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INTRODUCTION |
Cardiovascular disease is a leading cause of death in Western
societies with over 50% of the cases due to coronary heart disease (CHD)1 (1). Some patients who
develop CHD prematurely (before age 45 in men and before age 50 in
women) have a family history of the disease, suggesting genetic bases
for premature CHD. A recent case control study (2) identified a single
nucleotide polymorphism in thrombospondin-1 (TSP-1) that was strongly
associated with familial premature CHD in patients homozygous for the
single nucleotide polymorphism. The single nucleotide polymorphism
results in the substitution of a serine for an asparagine at residue
700 of TSP-1. TSP-1 is a 450-kDa trimeric extracellular matrix
glycoprotein that previously has been observed in atherosclerotic
plaques and intimal hyperplasia (reviewed in Ref. 3). During arterial
injury or upon stimulation with growth factors in vitro,
TSP-1 expression in smooth muscle cells is increased (4-7), and TSP-1
and platelet-derived growth factors synergistically enhance smooth
muscle cell migration (8). Patients having two Ser-700 alleles also had
2-fold lower levels of plasma TSP-1 than control patients (2).
A TSP-1 monomer contains an N-terminal module, an oligomerization
sequence, a procollagen module, three properdin (type 1) modules, three EGF-like (type 2) modules, a number of
Ca2+-binding (type 3) repeats, and a long C-terminal
sequence (Fig. 1A). The Ca2+-binding and
C-terminal sequences are unique to TSPs and are highly conserved. For
instance, the alignment of human TSP-1 and Drosophila TSP
demonstrates exact spacing of 16 cysteines in the
Ca2+-binding repeats. TSP-1 has an additional cysteine in
the C-terminal globe that favors the isomerization of disulfides (9)
and the formation of disulfide-linked complexes with thrombin (10) and von Willebrand factor (11, 12). Ca2+ (~5 mM)
prevents formation of thrombin-TSP-1 complexes (10). In the absence of
Ca2+, rotary shadowing microscopy has shown that the C
terminus of TSP-1 adopts an extended conformation (13-16) that is
sensitive to proteolytic degradation. Thus, the structure,
stability, and cysteine reactivity of TSP-1 C-terminal sequences
are Ca2+-sensitive.
The Ser-700 polymorphism localizes to the beginning of the
Ca2+-binding repeats (Fig. 1B). The homologous
region in TSP-5 (cartilage oligomeric matrix protein (COMP)) is linked
to two related autosomal dominant syndromes of skeletal dysplasia,
pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1)
(17, 18). Mutations in COMP localize to the
Ca2+-binding repeats and C-terminal sequence and are
missense mutations or small insertions/deletions that often affect
aspartate and asparagine residues important for binding
Ca2+ (18-26). Two PSACH-causing missense mutations in COMP
have been identified within 10 residues of the aspartate that occupies
the position of the N700S polymorphism of TSP-1 (20) (Fig.
1B). Several studies on PSACH and EDM1 mutations have
shown via electron microscopy that mutant proteins have altered
structures (27) and bind a decreased number of Ca2+ ions
with altered affinity (27-30). Mutant COMP accumulates in the
endoplasmic reticulum (ER) of chondrocytes with type IX collagen (31)
and chaperone proteins (32, 33).
We hypothesized that, similar to COMP mutations, the Ser-700
polymorphism alters the conformation of the Ca2+-binding
repeats of TSP-1. We characterized segments of TSP-1 comprised
of the Ca2+-binding repeats (Ca) and the
Ca2+-binding repeats with the third EGF-like module (E3Ca)
as without (Asn-700) and with (Ser-700) the polymorphism associated
with familial premature CHD. We found that the Ser-700 polymorphism causes a subtle local change in conformation that destabilizes the
protein in response to lowering Ca2+ concentration or
increasing temperature.
