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-residue aspartate-rich, Ca(2+)-binding repeats; and an approximately 230-residue C-terminal sequence. The Ca(2+)-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 Ca(2+)-binding repeats were expressed using recombinant baculoviruses and purified from conditioned media of insect cells. As previously described for intact TSP1, E3CaG2 bound Ca(2+) in a cooperative manner as assessed by equilibrium dialysis, and its circular dichroism spectrum was sensitive to the presence of Ca(2+). Mass spectrometry of the recombinant proteins digested with endoproteinase Asp-N revealed that disulfide pairing of the 18 cysteines in the Ca(2+)-binding repeats and C-terminal sequence is sequential, i.e. a 1-2, 3-4, 5-6, etc., pattern.
Misenheimer
T M
TM
Department of Medicine and the Biotechnology Center, University of Wisconsin, Madison, Wisconsin 53706, USA. tmmisenh@facstaff.wisc.edu
Hahr
A J
AJ
Harms
A C
AC
Annis
D S
DS
Mosher
D F
DF
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D019699:Thrombospondins / Q000737:chemistry / Q000378:metabolism*; D000099262:Thrombospondin 2
D002118:Calcium; D004220:Disulfides; D011994:Recombinant Proteins; D019699:Thrombospondins; D000099262:Thrombospondin 2