Thrombospondin-4 binds specifically to both collagenous and non-collagenous extracellular matrix proteins via its C-terminal domains

Full-length and truncated forms of rat thrombospondin-4 (TSP-4) were expres sed recombinantly in a mammalian cell line and purified to homogeneity. Bio chemical analysis revealed a limited proteolytic processing, which detaches the N-terminal heparin-binding domain from the rest of the molecule and co nfirmed the importance of the heptad-repeat domain for pentamerization. In electron microscopy the uncleaved TSP-4 was seen as a large central particl e to which five smaller globules are attached by elongated Linker regions. Binding of TSP-4 to collagens and to non-collagenous proteins could be dete cted in enzyme-linked immunosorbent assay-style ligand binding assays, by s urface plasmon resonance spectroscopy, and in rotary shadowing electron mic roscopy. Although the binding of TSP-4 to solid-phase collagens was enhance d by Zn2+, that to non-collagenous proteins was not. The interactions of TS P-4 with both classes of proteins are mediated by C-terminal domains of the TSP-4 subunits but do not require an oligomeric structure. Major binding s ites for TSP-4 are located in or close to the N- and C-terminal telopeptide s in collagen I, but additional sites are detected in more central regions of the molecule.