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EXPERIMENTAL PROCEDURES |
Cloning of Ca Asn-700, Ca Ser-700, E3Ca Asn-700, and E3Ca Ser-700
into the pAcGP67.coco Transfer Vector--
To facilitate
baculovirus-mediated protein expression, we used the pAcGP67.coco
transfer vector, in which cloning sites are flanked by 5' DNA encoding
a signal sequence and 3' DNA encoding a polyhistidine tag (34). The
Ser-700 polymorphism was introduced by PCR mutagenesis into a construct
called E3CaG Asn-700 that consisted of the last EGF-like module (E3),
the Ca2+-binding repeats (Ca), and the C-terminal globe and
contained residues 648-1170 (12). Using DNA encoding E3CaG Asn-700 as a template, Primer 1 and Primer 2, containing a 3' AvrII
site, were used to amplify by PCR a product that encoded residues
648-700. Primer 3, containing a 5' SpeI site, and Primer 4 were used to generate a PCR product encoding residues 700-1170. The
PCR products were digested with AvrII or SpeI and
were ligated together using the compatible cohesive ends of
AvrII and SpeI to generate DNA that encodes E3CaG
containing serine at residue 700 (E3CaG Ser-700). A second
amplification of DNA encoding E3CaG Ser-700 employed Primers 1 and 4 with BamHI and NsiI sites, respectively. This PCR
product was digested with BamHI and NsiI and
inserted into the pAcGP67.coco baculovirus vector (34) linearized with
BamHI and PstI using compatible cohesive ends of
NsiI/PstI.
DNA encoding Ca Asn-700 or Ser-700 (residues 689-945) then was
generated by PCR amplification using a template that encoded E3CaG
Asn-700 or E3CaG Ser-700. The forward primer contained an XmaI site, and the reverse primer contained an
NsiI site. E3Ca (residues 648-945) constructs also were
generated by PCR amplification using the previously mentioned Primer 1 containing restriction site BamHI and the reverse primer
described for the Ca constructs. The PCR products were inserted into
XmaI (for Ca constructs) or BamHI (for E3Ca
constructs) and PstI (using compatible cohesive ends of
NsiI) sites of pAcGP67.coco. Correct sequencing of
PCR-amplified DNAs was verified by automated DNA sequencing.
Expression and Purification of Recombinant
Proteins--
Recombinant infectious viruses were generated as
described (34). Passage 3 of the virus (>108
plaque-forming units/ml) was used to infect High-Five insect cells
(Invitrogen) at a multiplicity of infection of 5 in SF-900 II
serum-free medium at 22 °C. Conditioned medium, after 60-65 h, was
harvested and dialyzed into 10 mM MOPS, 0.3 M
NaCl, and 2 mM Ca2+ (pH 7.5). Dialyzed medium
was incubated with Ni2+-nitrilotriacetic acid resin
overnight at 4 °C, a column was poured with protein-bound resin, and
the protein was eluted in a buffer containing 300 mM
imidazole. Purified protein was dialyzed into 10 mM MOPS,
0.15 M NaCl, and 2 mM Ca2+, pH 7.5. The protein was stored in aliquots at
80 °C and thawed at 25 °C
prior to use.
Intrinsic UV Fluorescence and Titration with
Ca2+--
Prior to fluorescence assays, all recombinant
proteins were treated with 4 mM EDTA to remove the
Ca2+. The protein then was dialyzed at 4 °C into a
buffer containing 5 mM MOPS and 0.1 M NaCl (pH
7.5). Dialyzed wild-type or polymorphic proteins were titrated with
Ca2+ at 37 °C and excited at 295 nm in an Aminco SLM
8100 fluorometer in 1-cm path length cells. Intrinsic
fluorescence was measured from 310 to 400 nm, and the spectra were
recorded at each Ca2+ concentration from 0 mM
to saturating Ca2+ concentration. Reversal of the change
also was determined by the addition of EDTA in excess of the saturating
Ca2+ concentration. The change in fluorescence intensity
(
F) relative to the Ca2+-depleted protein
from 0 mM to saturation was calculated as
F = (Fo
F)/(Fo), where
Fo is the total fluorescence at 0 mM
Ca2+ and F is the total fluorescence at a given
Ca2+ concentration. The EC50 of
calcium-sensitive structural transition was determined from the graph
of
F versus Ca2+ concentration.
The Hill coefficient was determined by calculating the slope of the
line generated by log[
F/(1
F)]
versus log[Ca2+], and the values used for
F were taken from 30-70% of saturation. The E3Ca
proteins in 0.2 mM Ca2+ also were excited at
295 nm at varying temperatures (25, 37, 42, 50, and 65 °C), and
emission from 310-400 nm was recorded at each temperature.
