations--
Fluorescence
was measured with a PerkinElmer Life Sciences LS50B spectrophotometer
in 10-mm pathlength rectangular cells at 25 °C. Intrinsic
fluorescence was excited at 280 nm for all recombinant proteins (1 µM). Emission maxima were between 346 and 353 nm. Spectra
were recorded in TBS (less than 5 µM Ca2+) in
the presence of 2 mM CaCl2 and after the
subsequent addition of 4 mM EDTA. The percentage change in
fluorescence intensity at 350 nm was calculated as
F350 = 100 × (FCa
FEDTA)/FCa,
with FCa representing the signal at Ca2+
saturation, and FEDTA representing the fluorescence signal
in the presence of excess EDTA. Calcium titrations were performed as
described above.
Equilibrium Dialysis--
The number of bound calcium ions per
COMP molecule was determined by equilibrium dialysis in microdialysis
chambers (Membrapure, Bodenheim, Germany). 150 µl of protein
(10 µM) dissolved in TBS was dialyzed against 150 µl of
TBS containing 0.2 µCi of 45Ca2+ (Amersham
Pharmacia Biotech) and different 40CaCl2
concentrations. After 16-20 h, 10-µl aliquots were removed from each
dialysis chamber and counted in a scintillation counter. The number of
bound calcium ions B was calculated as B =
cpm/cpmtotal × nCa/nprotein, with
cpm
representing the difference in radioactivity between both dialysis
chambers, cpmtotal representing the sum of the activities,
nCa representing the number of calcium ions, and
nprotein representing the number of protein
molecules in the system. The free calcium concentration was calculated
as the difference between the total and the bound calcium
concentration. Protein concentrations were determined according the
method of Gill and von Hippel (64).
Data Evaluation--
The degree of saturation Y was
calculated from the signals S, corrected for dilutions, as
Y = (S
S0)/(SCa
S0), where SCa and
S0 represent the signals at Ca2+
saturation and 0 Ca2+, respectively. The free
Ca2+ concentrations were calculated from Y and
the corresponding total calcium concentration
Ca2+tot to: Ca2+free = Ca2+tot
Y × n × Ptot, with n
representing the number of calcium binding sites/molecule,
Ptot representing the total protein
concentration, and n × Ptot representing
the total concentration of binding sites present. We initially used
n = 13 for the number of Ca2+ binding
sites/molecule, as determined for thrombospondin-1 (33). Because the
protein concentrations used were much lower than
Ca2+tot, n had only a negligible
effect on Ca2+free. Thus, variation of
n between 10 and 20 and between 3 and 10 for the mutant T3
repeats had no significant effect on the calculated
Kd values and Hill coefficients. The degree of
saturation Y in a model including two independent classes of
binding sites can be described by Y = a × Y1 + (1
a) × Y2, with Y1 and
Y2 representing the degrees of saturation of the
first and second class of binding sites, and the parameter a
representing the percentage contribution of the signal induced by
saturation of the first class of binding sites to the whole signal.
Using the Hill equation to describe cooperativity between the
sites within each class of binding sites Y is given
by
|
(Eq. 1)
|
where [Ca] represents the free Ca2+ concentration,
Kd1 and Kd2 represent the
equilibrium dissociation constants, and nH1 and
nH2 represent the Hill coefficients. The program
Grafit (Erithacus software) was used to fit the model to the
experimental data with nonlinear least-square fit procedures.
Electron Microscopy--
For visualization by electron
microscopy, wild-type and mutant COMP and collagens (0.1 µM) were dialyzed against 0.15 M ammonium formate, pH 7.4. ZnCl2 was added to give 0.5 mM
Zn2+. The sample was diluted 1:1 with 80% glycerol and
sprayed onto freshly cleaved mica, dried in vacuum, and rotary shadowed
with platinum/carbon (52). The replicas were floated off onto destilled water, picked up on 400-mesh copper grids, and examined in a Zeiss 902 electron microscope.
 |
RESULTS |
We expressed rCOMP as well as several fragments thereof in a
eukaryotic expression system to ensure proper disulfide bond formation
and posttranslational modifications. Because it was unclear whether the
thrombospondin T3 repeats are independently folded, a construct EGF-T3,
encompassing the fourth EGF domain plus the eight T3 repeats, and a
construct T3, encompassing only the eight T3 repeats, were produced
(Fig. 1). Furthermore, we introduced two
mutations in the cDNA for COMP. The point mutation G1106T results
on the amino acid level in the change of Asp-361 to Tyr (D361Y). This
mutation was originally found in a large family with seven affected
members suffering from MED (28). The second mutation was a deletion of
one of the five triplets (nucleotides 1430-1444) encoding five
consecutive aspartic acid residues 469-473 (D469
). This is the most
commonly found mutation in COMP and causes severe PSACH (4, 5, 21,
28-30).

