CHARACTERIZATION OF HUMAN THROMBOSPONDIN-4

The thrombospondins are a family of extracellular calcium binding prot eins that are involved in cell proliferation, adhesion, and migration. We have sequenced full-length human thrombospondin-4 and characterize d the recombinant protein. In contrast to Xenopus laevis thrombospondi n-4, the human protein contains an RGD cell binding sequence in the th ird type 3 repeat. Transfection of mouse NIH3T3 fibroblasts or C2C12 m yoblasts with a full-length human thrombospondin-4 cDNA results in the expression of a polypeptide with a reduced molecular weight of 140,00 0. In the absence of reducing agent, the expressed protein has an appa rent molecular weight of 550,000. Recombinant thrombospondin-4 has bee n purified from the culture supernatant by heparin-Sepharose and anti- thrombospondin-4 antibody-Affi-gel affinity chromatography. Electron m icroscopy indicates that thrombospondin-4 is composed of five subunits with globular domains at each end. The observation of a calcium-depen dent change in the electron microscopic appearance of thrombospondin-4 is consistent with limited tryptic digestion data that indicate that thrombospondin-4 is resistant to digestion in the presence of calcium. These data indicate that thrombospondin-4 is a pentameric protein tha t binds to heparin and calcium.