Far UV CD--
Prior to CD spectral analysis, all recombinant
proteins were treated with EDTA and dialyzed as described above. At
37 °C, dialyzed Asn-700 and Ser-700 proteins were titrated with
Ca2+, and CD spectra were collected in the far UV region
(260-200 nm) in an AVIV 62 DS CD spectrophotometer at
each Ca2+ concentration. The reversibility was determined
by the addition of excess EDTA. The mean residue weight ellipticity was
calculated using the mean residue weight for each protein. The
fractional change in mean residue weight ellipticity at 220 nm
(
E) was calculated using the equation
E = (E0
E)/(E0
E2),
where E0 is the ellipticity at 0 mM
Ca2+, E is the ellipticity at a given
Ca2+ concentration, and E2 is the
ellipticity at 2 mM Ca2+. The EC50
of the transition was determined from the graph of
E
versus Ca2+. The Hill coefficient was determined
by calculating the slope of the line generated by
log[
E/(1
E)] versus
log[Ca2+].
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RESULTS |
Expression of Recombinant C-terminal Constructs--
Baculovirus
expression of TSP-1 fragments E3Ca and Ca (Fig.
1A) with either asparagine or
serine at residue 700 resulted in a high yield of protein (30-50
mg/liter conditioned medium). The N-terminal secretion signal
targeted protein into the medium, and the C-terminal polyhistidine tag
allowed for purification over a Ni2+-nitrilotriacetic acid
column. The polyhistidine tag was not removed for subsequent studies.
All proteins migrated more slowly than predicted by molecular weight
standards (Fig. 1C). This is likely because of the high
aspartate content (17%). The presence of the Ser-700 polymorphism did
not alter the migration of the protein by SDS-PAGE. There were no
observable differences in the expression levels of Asn-700 and Ser-700
proteins at 22 °C, the temperature used to infect High-Five
insect cells with baculovirus.

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Fig. 1.
Schematic of TSP-1 modules and expressed
proteins (A), localization of key residues in E3Ca
(B), and SDS-PAGE of expressed proteins
(C). A, modules E3Ca and Ca containing
either asparagine or serine at residue Ser-700 were used in this study.
N, N-terminal module; O, oligomerization
sequence; P123, properdin or TSP type 1 modules;
E123, EGF-like modules; G, C-terminal globe.
B, schematic of E3Ca. A line denotes the junction
between the last EGF repeat, E3, and the Ca2+-binding
repeats. The sequence of the first loop containing residue
Trp-698 (underlined "W") and the Ser-700
polymorphism is shown in alignment with the homologous sequence in COMP
along with the PSACH and EDM1 mutations that have been identified in
this sequence. Arrows denote Trp-344
(W344) of COMP as well as the PSACH mutation D469 and the
EDM1 mutation D361Y studied by Thur et al. (28). Disulfides
(thick lines) are depicted as deduced for TSP-2 (44).
C, Ca Asn-700 (N700), Ca Ser-700
(S700), E3Ca Asn-700 (N700), and E3Ca Ser-700
(S700) were run on a 12% polyacrylamide gel and stained
with Gel-Code Blue. The Ca and E3Ca proteins had predicted molecular
masses of 30 and 34 kDa, respectively, but migrated very close to the
ovalbumin 43-kDa molecular mass marker.
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Ca2+ Titration of Protein Secondary Structure as
Assessed by Far UV CD--
We performed far UV CD on Ca and E3Ca to
determine whether the Ser-700 polymorphism conferred a change in
secondary structure. Similar to what was reported for full-length TSP-1
(35), we observed that two conformations exist for both Ca Asn-700 and E3Ca Asn-700 (one conformation in the presence of Ca2+ and
one in its absence (Fig. 2, A
and C)). The shape of the CD spectra for both Ca and E3Ca
with a trough of negative ellipticity at 202 nm also was similar to
Ca2+-binding repeats of COMP (Fig. 2, A and
C) (28-30). The addition of 2 mM
Ca2+ resulted in greater negative ellipticity for Ca and
E3Ca (Fig. 2). This change in ellipticity was reversible upon addition
of excess EDTA. The presence of the Ser-700 polymorphism did not alter
greatly the shape of the CD spectra for either Ca Ser-700 or E3Ca
Ser-700 (Fig. 2, B and D) in the presence or
absence of Ca2+.

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Fig. 2.
