Immunochemical analysis of the structure of the signature domains of thrombospondin-1 and thrombospondin-2 in low calcium concentrations.
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.
Annis
Douglas S
DS
Department of Medicine, University of Wisconsin, Madison, Wisconsin 53706.
Gunderson
Kristin A
KA
Department of Medicine, University of Wisconsin, Madison, Wisconsin 53706.
Mosher
Deane F
DF
Department of Medicine, University of Wisconsin, Madison, Wisconsin 53706. Electronic address: dfmosher@wisc.edu.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002118:Calcium / Q000494:pharmacology*; D004815:Epidermal Growth Factor / Q000737:chemistry; D000939:Epitopes; D006801:Humans; D007120:Immunochemistry; D037102:Lectins / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D019700:Thrombospondin 1 / Q000737:chemistry* / Q000276:immunology; D019699:Thrombospondins / Q000737:chemistry* / Q000276:immunology; D000099262:Thrombospondin 2
D000911:Antibodies, Monoclonal; D002118:Calcium; D004815:Epidermal Growth Factor; D000939:Epitopes; D037102:Lectins; D019700:Thrombospondin 1; D019699:Thrombospondins; D000099262:Thrombospondin 2