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Fig. 1.
Domain composition of COMP and its
recombinant fragments. CC denotes the coiled-coil
domain at the amino terminus, and EGF denotes the epidermal
growth factor-like domains, with the asterisk indicating
calcium-binding EGF domains. TC represents the
carboxyl-terminal thrombospondin-like domain. The eight type 3 repeats
are shown as rectangles. The positions of the MED- and
PSACH-causing mutations D361Y and D469 , respectively, and the single
tryptophan (Trp-344; W) are indicated.
|
|
The Type 3 Repeats in COMP Bind 14 Calcium Ions with Moderate
Affinity and High Cooperativity--
A signal peptide targeted the
recombinant proteins into cell culture media used to purify the
proteins to homogeneity (Fig. 2). rCOMP
was present in the culture medium as a pentamer of about 550 kDa,
although bands migrating at lower molecular masses were also observed
under nonreducing conditions. A band at 100 kDa was isolated, and
amino-terminal sequencing revealed that the sequence started at amino
acid 78 within the linker between the coiled-coil domain and the first
EGF-like domain (results not shown). Apparently, a protease present
either within the cells or in the culture medium is able to cleave the
COMP subunits, yielding the cleaved carboxyl-terminal parts of 100 kDa
and fragments with one to five intact subunits connected via the
pentameric coiled-coil domain. The smaller fragments including the
100-kDa band could be removed chromatographically, and a preparation of pentameric COMP with a portion of COMP particles lacking the
carboxyl-terminal part of one subunit was obtained. Under reducing
conditions, this preparation showed a single band at 110 kDa (Fig. 2).
Amino-terminal sequencing confirmed that that this band represents the
full-length COMP. Electron microscopy after rotary shadowing confirmed
the mainly pentameric structure of rCOMP. As seen earlier for tCOMP (7), five globular domains are connected by thin flexible strands to a
central assembly domain (Fig. 3).

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Fig. 2.
SDS-PAGE of recombinant COMP and
fragments. Recombinant proteins were separated on polyacrylamide
gels (lanes 1-3, 3-15%; lanes 4-6, 12%;
lanes 7-10, 15%). Lanes were loaded with rCOMP
(lanes 1 and 4), D361Y (lanes 2 and
5), D469 (lanes 3 and 6), EGF-T3
(lane 7), T3 (lane 8), T3-D361Y (lane
9), and T3-D469 (lane 10). Nonreducing conditions
reveal the formation of pentamers for rCOMP and mutants (lanes
1-3); reducing conditions (lanes 4-10) reveal the
homogeneity and equal size of the proteins. The double bands
seen under nonreducing conditions result from proteolytic cleavage
(see "Results"). The migration position of calibrating
proteins is given in the left margins (in kDa).
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Fig. 3.
Electron microscopy of rCOMP and its
mutants. Proteins dialyzed against 0.2 M ammonium
formate, pH 7.4, were sprayed from glycerol and rotary shadowed. An
overview is shown for rCOMP (top). Panels of selected
particles are given for rCOMP (top panel), D361Y
(middle panel), and D469 (bottom panel). The
five globules in the periphery of each particle represent
the carboxyl-terminal domains, and the central rectangle
represents the coiled-coil domain. The bars represent 100 (overview) and 25 nm (panels).
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Folding of rCOMP was investigated by CD spectroscopy. CD spectra in the
far UV region for rCOMP and tCOMP were indistinguishable and
characteristic for a protein with a mixed
-sheet-random conformation and low
-helical content (Fig. 4).
Contents of 14%
-helix, 33%
-sheet, and 53%
-turns and
unordered conformation were calculated.

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Fig. 4.
Conformational changes in COMP and PSACH and
MED mutants. Circular dichroism spectra were recorded in the
absence (A and B) and presence of 1.5 mM calcium (C). A, rCOMP (solid
line), D361Y (dashed line), D469 (dotted
line). B and C, T3 (solid line),
T3-D361Y (dashed line), and T3-D469 (dotted
line). The decrease in ellipticity after calcium addition to T3,
T3-D361Y, and T3-D469 was completely reversible by addition of
excess EDTA.
