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 420,000-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 140,000-dalton proteins are bound to the column and are eluted with buffer containing 0.55 and 0.3 M NaCl, respectively. The 140,000-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Lawler
J
J
Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115.
Ferro
P
P
Duquette
M
M
D016475:3T3 Cells / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D002872:Chromosome Deletion; D004727:Endothelium / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006493:Heparin / Q000737:chemistry / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D019699:Thrombospondins; D014162:Transfection
D002352:Carrier Proteins; D010980:Platelet Membrane Glycoproteins; D019699:Thrombospondins; D006493:Heparin