Immunochemical Analysis of the Structure of the Signature Domains of Thrombospondin-1 and Thrombospondin-2 in Low Calcium Concentrations

Abstract

Thrombospondins (TSPs) undergo conformational changes upon removal of calcium. The eight C-type and five N-type calcium-binding repeats of TSP-2 form a circuitous wire that, in 2 mM calcium, interacts at its ends with more N-terminal epidermal growth factor (EGF)-like modules, EGF2 and EGF3, and the C-terminal lectin-like module. These components, along with the other EGF-like module(s), form the signature domain of TSPs. Characterization of conformation-sensitive epitopes of monoclonal antibodies to human TSP-2 and its TSP-1 homolog have given insights into the structure of the signature domain in the absence of calcium. The epitope for 4B6.13 anti-TSP-2 was localized to His-722 and Leu-703 in repeat 1C of the wire; recognition only occurred in constructs that included EGF3, the rest of the wire, and the lectin-like module and in the presence of calcium. The epitope for C6.7 anti-TSP-1 was localized to Glu-609 in the EGF2 module. The C6.7 epitope was preferentially recognized when EGF2 was expressed in the context of EGF1, EGF3, the wire, and the lectin-like module. Preferential recognition of the C6.7 epitope did not require calcium. Rotary shadowing electron microscopy of TSP-1 has shown elongation of the stalk and diminution of the C-terminal globule. We propose a model whereby at low calcium concentrations the lectin-like module drops away from EGF3 concomitant with changes in conformation of the wire and loss of the 4B6.13 epitope. A critical feature of the model is interaction of repeat 12N of the wire with EGF2 in both the presence and absence of calcium.

Calcium indirectly regulates immunochemical reactivity and functional activities of the N-domain of thrombospondin-1

“…This is consistent with previous reports that heparin binding does not require calcium (Dixit et al, 1984) and demonstrates that the epitopes in the N-module for all three antibodies are distinct from the heparin binding site and do not sterically interfere with heparin binding. Instead, we observed a significant enhancement of TSP1 binding to heparin in the presence of 4B6 and 2D11 relative to TSP1 alone or TSP1 complexed with a control TSP1 antibody ahTSP (HB8432), which binds to an epitope in the EGF-like repeats (Annis et al, 2006;Annis et al, 2007). Consistent with the finding that the heparin binding site overlaps with a sulfatidebinding site in the N-module (Yu et al, 2000), antibodies 4B6 and 2D11 and, to a lesser extent, 5H9 enhanced TSP1 binding to sulfatide (Fig.…”

“…Recent crystal structures of C-terminal constructs based on TSP1 and TSP2 identified a total of 30-31 calcium binding sites, one in the second EGF repeat, 26 in the so called wire modules, and three or four in the lectin-like G module (Kvansakul et al, 2004;Carlson et al, 2005). Removal of calcium alters the hydrodynamic properties and circular dichroism spectrum of TSP1 (Lawler and Simons, 1983;Vuillard et al, 1991), alters its structure as visualized by rotary shadowing electron microscopy (Galvin et al, 1985;Lawler et al, 1985), and produces local conformational changes in the wire modules as assessed by electron spin resonance (Slane et al, 1988), intrinsic fluorescence , and exposure of calcium-dependent epitopes recognized by two TSP1 antibodies recognizing epitopes in the wire module, A6.1 and D4.6 (Dixit et al, 1986;Annis et al, 2006) (Annis et al, 2007). Removing calcium markedly enhances binding of TSP1 to type V collagen (Galvin et al, 1987).…”

Thrombospondins: from structure to therapeutics

“…Shape and dimensions of the modules of THBS gained from the NMR and crystallographic structures are compatible with and allow for a more exact interpretation of images of intact THBS-2 obtained by rotary shadowing electron microscopy [61,62]. Models based on conformation-sensitive antibody-binding experiments suggest two possible results of calcium loss.…”

“…In the minimal model, wire repeat 13C loses its structure, and all interactions between the wire and the lectin-like module are lost. In the second case, repeats 11C and 13C lose their structure, but the interactions between repeats 8N and 9C of the wire and the lectin-like module are maintained [62]. In both models, interactions of the lectin-like module and EGF3 are lost, allowing the lectin-like module to fall away and elongating the stalk by 60 Å [62].…”

“…Models based on conformation-sensitive antibody-binding experiments suggest two possible results of calcium loss. These models are based on antibody evidence that wire repeat 12N remains bound to EGF2 when calcium is removed [62]. In the minimal model, wire repeat 13C loses its structure, and all interactions between the wire and the lectin-like module are lost.…”