CD spectra and Ca2+ titrations of
expressed proteins. CD spectra for E3Ca Asn-700 (N700,
panel A), E3Ca Ser-700 (S700, panel
B), Ca Asn-700 (N700, panel C), and Ca
Ser-700 (S700, panel D) were recorded in the
presence (solid line) and absence (dashed line)
of Ca2+ as well as in excess EDTA (dotted line).
Increasing concentrations of Ca2+ were added, and the
ellipticity at 220 nm was recorded. The fractional change in
ellipticity was calculated as described under "Experimental
Procedures" and plotted versus Ca2+
(panel E). E3Ca Asn-700 (N700) is represented by
closed circles ( ), E3Ca Ser-700 (S700) by
closed diamonds ( ), Ca Asn-700 (N700) by
open circles ( ), and Ca Ser-700 (S700) by
open diamonds ( ).
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The proteins were titrated with Ca2+ using ellipticity at
220 nm as a measure. The fractional change in ellipticity was
calculated for each protein and plotted versus
Ca2+ concentration (Fig. 2E). The
EC50 of Ca Asn-700 was determined to be 213 ± 14 µM (x ± S.E., n = 4).
The calculated Hill coefficient of 4.6 ± 0.1 indicates positive
cooperativity. Ca Ser-700 had an EC50 of 218 ± 13 µM and a Hill coefficient of 4.8 ± 0.1. E3Ca Asn-700 had an EC50 of 150 ± 15 µM with
a Hill coefficient of 5.1 ± 0.2. E3Ca Ser-700 had an
EC50 of 168 ± 7 µM with a Hill coefficient of 6.1 ± 0.5. Thus, the Ca proteins titrated at a significantly higher Ca2+ concentration than the E3Ca
proteins, but the polymorphism did not alter the Ca2+
concentration at which the secondary structure transition of the
Ca2+-binding repeats occurs. The presence of the Ser-700
polymorphism, however, caused a significant increase in the
cooperativity of the transition in E3Ca.
The Ser-700 Polymorphism Alters Conformation and Calcium
Sensitivity of a Region in the Ca2+-binding Repeats as
Assayed by UV Fluorescence--
A sole tryptophan (Trp-698), located 2 residues from the Ser-700 polymorphism in the Ca2+-binding
repeats (Fig. 1B), was used as a reporter for local changes in conformation. Ca Asn-700 and Ca Ser-700 were excited at 295 nm
specifically to excite the tryptophan, and emission spectra from 310 to
400 nm in varying concentrations of Ca2+ were collected for
both proteins (Fig. 3, A and
B). The addition of Ca2+ caused alterations in
two spectral parameters, fluorescence intensity and the wavelength of
peak intensity (
max). In the absence of Ca2+, Ca Asn-700 and Ser-700 both had a
max
of 354 nm. Upon addition of 2 mM Ca2+ to Ca
Asn-700, the fluorescence intensity was quenched 2.6-fold, and the
max underwent a blue shift to 340 nm (Fig.
3A). The addition of 2 mM Ca2+ to Ca
Ser-700 caused 1.7-fold quenching of fluorescence and a shift in the
max to 348 nm (Fig. 3B). The addition of 10 mM Ca2+ to Ca Ser-700 caused further quenching
(from 1.7- to 2.0-fold) and a further shift in the
max
to 344 nm.

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Fig. 3.
Fluorescence spectra and Ca2+
titrations of Ca Asn-700 and Ca Ser-700. Tryptophan 698 in Ca
Asn-700 (N700, panel A) and Ca Ser-700
(S700, panel B) was excited at 295 nm in 5 mM MOPS, 0.1 M NaCl in the absence
(dashed line) and presence (solid line) of 2 and
10 mM (dotted line) Ca2+. Intrinsic
fluorescence was measured from 310 to 400 nm, and spectra were recorded
at each Ca2+ concentration from 0 to 10 mM
Ca2+. Using the area under the curve and the equation
described under "Experimental Procedures," Ca2+
titration curves were generated (panel C) with Ca Asn-700
represented by closed circles ( ) and Ca Ser-700
represented by closed diamonds ( ).
