A cDNA construct of tissue inhibitor of metalloproteinases (TIMP) linked to the last exon of Thy-1 confers glycophospholipid anchorage on this naturally secreted protein.
A naturally secreted protein, tissue inhibitor of metalloproteinases (TIMP), has been transiently expressed on the surface of transfected COS cells and stably on transfected murine BW 5147 thymoma cells, by linkage of the entire coding sequence of the cDNA to the last exon of Thy-1. Thy-1 is a glycophospholipid-linked protein. In COS cells the chimaeric protein can be labelled by [3H]ethanolamine, which is a component of glycophospholipid anchors. Ltk- cells cannot anchor proteins by glycan phosphatidylinositol linkage and were found to be unable to express the engineered protein extracellularly on their plasma membranes. Phosphatidylinositol-specific phospholipase C treatment released 90% of the protein from all BW 5147 cells, but very little from the COS-1 cells. It is concluded that the last exon of Thy-1 has conferred the property of glycophospholipid anchorage on the normally secreted protein TIMP.
Clissold
P M
PM
MRC Molecular Immunopathology Unit, Medical Research Council Centre, Cambridge, U.K.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002678:Chimera; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D005091:Exons; D006017:Glycolipids / Q000378:metabolism*; D006023:Glycoproteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D017261:Glycosylphosphatidylinositols; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism*; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D018800:Thy-1 Antigens; D019714:Tissue Inhibitor of Metalloproteinases; D014158:Transcription, Genetic; D014162:Transfection
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D006017:Glycolipids; D006023:Glycoproteins; D017261:Glycosylphosphatidylinositols; D010716:Phosphatidylinositols; D012315:RNA Caps; D012333:RNA, Messenger; D011994:Recombinant Proteins; D018800:Thy-1 Antigens; D019714:Tissue Inhibitor of Metalloproteinases; D004247:DNA