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|
The addition of 0.25 mM calcium induced a small
conformational change, whereas higher calcium concentrations induced an
aggregation of rCOMP and tCOMP that leads to a loss of ellipticity due
to light scattering (results not shown). The
calcium-dependent conformational change was also observed
for EGF-T3 (results not shown) and the T3 repeats alone (Fig. 4). For
the smaller fragments, it could be reversed even in the presence of 1.5 mM calcium by the addition of excess EDTA. Equilibrium
dialysis revealed that 14 calcium ions bind to the T3 repeats at
calcium saturation (Fig. 5). A model of
binding sites without cooperativity failed to adequately describe the
binding curve. A good fit was achieved when cooperativity was allowed
in the model, and a Kd = 0.15 mM and a
Hill coefficient nH = 4.9 were evaluated. The CD
signal at 215 nm was used to monitor the calcium dependence of the
conformational change of the T3 repeats. A biphasic titration curve was
observed in which 80% of the signal were reached by the addition of 1 mM calcium, and further addition to 20 mM
resulted in a second signal change (Fig.
6). Again, a good fit was achieved when
cooperativity was allowed in the model and yielded an equilibrium
dissociation constant Kd1 = 0.3 mM and a
Hill coefficient nH1 = 3.7 that describes the
conformational change by calcium binding at low calcium concentrations. A second Kd2 = 4.1 mM with low
cooperativity (Hill coefficient nH2 = 1.2) was
obtained for the signal change at high calcium concentrations (Table
I). No significant difference in calcium affinity was observed for the EGF-T3 fragment (results not shown).

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Fig. 5.
Calcium binding of type 3 repeats and their
mutants. Equilibrium dialysis of T3 ( ), T3-D361Y ( ), and
T3-D469 ( ) at different calcium concentrations revealed different
numbers of calcium binding sites B. Best fit curves obtained
from a binding model including cooperativity are shown as solid
lines.
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Fig. 6.
Calcium affinity and cooperativity of type 3 repeats and their mutants. Increasing concentrations of calcium
were added to the proteins dissolved in TBS, and circular dichroism was
recorded at 215 nm. Ellipticity was converted into saturation
Y. A, T3-D361Y ( ) and T3 ( ). B,
T3-D469 ( ) and T3 ( ). Best fit curves obtained from a binding
model including cooperativity are shown as solid
lines.
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COMP Carrying Mutations that Cause PSACH or MED Is Folded and
Secreted by 293 Cells--
In serum-free cell culture media, we could
readily detect both mutated full-length COMP D361Y and D469
,
which were subsequently purified to homogeneity (Fig. 2). We observed
neither signs of intracellular inclusions or cell death nor significant
differences in protein yields as compared with the wild-type proteins.
Electron microscopy revealed a pentameric structure with five globular domains connected to a central assembly domain similar to that seen for
rCOMP and tCOMP (Fig. 3). We used CD spectroscopy to analyze whether
secondary structure is maintained in mutated COMP molecules. Compared
with tCOMP and rCOMP, only a very small difference in ellipticity could
be observed in the spectra (Fig. 4). Furthermore, the small
conformational change upon the addition of calcium could also be
observed for the D361Y and D469
mutant proteins, and higher calcium
concentrations induced precipitation as seen with tCOMP and rCOMP. The
small change in ellipticity and the precipitation precluded the
determination of calcium affinity in the full-length proteins.
Calcium Binding Is Affected in Mutated COMP--
To analyze the
conformational change in greater detail, we introduced the D361Y and
D469
mutations in the T3 repeats. Again, both proteins were
expressed and secreted by the 293 cells (Fig. 2). The number of calcium
binding sites in T3-D361Y and T3-D469
was reduced to 8 and 10, respectively, but affinity and cooperativity of the remaining sites
were only slightly affected (Fig. 5; Table I). Analysis of the CD
spectra revealed pronounced conformational differences in the presence
and absence of calcium compared with wild-type T3 repeats (Fig. 4).
Although both T3-D361Y and T3-D469
were able to reversibly bind
calcium, titrations showed clear differences compared with wild-type T3
repeats (Fig. 6). The total change of ellipticity was slightly
decreased in both mutants. 70% of signal of the D469
mutation
corresponded to the cooperative sites with moderate calcium affinity
(Kd1 = 0.28 mM, Hill coefficient
nH1 = 3.7), whereas only 55% of the signal was induced in T3-D361Y by the occupation of these calcium sites
(Kd1 = 0.25 mM,
nH1 = 4.5). The loss of signal induced by the
low calcium concentrations was accompanied by a larger signal change
induced at millimolar calcium concentrations (Kd2 = 1.5 and 2.7 mM, respectively; Table I). Thus, both
mutations shift part of the calcium binding from moderate affinity to
low affinity, which results in a decreased number of bound calcium ions
at the physiological calcium concentration of 1 mM.
