Disulfide connectivity of recombinant C-terminal region of human thrombospondin 2

The thrombospondin (TSP) family of extracellular glycoproteins consists of five members in vertebrates, TSP1 to -4 and TSP5/cartilage oligomeric matrix protein, and a single member in Drosophila. TSPs are modular multimeric proteins. The C-terminal end of a monomer consists of 3-6 EGF-like modules; seven tandem 23-, 36-, or 38-residne aspartate-rich, Ca2+-binding repeats; and an similar to 230-residue C-terminal sequence. The Ca2+-binding repeats and C-terminal sequence are spaced almost exactly the same in different TSPs and share many blocks of identical residues. We studied the C-terminal portion of human TSP2 from the third EGF-like module through the end of the protein (E3CaG2). E3CaG2, CaG2 lacking the EGF module, and Ca2 composed of only the Ca2+. binding repeats were expressed using recombinant baculoviruses and purified from conditioned media of insect cells. As previously described for intact TSP1, E3CaG2 bound Ca2+ in a cooperative manner as assessed by equilibrium dialysis, and its circular dichroism spectrum was sensitive to the presence of Ca2+. Mass spectrometry of the recombinant proteins digested with endoproteinase Asp-N revealed that disulfide pairing of the IS cysteines in the Ca2+-binding repeats and C-terminal sequence is sequential, i.e. a 1-2, 3-4, 5-6, etc., pattern.