EXPRESSION AND MUTAGENESIS OF THROMBOSPONDIN

Thrombospondin is a 420 000-dalton adhesive glycoprotein that is composed of three subunits of equivalent molecular weight. When the cDNA for the complete coding region of the human endothelial cell thrombospondin subunit is expressed in mouse NIH 3T3 cells, a 420000-dalton protein is synthesized and secreted. The expressed protein comigrates with human platelet thrombospondin both in the presence and in the absence of a reducing agent. The expressed protein binds to a monoclonal anti-thrombospondin antibody, heparin, and calcium. In addition to the 420 000-dalton protein, the transfected cell lines also express a variable amount of a 140 000-dalton polypeptide. When the culture supernatants that are produced by cells that are expressing thrombospondin are applied to heparin-Sepharose, the 420 000-dalton and the 140000-dalton proteins are bound to the column and are eluted with buffer containing 0.55 and 0.3 M NaCl, respectively. The 140000-dalton protein only binds to heparin-Sepharose in the presence of calcium. Deletion of the region of homology with procollagen results in defective assembly of the trimer. Deletion of the type 1 or type 2 repeats results in decreased stability of the subunit with the predominant polypeptides that are expressed having molecular weights of 127 000 and 130 000, respectively. These polypeptides retain low-affinity heparin-binding activity. High-affinity heparin binding is markedly diminished by mutations in either of two sequence motifs that include clusters of lysines and arginines. Decreased high-affinity heparin binding is observed when (1) K(80) and K(81) are mutated to Q(80) and N(81), (2) R(23) and K(24) are mutated to Q(23) and N(24), or (3) R(28) and R(29) are mutated to N(28) and Q(29). These results indicate that both of these regions of positively charged amino acids are required for high-affinity heparin binding.