Synthesis of truncated amino-terminal trimers of thrombospondin.
Thrombospondin (TSP) is a 450-kDa glycoprotein that is comprised of three identical disulfide-bonded subunits (1152 amino acids) held together near the heparin-binding amino-terminal globular domains. TSP truncated at residue 277 (TSP-277) or 381 (TSP-381) consisted largely of disulfide-bonded trimers when expressed in COS cells or insect cells. In addition, TSP-381 formed heterotrimers with endogenous COS cell TSP. Cleavage of TSP and the truncated mutants in the proteolytically sensitive region between residues 220 and 237 yielded monomeric amino-terminal fragments. Cys-252 and Cys-256 are the only cysteines between residues 238 and 277 and therefore must bridge among subunits. TSP-381 in which Cys-252 and Cys-256 were changed to glycine was secreted efficiently by COS cells but with only a minor portion of the protein in the form of disulfide-bonded trimers. The sequence of TSP between residues 258 and 283 is predicted to form an amphiphatic alpha-helix. We suggest that assembly of TSP trimers involves formation of an alpha-helical coiled-coil structure which is stabilized by formation of disulfides.
Sottile
J
J
Department of Physiological Chemistry, University of Wisconsin, Madison 53706.
Selegue
J
J
Mosher
D F
DF
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001792:Blood Platelets / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D004220:Disulfides / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D007313:Insecta; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010957:Plasmids; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D013728:Terminator Regions, Genetic; D019699:Thrombospondins
D004220:Disulfides; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D019699:Thrombospondins