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In the absence of Ca2+, both E3Ca Asn-700 and E3Ca Ser-700
(Fig. 4, A and B)
had a
max of 353 nm, very similar to the
max of the Ca proteins in 0 mM
Ca2+. In the presence of 2 mM Ca2+,
both proteins had a
max of 334 nm. This value, lower
than the
max of the Ca proteins in the presence of
Ca2+, was not influenced by the polymorphism. Also in
contrast to Ca proteins, the fold changes (~4-fold) in fluorescence
of E3Ca Asn-700 and E3Ca Ser-700 were similar. Changes in the
fluorescence of Ca and E3Ca upon addition of Ca2+ were
reversible (data not shown) when excess EDTA was added.

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Fig. 4.
Fluorescence spectra and Ca2+
titrations of E3Ca Asn-700 and E3Ca Ser-700. Tryptophan 698 in Ca
Asn-700 (N700, panel A) and Ca Ser-700
(S700, panel B) was excited at 295 nm in 5 mM MOPS, 0.1 M NaCl in the absence
(dashed line) and presence (solid line) of 2 mM Ca2+. Intrinsic fluorescence was measured
from 310 to 400 nm, and spectra were recorded at each Ca2+
concentration from 0 to 2 mM Ca2+. Using the
area under the curve and the equation described under "Experimental
Procedures," Ca2+ titration curves were generated
(panel C) with E3Ca Asn-700 represented by closed
circles ( ) and E3Ca Ser-700 represented by closed
diamonds ( ).
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The fractional change in total fluorescence for each protein was
calculated at each Ca2+ concentration from 0 to saturating
Ca2+ and then plotted versus Ca2+
concentration (Figs. 3C and 4C, mM
[Ca2+]). The titration curves were
sigmoidal and exhibited positive cooperativity. The titrations were
influenced by the presence or absence of E3 and the Ser-700
polymorphism. The EC50 for Ca Asn-700 was 390 ± 20 µM (x ± S.E., n = 4).
The presence of the Ser-700 polymorphism caused the EC50 to
more than double to 950 ± 10 µM. The Hill
coefficient for Ca Asn-700 was calculated to be 2.5 ± 0.2. The
calculated Hill coefficient for Ca Ser-700 was lower, 1.4 ± 0.1. The altered EC50 and cooperativity suggest that the Ser-700
polymorphism causes a local structural change in the Ca2+-binding repeats. Inspection of the titration curve
also suggests that the fluorescence characteristics of Ca Asn-700 and
Ser-700 proteins at saturating Ca2+ would be different even
at very high Ca2+ concentration.
Although the presence of the Ser-700 polymorphism did not cause a
detectable conformation change in E3Ca in the absence or presence of 2 mM Ca2+ as assessed by intrinsic UV
fluorescence (Fig. 4, A and B), the two proteins
titrated at different Ca2+ concentrations (Fig.
4C). The EC50 of the calcium-sensitive
structural transition was 70 µM ± 2 for E3Ca Asn-700
(x ± S.E., n = 4) and 110 µM ± 6 for E3Ca Ser-700. The Hill coefficient for E3Ca
Ser-700 was 5.3 ± 0.4, greater than the Hill coefficient for E3Ca
Asn-700, 3.8 ± 0.3.
The Ser-700 Polymorphism Causes Thermal Instability--
The
intrinsic UV fluorescence of E3Ca Asn-700 and E3Ca Ser-700 in a
Ca2+ concentration of 0.2 mM was measured at
varying temperatures (25, 37, 42, 50, and 65 °C). The concentration
of 0.2 mM was selected because this is the lowest
Ca2+ concentration at which both proteins are in a
Ca2+-replete conformation at 37 °C as assessed by
intrinsic UV fluorescence (Fig. 3C). E3Ca Asn-700 in 0.2 mM Ca2+ had a
max of 333-334 nm
at 25 and 37 °C. E3Ca Ser-700 had a
max of 334 and
336 nm at 25 and 37 °C, respectively, slightly higher than E3Ca
Asn-700. Increasing the temperature resulted in an increase in
fluorescence and a red shift in the
max to ~352 nm
(Fig. 5, A and B).
These changes occurred at lower temperatures for E3Ca Ser-700, for
which the red shift was apparent at 42 °C. An increase in
fluorescence and a further shift in the
max were noted
for E3Ca Ser-700 at 50 and 65 °C, respectively. The
max and fluorescence intensity of E3Ca Asn-700 remained
constant through 50 °C. At 65 °C, the
max of E3Ca
Asn-700 underwent a red shift to 352 nm, and the fluorescence intensity
increased.

