Identification and characterization of a tumor cell receptor for CSVTCG, a thrombospondin adhesive domain.

Abstract

Abstract. 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 Mr = 50,000 under nonreducing conditions and as two protein bands of Mr = 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 [1251] Bolton-Hunter reagent was taken up by cells in a dose-dependent manner and the cell association was inhibited with a monospecific polyclonal anfi-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.T HROMBOSPONDIN (TSP) 1 is a large multi-domain protein that was originally purified from platelets (15) but has since been found in many tissues including bone (23), muscle (39), skin (38, 39), and brain (20). A major physiological function of this protein appears to involve cell-extracellular matrix interaction because purified preparations of TSP promote cell-substratum interaction of a variety of cell types (34), including platelets (30, 33). Support for a role of TSP in matrix-cell interaction was demonstrated in a recent study by Arbeille et al. (3) who showed that TSP was localized in microfibrils at the junction between basement membrane and connective tissue in sections of human placenta, porcine arteries and skin. Additional support comes from several in vitro studies which show that TSP has a high binding affinity for components of the basement membrane such as collagen (8), fibronectin (13), and heparansulfate containing macromolecules (9).Recently, our group (32) and two others (21, 22) independently demonstrated that peptides containing the sequence VTCG promoted cell attachment. Rich et al. (22) that several peptides containing VTCG from region H of the circumsporozoite protein isolated from the malarial parasite Plasmodium vivax strongly promoted the attachment of T cells and myeloid cells. Prater et ...

Endothelial apoptotic activity of angiocidin is dependent on its polyubiquitin binding activity

“…We previously isolated a protein from lung tumour extracts by peptide affinity chromatography using the type 1 TSP-1 repeat peptide CSVTCG (Tuszynski et al, 1993). The protein was cloned from a prostate cell cDNA library and the full-length cDNA was nearly identical to that reported for S5a, a ubiquitin recognition subunit of the 19S proteasome subunit and antisecretory factor, a secreted protein that regulates water secretion by the large bowel (Johansson et al, 1997).…”

The role of angiocidin in sarcomas

“…Our laboratory and others have shown that TSP-1 up-regulates tumor-associated proteases and promotes tumor cell invasion and metastases in epithelial malignancies. 9,[15][16][17][25][26][27][28][29][30][31][32][33][34] We have recently shown that TSP-1 promotes tumor cell invasion in pancreatic and gastric cancer through a mechanism that involves up-regulation of gelatinases, 16,25,29,35 key enzymes in the metastatic cascade. 36,37 The potential role of TSP-1 in mesenchymal tumors such as sarcomas is presently unknown.…”

“…The CSVTCG peptide, homologous to the CSVTCG sequence in the type I domain of TSP-1, binds with high specificity to the TSP-1-binding domain on angiocidin and inhibits A549 human lung adenocarcinoma tumor cell metastasis. 15 The 25-mer peptide containing a 20 amino acid sequence from angiocidin mediates an antitumor effect by competitively binding to TSP-1. 10 We measured the inhibitory effects of our peptides on TSP-1-mediated gelatinase expression and sarcoma tumor cell invasion.…”

“…17 Polyclonal antiangiocidin antibody was raised in a rabbit against angiocidin protein isolated from a human lung carcinoma and purified as previously described. 15 Sheep antihuman matrix metalloproteinase (MMP)-9 immunoglobulin G (IgG) was purchased from The Binding Site Co. (Birmingham, UK), and goat antihuman MMP-2 IgG was purchased from R&D Systems (Minneapolis, Minn). For each antibody, the species-respective nonspecific IgG was used as a control.…”

“…9,12,13 The protumor effects of angiocidin are the result of highaffinity binding interactions with extracellular matrix proteins including thrombospondin-1 (TSP-1), a matrix protein produced in tumors by activated stromal cells of mesenchymal origin. 14,15 These angiocidin-stromal protein binding interactions lead to an up-regulation in gelatinase expression and matrix remodeling, critical steps in the metastatic cascade. 9,16 We have recently synthesized 2 angiocidin-inhibitory peptides that block the activity of angiocidin through competitive inhibition of its stromal protein-binding interactions.…”