Calcium binding was also monitored by intrinsic fluorescence. Upon
excitation at 280 nm, an emission maximum at 346 nm was observed for
the calcium-saturated T3 repeats. This fluorescence is caused by
Trp-344 within the third T3 repeat because no additional Trp or Tyr is
present within the T3 repeats. Removal of calcium shifted the
wavelength at which maximal emission occurred to 353 nm and was
accompanied by a 4.3-fold increase in fluorescence intensity for
wild-type T3. In contrast, only a 2.8-fold increase was observed for
T3-D469
(Fig. 7, A and
C; Table I). The intrinsic fluorescence was also used to
determine calcium affinity and cooperativity of the sites neighboring
Trp-344. No significant differences in calcium affinity
(Kd1 = 0.25 mM) and cooperativity
(nH1 = 6-8) were observed between T3 and
T3-D469
(Fig. 7D). The cooperativity demonstrates that
the Trp-344 fluorescence is influenced by the binding of several
calcium ions. The Hill coefficients obtained from fluorescence
titrations were larger than the ones from CD and point to a somewhat
higher cooperativity of calcium binding to the amino terminus of the T3
repeats. Low affinity binding sites were hardly detectable by
fluorescence titrations. The introduction of Tyr-361 for Asp
drastically reduced the intrinsic fluorescence change at 350 nm upon
excitation at 280 nm to only 20% (Fig. 7B). To analyze the
contributions of Tyr and Trp to the fluorescence, we measured calcium
binding upon excitation at 297 nm, at which only Trp is excited. The
same 20% decrease of fluorescence was observed. This rules out
a compensatory effect of the introduced Tyr and demonstrates that the
local environment of Trp-344 is affected by the mutation. In addition,
calcium titrations showed a 5-fold decreased affinity
(Kd = 1.1 mM) and a decreased cooperativity in T3-D361Y (Fig. 7D; Table I). The difference in affinities for T3-D361Y obtained by CD and fluorescence is only
apparent. CD monitors the change in overall secondary structure caused
by the remaining intact binding sites, whereas the intrinsic fluorescence monitors the surrounding of the single Trp-344 that is severely affected by the neighboring mutation.

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Fig. 7.
Fluorescence spectra and calcium titrations
of wild-type and mutant type 3 repeats. Fluorescence emission
spectra were recorded at an excitation at 280 nm in TBS in the absence
of calcium (solid lines) and in the presence of 1.5 mM calcium (dotted lines) for T3 (A),
T3-D361Y (B), and T3-D469 (C). Calcium
titrations were monitored upon excitation at 280 nm and emission at 350 nm (D). Fluorescence signals were converted into saturation
Y with T3 represented by , T3-D361Y represented by ,
and T3-D469 represented by . Best fit curves obtained from a
binding model including cooperativity are shown as solid
lines.
|
|
COMP Binds to Collagen IX--
COMP binding to collagen I and II
was reported recently (20). This prompted us to investigate whether
COMP also binds to collagen IX, which is found in cartilage as well as
the fibrocartilaginous insertion sites of tendons and ligaments to bone
and which accumulates together with COMP in the ER inclusions in MED
and PSACH patients (24, 43, 53, 54). We coated microtiter plates with
collagen IX and detected COMP binding in an enzyme-linked immunosorbent assay-style ligand-binding assay. Both tCOMP and rCOMP showed concentration-dependent saturation curves, and half-maximal
binding was obtained at 3.5 and 1.5 nM (Fig.
8, A and B).
Control experiments with COMP added to bovine serum albumin-coated
wells resulted in less than 5% of the absorbances obtained with
collagen-coated wells even at high COMP concentrations. Binding was
confirmed by surface plasmon resonance detection with COMP immobilized
on the chip and collagen in solution (Fig.
9) as well as with immobilized collagens
and COMP in solution (results not shown). To obtain Kd values from the surface plasmon resonance curves, we fitted a Langmuir model for one-to-one interaction to the curves measured at different collagen concentrations. Multiple binding sites
are present on both the collagens and the pentameric COMP, and the
Kd values are therefore an average of several binding sites. The average Kd value for collagen IX
binding was 32 nM (Table II).