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Fig. 5.
The effect of temperature on E3Ca Asn-700 and
E3Ca Ser-700 in 0.2 mM Ca2+.
The effects of temperature were measured using intrinsic fluorescence.
E3Ca Asn-700 (N700, panel A) and E3Ca Ser-700
(S700, panel B) were excited at 295 nm, and
emission spectra were recorded from 310 to 400 nm at the following
temperatures: 25, 37, 42, 50, and 65 °C.
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DISCUSSION |
The Ser-700 polymorphism that localizes to the
Ca2+-binding repeats of TSP-1 has been associated with
familial premature CHD (2), raising the question of if or how this
polymorphism alters the structure and function of TSP-1. The results
described above indicate that the Ser-700 polymorphism is associated
with a perturbation in the local conformation of the
Ca2+-binding repeats of TSP-1 at low concentrations of
Ca2+ or high temperatures.
The Ca2+-binding repeats are highly conserved across TSP
family members and species. The importance of this conservation is demonstrated by the finding that missense mutations or minor
expansions/deletions in the Ca2+-binding repeats of COMP
(TSP-5) cause two forms of autosomal dominant skeletal dysplasias,
pseudoachondroplasia and multiple epiphyseal dysplasia (17, 18). These
mutations often change aspartate or asparagine residues that are likely
important for binding Ca2+. The Ser-700 polymorphism is
similar to COMP mutations in that it localizes to the
Ca2+-binding repeats of TSP-1 and changes an asparagine to
serine at a site where there is either an aspartate or asparagine in all TSPs (36, 37). The structural consequences of the Ser-700 polymorphism on the Ca2+-binding repeats were studied by
intrinsic fluorescence and far UV CD to compare
Ca2+-sensitive structural changes with and without the
polymorphic residue.
Intrinsic fluorescence was due to a single tryptophan, Trp-698, within
the first presumptive Ca2+-binding loop of the Ca region
and fortuitously only 2 residues away from the Ser-700 polymorphism
(Fig. 1B). A construct similar to our Ca construct but
derived from COMP and containing PSACH and EDM1 mutations (Fig.
1B) has been expressed in bacterial or mammalian human
embryonic kidney cells and assessed for altered protein
structure using similar techniques (28-30). The single tryptophan,
Trp-344, of the Ca construct from COMP is in the fourth presumptive
Ca2+-binding loop, not in the same Ca2+-binding
loops that harbor the D361Y EDM1 mutation and the D469
PSACH
mutation tested (Fig. 1B) (28). Comparing the Ca constructs of both TSPs, Trp-698 of TSP-1 titrated at a higher Ca2+
concentration than Trp-344 of COMP (0.4 versus 0.2 mM) (28). Ca Ser-700 had its fluorescence transition at
0.95 mM. This increase is similar to the
Ca2+-binding repeats of COMP with the D361Y EDM1 mutation
that had a transition at 1.1 mM (28). Interestingly, the
Ca2+-binding repeats harboring the more severe PSACH
mutation D469
had a fluorescence transition similar to Ca of
wild-type COMP, indicating that the location of the tryptophan may be
critical for detection of conformational changes by intrinsic fluorescence.
The far UV CD spectra of Ca and E3Ca had sharp minima at 202 nm and
were similar to the Ca2+-binding repeats of COMP (28-30).
The Ca2+-binding repeats of TSP-1 and COMP exhibited
transitions at 0.2 mM Ca2+ in this study and
0.3 mM Ca2+ in a study by Thur et
al. (28), respectively. The Ser-700 polymorphism did not alter the
Ca2+ concentration at which the far UV CD transition for Ca
occurs. Similarly, the COMP Ca2+-binding repeats with and
without COMP mutations D361Y, D469
, and D446N have been analyzed by
far UV CD (28, 29). In both such studies, the COMP mutations did not
alter greatly how the spectral changes were titrated by
Ca2+. Therefore, in the absence of a major change in
the CD, the Ser-700 polymorphism of TSP-1 and the D361Y disease-causing
COMP mutation both cause a local structural change as assessed by fluorescence.
The adjacent EGF-like module influenced the conformation of the
Ca2+-binding repeats. The module affected the secondary
structure at intermediate Ca2+ concentrations (Fig.
