Thrombospondins (TSPs) are secreted glycoproteins that play key roles in interactions between cells and the extracellular matrix. Here, we describe the 2.6 Å resolution crystal structure of the glycosylated signature domain of human TSP-2, which includes three epidermal growth factor-like (EGF-like) modules, 13 aspartate-rich repeats, and a lectin-like module. These elements interact extensively to form three striking structural regions termed the stalk, wire, and globe. The TSP-2 signature domain is stabilized by these interactions and by a network of 30 bound Ca 2+ ions and 18 disulfide bonds. The structure suggests how genetic alterations of TSPs result in disease.Keywords calcium binding; extracellular matrix; lectin-like module; skeletal dysplasia; epidermal growth factor-like module; protein crystallography Thrombospondins (TSPs) are a family of five secreted multimodular metallo/glycoproteins that have diverse roles involving interactions between cells and the extracellular matrix {Adams, 2004 #3568}. For TSP-2, these functions are critical for such processes as synaptogenesis{Christopherson, 2005 #3578}, megakaryocytopoiesis{Kyriakides, 2003, and the foreign body reaction{Kyriakides, 2001 #3588}. All TSPs contain a highly conserved "signature domain" consisting of tandem epidermal growth factor-like (EGF-like) modules, aspartate-rich repeats, and a lectin-like module at their C-termini ( Fig. 1a). These modules are conserved with remarkable fidelity as is evidenced by a 458-residue stretch of fly TSP and human TSP-2 in the signature domain that are 60% identical without an insertion or deletion{Adams, 2003 #3571;LaBell, 1993 #340}. The signature domain contains the sites of polymorphisms and mutations linked to familial coronary artery disease and two skeletal disorders, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1){Topol, 2001 #3537; Kennedy, 2005 #3585;Posey, 2004 #3579}. Here, we describe the 2.6 Å resolution crystal structure of the complete glycosylated signature domain of human TSP-2, which includes three epidermal growth factor-like (EGF-like) modules, 13 aspartate-rich repeats, and a lectin-like module, along with 30 bound Ca 2+ ions. The structure reveals the highly intertwined nature of the signature domain that includes major interactions among the different parts of the molecule. The structure furthermore establishes a molecular explanation of how known genetic alterations of TSPs result in disease.Correspondence and requests for materials should be addressed to D. NIH Public Access Results Structure of the human TSP-2 signature domainThe crystal structure of the signature domain of human TSP-2 was solved using a combination of anomalous scattering and molecular replacement approaches. Electron-density maps, particularly those derived from molecular replacement using the structure of a fragment of the signature domain from TSP-1{Kvansakul, 2004 #3561}, were of high quality ( Fig. 1b), and allowed modelling of the entire domain structure.The overall structu...

A single nucleotide polymorphism that results in substitution at residue 700 of a serine (Ser-700) for an asparagine (Asn-700) in thrombospondin-1 is associated with familial premature coronary artery disease. The polymorphism is located in the first of 13 Ca 2؉ -binding motifs, within a consensus sequence in which Asn-700 likely coordinates Ca 2؉ . Equilibrium dialysis of constructs comprised of the adjoining epidermal growth factor-like module and the Ca 2؉ -binding region (E3Ca) demonstrated that E3Ca Ser-700 binds significantly less Ca 2؉ than E3Ca Asn-700 at low [Ca 2؉ ]. The hypothesis that this difference is due to loss of a binding site in Ser-700 protein was tested with truncations of E3Ca containing four (Tr4), three (Tr3), two (Tr2), or one (Tr1) N-terminal Ca 2؉ -binding motifs. The Ser-700 truncation constructs bound 1 fewer Ca 2؉ than matching Asn-700 constructs and exhibited decreased binding affinities. Intrinsic fluorescence of a tryptophan at residue 698 (Trp-698) in the most N-terminal motif was cooperatively quenched by the addition of Ca 2؉ to Asn-700 Tr2, Tr3, and Tr4 constructs. In Ser-700 constructs, quenching of Trp-698 was incomplete in the Tr2 and Tr3 constructs and complete only in the Tr4 construct. Ca 2؉ -induced quenching of Ser-700 constructs required higher [Ca 2؉ ] and was slower as shown in stopped-flow experiments than quenching of Asn-700 constructs. Such differences were not found with Tb 3؉ , which quenched the fluorescence of Asn-700 and Ser-700 constructs equivalently. Thus, the Ser-700 polymorphism alters a rapidly filled, high affinity Ca 2؉ -binding site in the first Ca 2؉ -binding motif. Slower Ca 2؉ binding to adjoining motifs partly compensates for the change.