Furthermore, complex formation between COMP and collagen IX could be
observed by electron microscopy (Fig. 10). The recombinant collagen IX forms
a filamentous structure with a globular end representing the
amino-terminal NC4 domain of the
3 chain. A sharp kink within the
collagenous domain represents the NC3 domain, as shown for collagen IX
isolated from tissue (55). COMP is bound to both ends of the collagen
IX molecule and to two additional internal sites. One is located at the
kink representing the NC3 domain at a distance of 50 nm from the
amino-terminal globule. The second internal site is found at a distance
of about 160 nm and is thus in the vicinity of the NC2 domain. In some complexes, resolution was sufficient to reveal that COMP interacts by
its globular domains with the collagen IX (Fig. 10). Identical saturation curves for tCOMP and rCOMP were also obtained with collagen
I (Fig. 8, A and B), corroborating the
functionality of the recombinant proteins.

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Fig. 8.
Binding of COMP and mutant COMP to collagens
I and IX in a solid-phase assay. Collagen I (solid
lines) and IX (dashed lines) were coated overnight at 2 µg/ml in 0.18 M acetic acid onto plastic wells and
incubated with 0-20 nM tCOMP (A), rCOMP
(B), D361Y (C), and D469 (D) in TBS
with 0.5 mM ZnCl2 for 1 h. Bound COMP was
detected using an antiserum to COMP followed by peroxidase-coupled
antiserum against rabbit IgG using TMB as the chromogenic
substrate and recording absorbance at 450 nm. Data are the means of
quadruplicate determinations, and error bars show the mean
absolute deviations.
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Fig. 9.
COMP binding to collagen IX detected by
surface plasmon resonance assay. A, concentration
dependence of collagen IX (11, 22, 44, and 88 nM) binding to immobilized rCOMP. B, rCOMP
(solid lines), D361Y (dashed line), and D469
(dotted line) immobilized on a CM5 sensorchip were allowed
to interact with 88 nM collagen IX in 20 mM
Hepes, pH 7.4, 150 mM NaCl in the presence of 0.05 mM ZnCl2. A flow rate of 80 µl/min was used,
and a volume of 40 µl of ligand was injected at 120 s.
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Fig. 10.
Binding of wild-type COMP to collagen IX
visualized by electron microscopy. COMP was incubated with
collagen IX during dialysis overnight against 0.2 M
ammonium formate, pH 7.4, with 0.5 mM zinc for glycerol
spraying/rotary shadowing. The molar ratio of COMP:collagen was 1. The
amino-terminal NC4 domain of collagen IX forms a globular structure
(arrowhead), and a kink is present at the NC3 domain. COMP
binds via a globular end (arrow) to several sites on
collagen IX.
|
|
Binding of COMP to Collagens I, II, and IX Is Affected by PSACH and
MED Mutations--
Collagen binding of COMP was previously shown to
depend on the presence of zinc ions (20). We therefore compared the
binding to collagens at different zinc concentrations. Whereas
half-saturation binding of rCOMP to collagen I was observed at 10 µM zinc, significantly more zinc was needed to induce
binding of D361Y and D469
(K0.5 = 20 and 40 µM, respectively; Fig.
11A). Similarly, higher zinc concentrations were needed to induce binding of D361Y and D469
to
collagen II and IX compared with wild-type COMP (Fig. 11, B and C). However, when collagen binding was measured at
higher zinc concentrations, similar binding profiles and affinities of wild-type and mutated COMP for collagen were obtained (Figs. 8 and 9;
Table II). We also quantitated COMP binding using electron microscopy.
Analysis of more than 300 COMP molecules showed that in 1 mM EDTA, only 28% were bound to collagen IX, whereas 51% were bound in the presence of 1 mM zinc. The only partial
binding in zinc results from low concentrations of both binding
partners used to avoid nonspecific juxtaposition of molecules on the
grid and is in agreement with Kd values obtained by
the surface plasmon resonance studies.

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Fig. 11.
Zinc dependence of collagen binding to
wild-type and mutant COMP. Collagens I (A), II
(B), and IX (C) were coated overnight at 2 µg/ml in 0.18 M acetic acid onto plastic wells and
incubated with 4 nM rCOMP ( ), D361Y ( ), and D469
( ) in TBS at different zinc concentrations for 1 h. Bound COMP
was detected using an antiserum to COMP followed by peroxidase-coupled
antiserum against rabbit IgG using tetramethyl-benzidine as the
chromogenic substrate and recording absorbance at 450 nm. Data are the
means of quadruplicate determinations, and error bars show
the mean absolute deviations.
|
|
The zinc dependence was further analyzed using proteolysis of COMP.