2E) such that the far UV CD transition for E3Ca occurred at
1.5-fold lower Ca2+ than the transition for Ca. E3 also
caused a blue shift in the
max of Trp-698 in the
presence of Ca2+. The fluorescence transition for E3Ca
occurred at 6-fold lower Ca2+ concentrations and was
associated with increased positive cooperativity compared with Ca. The
fluorescence transition of E3Ca also occurred at a lower
Ca2+ concentration than the far UV CD transition, whereas
the opposite is true for Ca, suggesting that E3 influences the
structure of the adjacent first Ca2+-binding repeat (Repeat
1) that contains both Trp-698 and the Ser-700 polymorphism (Fig.
1B). Despite the stabilizing effect of the third EGF-like
module on the Ca2+-binding repeats, the presence of the
Ser-700 polymorphism still altered the titration of Trp-698. This
finding suggests that the Ser-700 polymorphism alters the affinity and
cooperativity of Ca2+ binding to Repeat 1 in intact
TSP-1.
Alteration of a potential Ca2+-binding residue not only
alters local Ca2+ binding but also sensitizes the
Ca2+-binding repeats to heating. Temperature has a
quenching effect on tryptophan fluorescence regardless of protein
structure (38). Although decreasing the temperature below 20 °C
causes a blue shift in the
max of
N-acetyl-L-tryptophanamide in viscous solvents (39), a red shift is not seen when the temperature is increased above
20 °C (39). Therefore, in MOPS-buffered saline at the temperature
range studied, a change in temperature should not alter the
max unless there is a change in protein structure. Our
studies were carried out in 0.2 mM Ca2+, the
lowest calcium concentration at which both E3Ca Asn-700 and E3Ca
Ser-700 are found in calcium-replete conformations at 37 °C as
assessed by intrinsic fluorescence. Increasing the temperature caused
the Trp-698 in the E3Ca proteins to have increased fluorescence intensity that underwent a red shift, thereby mimicking the
fluorescence pattern seen in the absence of Ca2+. The
temperature-induced change occurred at lower temperatures for
polymorphic E3Ca Ser-700 than wild-type E3Ca Asn-700.
The pathophysiology of PSACH is likely related to the instability of
COMP and its accumulation with type IX collagen and chaperone proteins
in ER vesicles of chondrocytes (31-33). Familial premature CHD
associated with the Ser-700 polymorphism requires that both alleles
encode the polymorphism, whereas PSACH and EDM1 are autosomal dominant
syndromes. Because COMP is a pentamer, a mutation in one allele
statistically is predicted to result in only 3.1% of the homopentamers
containing all wild-type subunits. TSP-1 is a trimeric protein. If only
one allele of TSP-1 encoded the Ser-700 polymorphism, then
statistically 12.5% of the homotrimers would contain asparagine as
residue 700 in all three subunits. TSP-1 also can heterotrimerize with
TSP-2 (40), thereby potentially further diluting the number of TSP
trimers composed of subunits containing the Ser-700 polymorphism. One
may speculate that a threshold of "normal" TSP molecules protects
against disease by proteins carrying destabilizing subunits.
Alternatively, the Ser-700 polymorphism in TSP-1 may be less
destabilizing than any of the PSACH and EDM1 mutations. It is unknown
if trimeric TSP-1 containing residue Ser-700 in all subunits is
destabilized in the ER. However, the observation that patients with
both Ser-700 alleles have significantly lower levels of plasma TSP-1
than control patients (2) suggests that a secretion defect is present
in cells that contribute to the pool of plasma TSP-1. The
Ca2+ concentration of the ER has been estimated to be
~100-800 µM depending upon the cell type and the
method of measurement (41-43). Our fluorescence results demonstrate
that the Ser-700 polymorphism causes a local change in conformation at
the lower end of this Ca2+ range. Therefore, it is possible
that, similar to mutant COMP, TSP-1 Ser-700 is retained in the ER
because of protein aggregation with other extracellular matrix
molecules and chaperone proteins. Future studies are needed both to
determine whether structural alterations caused by the Ser-700
polymorphism alter the secretion of intact TSP-1 and to relate such
results to coronary artery lesions of patients with familial premature
CHD associated with the Ser-700 polymorphism.