Identification and characterization of a tumor cell receptor for CSVTCG, a thrombospondin adhesive domain.
We have previously shown that peptides derived from the thrombospondin sequence, CSVTCG, promoted tumor cell adhesion. To further investigate this observation, the CSVTCG-tumor cell adhesion receptor from A549 human lung adenocarcinoma cells was isolated and characterized. A single protein peak was isolated by CSVTCG affinity chromatography which also analyzed as a single peak by anion exchange chromatography. The purified protein had a pI of 4.7 and analyzed on SDS-gels as a single band of M(r) = 50,000 under nonreducing conditions and as two protein bands of M(r) = 50,000, and 60,000 under reducing conditions. Purified CSVTCG binding protein (CBP) bound either CSVTCG- or TSP-Sepharose but showed little interaction with either VCTGSC- or BSA-Sepharose. CBP was cell surface exposed. CSVTCG derivatized with [125I] Bolton-Hunter reagent was taken up by cells in a dose-dependent manner and the cell association was inhibited with a monospecific polyclonal anti-CBP antibody. Examination of the cell proteins crosslinked to labeled CSVTCG by SDS-gel electrophoresis revealed one band that comigrated with purified CPB. Using an in vitro binding assay, purified CBP bound mannose, galactose, and glucosamine-specific lectins. CBP bound TSP saturably and reversibly. The binding was Ca+2/Mg+2 ion dependent and inhibited with fluid phase TSP and anti-CBP. Little or no binding was observed on BSA, fibronectin, GRGES, and GRGDS. Heparin, but not lactose, inhibited binding. Anti-CBP IgG and anti-CSVTCG peptide IgG inhibited A549 cell spreading and adhesion on TSP but not on fibronectin and laminin. These results indicate that CBP and the CSVTCG peptide domain of TSP can mediate TSP-promoted tumor cell adhesion.
Tuszynski
G P
GP
Department of Medicine, Medical College of Pennsylvania, Philadelphia 19129.
Rothman
V L
VL
Papale
M
M
Hamilton
B K
BK
Eyal
J
J
D000230:Adenocarcinoma; D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000032:analysis; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D016023:Integrins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D037102:Lectins; D008175:Lung Neoplasms; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D019699:Thrombospondins; D014407:Tumor Cells, Cultured
D002241:Carbohydrates; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D016023:Integrins; D037102:Lectins; D010980:Platelet Membrane Glycoproteins; D019699:Thrombospondins