Chymotrypsin completely degraded COMP when no cations were present.
Calcium and zinc were able to partially protect cleavage sites.
However, different fragments were obtained in the presence of zinc
compared with calcium (Fig. 12). To
further investigate the cleavage site obtained in the presence of zinc, the corresponding band was excised from the gel, digested with trypsin,
and analyzed by sequencing of some of the resulting peptides using
ESI-Q-TOF (electrospray ionization quadruple time-of-flight) mass spectrometry. The peptide encompassing the amino terminus of COMP
and the peptides from the T3 repeats were identified, whereas
carboxyl-terminal peptides were lacking. This demonstrates a
carboxyl-terminal cleavage presumably in the carboxyl-terminal domain.
An effect of zinc on the conformation of the carboxyl-terminal domain
is also in agreement with CD titrations, showing that, in contrast to
calcium, 1 mM zinc did not affect the conformation of the
T3 repeats (results not shown).

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Fig. 12.
Cation-dependent proteolytic
susceptibility of COMP. rCOMP was digested with chymotrypsin in
the presence of 0.5 mM zinc, 1 mM calcium, or 2 mM EDTA for 20 h at room temperature. Proteins
were subjected to SDS-PAGE on 4-15% gels under reducing conditions
and stained with Coomassie Brilliant Blue. The migration position of
calibrating proteins is given in the left margin (in
kDa).
|
|
 |
DISCUSSION |
COMP is a modular protein composed of different types of domains.
Thus far, the three-dimensional structure has been characterized only
for the coiled-coil domain of COMP (9). The structures of the EGF-like
domains can be predicted from homologous domains in other proteins. On
the other hand, reasonable structural models are available for neither
the T3 repeats nor the carboxyl-terminal domain. Also, the knowledge of
COMP function is limited to the recent demonstration of interactions
with collagens I and II (20). Due to this lack of structural and
functional data, the consequences of PSACH- and MED-causing mutations
and hence the molecular pathogenesis of the disease are unknown. To
learn more about structural and functional alterations, we expressed
recombinant T3 repeats either alone, in connection with the fourth
EGF-like domain, or within the full-length protein. CD spectra,
electron micrographs, and collagen binding measurements clearly
demonstrate that the recombinant wild-type COMP is structurally and
functionally equivalent to tissue-derived COMP. When attempting to
analyze the calcium-dependent conformations, we found that
both rCOMP and tCOMP precipitate at the high protein concentrations
used for CD spectroscopy when more than 0.25 mM calcium is
present. It is unclear whether this precipitation is of physiological
relevance, and the phenomenon was not further investigated. The major
elements of secondary structure in COMP are
-sheets and unordered
conformations, indicating that the T3 repeats and the carboxyl-terminal
domain have only a low content of
-helix.
To analyze the effects of COMP mutations, we genetically engineered the
COMP cDNA in such a manner that COMP with a D469
or a D361Y
substitution was produced. The mutations were chosen according to the
severity of phenotypes observed for PSACH and MED patients. The D469
mutation causes PSACH with severe dwarfism, whereas the D361Y mutation
causes MED with mild to severe osteoarthritis (4, 5, 21, 28-30). Both
mutants were secreted by the human kidney cells used for expression,
formed pentamers, and showed a folding similar to that of wild-type
rCOMP and tCOMP. In addition, a similar conformational change
upon calcium addition was observed at low calcium concentrations. Thus,
in the context of full-length COMP, mutations in the T3 repeats have
only subtle effects and do not produce an instability of the protein
per se.
