Properties of recombinant mouse thrombospondin 2 expressed in Spodoptera cells.
A baculovirus system was used to express full-length recombinant mouse thrombospondin 2 (rTSP2) as a disulfide-bonded homotrimer with an NH2 terminus beginning with Asp20.rTSP2, like TSP1, was more sensitive to trypsin digestion if depleted of calcium ion. The trypsin digestion pattern of rTSP2 and TSP1 differed in that trypsin cut between the first and second type 1 modules of rTSP2. For bovine aortic endothelial cells adhering to TSP-coated polystyrene plates, reduction after coating caused both TSPs to be much more adhesive; these adhesions were blocked completely by RGDS peptide or antibody to alpha v beta 3 integrin.rTSP2 and TSP1 also mediated the adhesion of HT-29 human colon adenocarcinoma cells that carry alpha v beta 5 but not alpha v beta 3 integrin. Antibody to alpha v beta 5 did not inhibit adhesion of HT-29 cells to TSP1 or rTSP2. Rather, adhesion of HT-29 cells was decreased by treatment of TSPs with EDTA, abolished by reduction of the TSPs, and, in the case of rTSP2, blocked by heparin. Adhesion of MG63 cells to both TSPs was complex. Treatment with EDTA enhanced the adhesive activity of rTSP2 but decreased the adhesive activity of TSP1. These results show that TSP2 can be processed and secreted when overexpressed using baculovirus, TSP1 and rTSP2 differ in protease susceptibility in the type 1 module region, and TSP1 and rTSP2 mediate cell adhesion by complex and similar but not identical mechanisms.
Chen
H
H
Department of Medicine, University of Wisconsin, Madison 53706.
Sottile
J
J
O'Rourke
K M
KM
Dixit
V M
VM
Mosher
D F
DF
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002417:Cattle; D002448:Cell Adhesion; D002478:Cells, Cultured; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D019699:Thrombospondins; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D019699:Thrombospondins; D014357:Trypsin