The C-terminal regions of thrombospondins (TSPs) contain three elements, EGF-like modules (E), a series of Ca(2+)-binding repeats (Ca), and a C-terminal sequence (G). We have looked for interactions among these elements in four recombinant proteins based on human TSP-2: E3CaG-2, CaG-2, E3Ca-2, and Ca-2. When bound Ca(2+) was assayed by atomic absorption spectroscopy or an equilibrium dialysis protocol in which Ca(2+) was removed from the proteins prior to equilibrium dialysis, E3CaG-2 bound 22-27 Ca(2+), CaG-2 bound 17-20 Ca(2+), and E3Ca-2 and Ca-2 bound 14-20 Ca(2+). Approximately 10 of the bound Ca(2+) in E3CaG-2 were exchangeable. The far UV circular dichroism (CD) spectrum of Ca(2+)-replete E3CaG-2 contained a strong negative band at 203 nm attributable to Ca and a less intense negative band at 218 nm attributable to Ca and G. Chelation of Ca(2+) with EDTA shifted the 203 nm band of all four proteins and the 218 nm band of E3CaG-2 and CaG-2 to less negative positions. The apparent EC50 for the far UV CD transition was 0.22 mM Ca(2+) for all proteins, indicating that Ca(2+) binding to Ca is primarily responsible for the CD change. Near UV CD and intrinsic fluorescence revealed that the tryptophan residues in G are sensitive to changes in Ca(2+). Differential scanning calorimetry of the proteins in 2 mM Ca(2+) showed that E3CaG-2 melts with two transitions, 44-51 degrees C and 75-83 degrees C. The lower transition required G, while the higher transition required Ca. Both transitions were stabilized in constructs containing E3. These results indicate that E3, Ca, and G function as a complex structural unit, and that the structures of both Ca and G are influenced by the presence or absence of Ca(2+).

A single nucleotide polymorphism that substitutes a serine for an asparagine at residue 700 in the Ca 2؉ -binding repeats of thrombospondin-1 is associated with familial premature coronary heart disease. We expressed the Ca 2؉ -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 Ca 2؉ . The third epidermal growth factor-like repeat dramatically altered the Ca 2؉ -dependent fluorescence transition for the Asn-700 constructs; the half-effective concentration (EC 50 ) of Ca Asn-700 was 390 M, and the EC 50 of E3Ca Asn-700 was 70 M. The Ser-700 polymorphism shifted the EC 50 to higher Ca 2؉ concentrations (Ca Ser-700 EC 50 of 950 M and E3Ca Ser-700 EC 50 of 110 M). 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 M Ca 2؉ , in which both E3Ca Asn-700 and Ser-700 are in the Ca 2؉ -replete conformation at 37°C, the fluorescence of E3Ca Ser-700 reverted to the Ca 2؉ -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 Ca 2؉ and thermal denaturation.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 Ca 2ϩ -binding (type 3) repeats, and a long C-terminal sequence (Fig. 1A). The Ca 2ϩ -binding and C-terminal sequences are unique to TSPs and are highly conserved. For instance, the alignment of human TSP-1 and Drosophila TSP de...

The splenic pancreas of 165 day old diabetic KKAy and age-matched nondiabetic C57BL/6 mice was examined by morphometry and immunocytochemistry at the light microscopic level and by radioimmunoassay to evaluate the morphology, surface area, endocrine cell composition and hormone content of the pancreatic islets. The insulin cells of the diabetic mice were severely degranulated and many of the glucagon, somatostatin and pancreatic polypeptide cells were displaced from the mantle to the core of the islet tissue where the non-insulin cells appeared to lose their continuity. The topography of some of the islets of KKAy mice was further deranged by acinar cells among the endocrine tissue. Morphometric analysis revealed that the surface area of the islets of KKAy mice was significantly expanded in comparison with that of C57BL/6 mice. The volume and numerical percents of the insulin cells were significantly increased whereas those of the glucagon and somatostatin cells were decreased in the KKAy mice. Since only the mean absolute number of insulin cells was elevated in the diabetic mice, the alteration in the relative proportions of the non-insulin cells and hypertrophy of the islets seemed to be a manifestation of insulin cell hyperplasia. Pancreatic insulin and somatostatin contents were markedly diminished in the islets of KKAy compared with those of C57BL/6 mice. These results demonstrate that the microscopic anatomy, endocrine cell populations and hormone content of the pancreatic islets are deranged in the KKAy mouse with severe hyperinsulinemia and hyperglycemia.