More detailed insight was obtained by the analysis of the recombinant
T3 repeats. The findings that the T3 repeats can be expressed
independently and that they bind calcium with high cooperativity indicate that they constitute an autonomous folding unit. Despite the
uneven number of 17 cysteines, oligomer formation could not be
detected. A mobility shift in SDS-PAGE between reducing and nonreducing
conditions indicates the formation of intramolecular disulfide bonds
within the T3 repeats (results not shown). CD and fluorescence
spectroscopy revealed that calcium but not zinc can induce a reversible
change of the conformation. Analysis of secondary structures of the T3
repeats indicated the presence of
-helices and
-sheets. However,
their elongated structure, multiple disulfide bonds, and acidic nature
may invalidate these secondary structure predictions, which depend on
the chosen standard protein data set (56). Attempts to crystallize the
T3 repeats in the presence or absence of calcium have failed, perhaps
indicating a highly flexible
conformation.2 Calcium
binding is strongly cooperative, and the affinity is high enough that
the T3 repeats should be in the calcium-saturated form in the presence
of physiological extracellular calcium concentrations. Interestingly,
the mutations D469
and D361Y did not induce a breakdown of the
cooperative system, and only 4 or 6 of the 14 calcium binding sites
were lost. 14 calcium binding sites for wild-type COMP are in good
agreement with the 13 sites reported for the homologous
thrombospondin-1 (33). During the course of our study, Maddox et
al. (65) reported that the mutation D446N in COMP, which
causes PSACH, reduced the binding from 17 calcium ions to 8 calcium
ions. In an additional study, Chen et al. (66) also
introduced the deletion of D469 in COMP. Their mutant protein bound
seven calcium ions, compared with nine calcium ions bound to the
wild-type protein. The relative loss of about 25% of the calcium
binding sites is similar to our results. Equilibrium dialysis results
depend strongly on exact protein concentrations, and this is presumably
the reason for the discrepancy in the absolute numbers. We
experimentally determined extinction coefficients and used these to
measure protein concentrations by UV spectroscopy, thus avoiding many
of the pitfalls of other methods (64).
To gain more insight into the effect of mutations on the conformation,
we used circular dichroism and fluorescence spectroscopy. Because
Trp-344 fluorescence changes cooperatively upon calcium binding with
nH
6, at least six calcium ions
influence the local conformation of Trp-344, in agreement with the loss
of six sites when the mutation is present in the neighboring repeat.
However, affinity and cooperativity of the remaining intact sites are
only slightly affected. The interference of the fluorescence change in
repeat 3 by the D469
mutation, which is located in repeat 7, corroborates the model in which the eight T3 repeats do not represent
individual autonomous repeats but form a larger disulfide-bonded folding unit. The point mutations causing PSACH and MED locally affect
the affinity of several calcium ions but also influence the
conformation of distant repeats. Thus far, no correlation between the
type or location of the mutation and the severity of the disease has
been found. The fact that the D361Y mutation, which causes mild MED,
binds fewer calcium ions than the D469
mutation, which causes severe
PSACH, demonstrates that the number of bound calcium ions does not
correlate with the PSACH or MED phenotype.
We show that the mutations affect the binding of COMP to collagens I
and II, the only COMP ligands described previously (20). Because the
binding site for collagens is located in the carboxyl-terminal domain
of COMP, the affected collagen binding of COMP with mutations in the T3
repeats suggests that the conformation of the carboxyl-terminal domain
is influenced by the type 3 repeats and that this interaction is
disturbed by PSACH and MED mutations. This is in agreement with the
finding that mutations in the carboxyl-terminal domain can also cause
PSACH and MED, respectively. Decreased arm length and different stokes
radius seen in the mutant COMP also support this notion (65, 66). The
interaction of COMP and collagens is dependent on the presence of zinc
ions, and higher zinc concentrations are needed for efficient binding
of mutant COMP. We also showed that zinc can directly affect the
conformation of COMP, presumably by binding to the carboxyl-terminal
domain. Zinc concentrations in synovial fluid are 9-25
µM (57), whereas the total zinc concentration in
cartilage varies with age between 15 and 120 µM (58). In different layers and compartments of cartilage, such as the
hypertrophic zone of the growth plate, a concentration of up to 1400 µM total zinc was detected (59). The mutations in COMP
might thus shift the proportion of COMP molecules bound to collagens.
We propose that the affected collagen binding of mutant COMP may
contribute to the development of phenotypes of PSACH and MED patients.
Although the precise mechanisms are still unknown, secreted mutant COMP might interfere with proper formation and organization of collagen I
and II fibrils and thus affect structure and stability of cartilage, tendons, and ligaments. Such a dominant negative effect of secreted mutant COMP is also in agreement with the observation that mice deficient in COMP grow to normal length and do not develop severe osteoarthritis.3
The presence of inclusions in the ER points to an alternative mechanism
for the pathogenesis of PSACH and MED. According to this hypothesis,
COMP and additional proteins destined for secretion are retained in the
ER, and the lack of these proteins in the extracellular matrix,
together with an affected cellular function, could cause the phenotypes
(5, 60). Thus far, the lack of antibodies specific for mutant COMP
molecules has hampered the study of the fate of such COMP molecules.
