A Polymorphism in Thrombospondin-1 Associated with Familial Premature Coronary Heart Disease Causes a Local Change in Conformation of the Ca2+-binding Repeats*

Blue-leaf A. HannahDagger §, Tina M. MisenheimerDagger , Douglas S. AnnisDagger , and Deane F. MosherDagger

From the Dagger  Department of Medicine, Medical Sciences Center and the § Molecular and Cellular Pharmacology and Medical Scientist Training Programs, University of Wisconsin, Madison, Wisconsin 53706

Received for publication, October 31, 2002, and in revised form, January 3, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A single nucleotide polymorphism that substitutes a serine for an asparagine at residue 700 in the Ca2+-binding repeats of thrombospondin-1 is associated with familial premature coronary heart disease. We expressed the Ca2+-binding repeats alone (Ca) or with the third epidermal growth factor-like module (E3Ca), without (Asn-700) or with (Ser-700) the disease-associated polymorphism. The intrinsic fluorescence of a single tryptophan (Trp-698) adjacent to the polymorphic residue was quenched cooperatively by adding Ca2+. The third epidermal growth factor-like repeat dramatically altered the Ca2+-dependent fluorescence transition for the Asn-700 constructs; the half-effective concentration (EC50) of Ca Asn-700 was 390 µM, and the EC50 of E3Ca Asn-700 was 70 µM. The Ser-700 polymorphism shifted the EC50 to higher Ca2+ concentrations (Ca Ser-700 EC50 of 950 µM and E3Ca Ser-700 EC50 of 110 µM). This destabilizing effect is due to local conformational changes, as the Ser-700 polymorphism did not influence the secondary structure of E3Ca or Ca as assessed by far UV circular dichroism. At 200 µM Ca2+, in which both E3Ca Asn-700 and Ser-700 are in the Ca2+-replete conformation at 37 °C, the fluorescence of E3Ca Ser-700 reverted to the Ca2+-depleted spectrum at 50 °C compared with 65 °C for E3Ca Asn-700. These findings indicate that the Ser-700 polymorphism subtly but significantly sensitizes the calcium-binding repeats to removal of Ca2+ and thermal denaturation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (Delta F) relative to the Ca2+-depleted protein from 0 mM to saturation was calculated as Delta 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 Delta F versus Ca2+ concentration. The Hill coefficient was determined by calculating the slope of the line generated by log[Delta F/(1 - Delta F)] versus log[Ca2+], and the values used for Delta 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 (Delta E) was calculated using the equation Delta 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 Delta E versus Ca2+. The Hill coefficient was determined by calculating the slope of the line generated by log[Delta E/(1 - Delta E)] versus log[Ca2+].

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 D469Delta 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.

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 (black-diamond ), Ca Asn-700 (N700) by open circles (open circle ), and Ca Ser-700 (S700) by open diamonds (diamond ).

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 (lambda max). In the absence of Ca2+, Ca Asn-700 and Ser-700 both had a lambda max of 354 nm. Upon addition of 2 mM Ca2+ to Ca Asn-700, the fluorescence intensity was quenched 2.6-fold, and the lambda 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 lambda 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 lambda 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 (black-diamond ).

In the absence of Ca2+, both E3Ca Asn-700 and E3Ca Ser-700 (Fig. 4, A and B) had a lambda max of 353 nm, very similar to the lambda max of the Ca proteins in 0 mM Ca2+. In the presence of 2 mM Ca2+, both proteins had a lambda max of 334 nm. This value, lower than the lambda 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 (black-diamond ).

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 lambda max of 333-334 nm at 25 and 37 °C. E3Ca Ser-700 had a lambda 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 lambda 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 lambda max were noted for E3Ca Ser-700 at 50 and 65 °C, respectively. The lambda max and fluorescence intensity of E3Ca Asn-700 remained constant through 50 °C. At 65 °C, the lambda 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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 D469Delta 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 D469Delta 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, D469Delta , 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 lambda 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 lambda 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 lambda 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.

    ACKNOWLEDGEMENTS

We thank Darrel McCaslin for help with the circular dichroism and Kristin Huwiler for advice and helpful discussions.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL54462 and T32 GM08688 and by American Heart Association Grant 0215322Z. Circular dichroism data were obtained at the University of Wisconsin-Madison Biophysics Instrumentation Facility, which is supported by the University of Wisconsin-Madison and by National Science Foundation Grant S10 RR1370.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.

To whom correspondence should be addressed. Tel.: 608-262-4455; Fax: 608-263-4969; E-mail: bahannah@students.wisc.edu.

Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M211185200

    ABBREVIATIONS

The abbreviations used are: CHD, coronary heart disease; TSP, thrombospondin; COMP, cartilage oligomeric matrix protein; PSACH, pseudoachondroplasia; EDM1, multiple epiphyseal dysplasia; ER, endoplasmic reticulum; MOPS, 4-morpholinepropanesulfonic acid; EGF, epidermal growth factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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