Cell attachment activity of the carboxyl-terminal domain of human thrombospondin expressed in Escherichia coli.
Thrombospondin (TS) is a multidomain, adhesive glycoprotein that associates with cells through multiple cell attachment sites. One of these has been located in or near the globular COOH-terminal region of TS by the monoclonal antibody (mAb) C6.7, which inhibits the attachment of human melanoma cells (G361) to TS. The epitope for C6.7 lies within the last 122 residues of the COOH-terminal domain of TS. This domain is distant from two known cell attachment sites in TS, namely the NH2-terminal heparin-binding domain and the CSVTCG sequences in the type I repeats, but is close to the RGDA sequence, an integrin-dependent cell attachment site. In order to separate the adhesive activity of the TS COOH-terminal domain from that of the RGD sequence, we have expressed the COOH-terminal 212 amino acids (residues 941-1152) of TS in Escherichia coli using the expression vector pRIT2T. The resultant fusion protein is effective in supporting G361 cell attachment even though it lacks the RGD sequence. In addition, the expressed protein inhibits adhesion of G361 cells to intact TS. mAb C6.7 blocks adhesion to the expressed TS COOH-terminal domain whereas GRGDSP and VTCG peptides are not inhibitory. These results show that the TS COOH-terminal domain contains a separate cell adhesion site, defined by mAb C6.7, that is distinct from the other adhesion sites of TS.
Kosfeld
M D
MD
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Pavlopoulos
T V
TV
Frazier
W A
WA
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002448:Cell Adhesion; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008545:Melanoma; D008969:Molecular Sequence Data; D010957:Plasmids; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019699:Thrombospondins; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D010980:Platelet Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D019699:Thrombospondins