Patient chondrocytes, tenocytes, and ligament cells are able to secrete
pentameric COMP in vitro (61, 62). For chondrocytes, this is
presumably related to their dedifferentiation in monolayer cell culture
(60). In vivo, COMP has been shown to be present
extracellularly in tendons of a PSACH patient, in agreement with the
lack of inclusions in tenocytes (24). Thus, at least in tendons, mutant
COMP is secreted and might cause a dominant negative effect. In
contrast, COMP was not found (24) or was found in reduced amounts (62)
in the extracellular matrix of patient cartilage in immunohistochemical studies. This was interpreted as being due to the retention in the
endoplasmic reticulum of chondrocytes but could also be caused by an
only weak anchorage of secreted mutant COMP in the extracellular matrix
and subsequent loss of COMP during the staining procedure. Additional
mechanisms, such as enhanced degradation of mutant COMP by endogeneous
proteases, may also contribute to this phenomenon.
We now demonstrate for the first time that COMP interacts with collagen
IX in a zinc-dependent manner, as observed previously for
collagens I and II. The binding sites are present at the termini and
two internal sites of the collagen IX molecule. Interestingly, inclusions in the ER have been found in all cell types in which COMP
and collagen IX are coexpressed, namely, chondrocytes and fibrocartilaginous cells at the insertion site of ligaments (34, 53,
63). Tenocytes and ligament cells distant from the insertion sites
express COMP but lack collagen IX, and no inclusions have been found in
these cells (24, 34). Thus, we suspected that binding of mutant COMP to
collagen IX could be the cause of the inclusions. Our results show that
mutant COMP binds to collagen IX, albeit with an affinity similar to
that of wild-type COMP. Thus, in the ER of chondrocytes and
fibrocartilaginous cells, mutant COMP molecules might be recognized by
a specific factor such as a chaperone, and subsequent binding of
collagen IX to the COMP-chaperone complex might induce the formation of
inclusions in the ER. A similar scenario is also likely for MED caused
by mutations in collagen IX genes, which also show inclusions in chondrocytes (40).
Nonetheless, the binding of wild-type COMP to specific sites on
collagen IX points to a physiological function of this interaction in
the extracellular matrix. It was proposed that collagen IX, which is
located on the surface of fibrils, represents a molecular bridge
between fibrils and other matrix components and increases the stability
of fibrillar network. This is in agreement with the late-onset
osteoarthritis seen in collagen IX transgenic and deficient mice
(44-47). The reduced binding of PSACH and MED mutant COMP to collagen
IX might thus affect the stability of the fibrillar network.
The contribution of a disturbed collagen IX-COMP interaction to the
molecular basis of the similar clinical phenotypes seen in MED patients
with mutations in either collagen IX chains or COMP remains to be
determined. We did not find pronounced differences between the COMP
mutations that cause PSACH and MED, respectively, suggesting that
additional factors contribute to the severity of these diseases.
 |
ACKNOWLEDGEMENTS |
We are grateful to S. Gösling for
excellent technical assistence and Drs. F. Zaucke and R. Dinser for
fruitful discussions. We thank Dr. M. Mörgelin for
introducing D. P. N. into the methods for electron microscopy of macromolecules.
 |
FOOTNOTES |
*
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.
§
Supported by the Deutsche Forschungsgesellschaft (Kr 558/10).
Supported by the Swedish Medical Research Council and the
Swedish League against Rheumatism.

Recipient of an award from the Humboldt Foundation.
§§
To whom correspondence should be addressed: Institut für
Biochemie II, Medizinische Einrichtungen der Universität zu
Köln, Joseph-Stelzmann-Str. 52, D-50931 Köln, Germany.
Phone: 49-221-478-6943; Fax: 49-221-478-6977; E-mail:
patrik.maurer@uni-koeln.de.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M009512200
2
E. Hohenester and P. Maurer, unpublished observations.
3
L. Svensson and Å. Oldberg, personal communication.
 |
ABBREVIATIONS |
The abbreviations used are:
PSACH, pseudoachondroplasia;
MED, multiple epiphyseal dysplasia;
COMP, cartilage oligomeric matrix protein;
EGF, epidermal growth factor;
T3, thrombospondin type 3;
PCR, polymerase chain reaction;
rCOMP, recombinant full-length COMP;
PAGE, polyacrylamide gel electrophoresis;
TBS, Tris-buffered saline (50 mM Tris, pH 7.4, 150 mM NaCl);
tCOMP, COMP isolated from tissue;
ER, endoplasmic
reticulum.
 |
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