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Dissertation
Cloning and characterization of CSVTCG-specific receptor of thrombospondin-1
Doctor of Philosophy (Ph.D.), Medical College of Pennsylvania and Hahnemann University
Apr 2002
DOI:
https://doi.org/10.17918/00008964
Abstract
Thrombospondin-1 (TSP-1) is a 450 kDa glycoprotein involved in a variety of processes, including cell adhesion, tumor progression, and angiogenesis. Our laboratory previously showed that the CSVTCG sequence of TSP-1 functions as a tumor cell adhesion domain and CSVTCG peptides as well as an anti-peptide antibody had anti-metastasic activity in a murine model of lung metastasis. A novel tumor cell receptor was isolated from A549 human lung carcinoma by CSVTCG peptide affinity chromatography. A polyclonal antibody prepared against purified receptor stained human tumors and inhibited breast cancer progression both in vitro and in vivo. The full-length receptor cDNA was isolated from a lambda Zap-express prostate cancer cell (PC3-NII) cDNA library using the polyclonal receptor antibody. The full-length cDNA was then transferred from phage to a pBK-CMV vector and finally to a His tag expression vector. Recombinant receptor containing six histidine residues linked to the amino terminus was expressed in bacteria and isolated by Ni-chelation chromatography. The purified recombinant protein has a molecular weight of 60 kDa under reducing conditions and bound polyclonal receptor antibody, CSVTCG peptide and TSP-l. Recombinant protein (angiocidin) bound TSP-1 saturably with high affinity and specificity. To localize the region, we expressed truncated forms of receptor and examined their binding activity on TSP-1. Seven peptides were synthesized that spanned the active TSP-1 binding fragment. One of these peptides proved to have a higher binding activity than the others using the Affinity Sensor assay and ELISA competition assay. Our results suggest that the binding site resides in a small region FCTGIRVAHLALKHR between position 86 and 100 of the receptor molecule. Recombinant angiocidin specifically inhibited endothelial cell adhesion, and viability, while having no effect on a variety of cells including fibroblasts, smooth muscle cells, and tumor cells. Our results suggested that the site also resides in a small region between position 86 and position 100 of the angiocidin molecule. The TSP-1 binding site of angiocidin co-localized with the anti-angiogenic site. These experiments and other data from our laboratory should provide the basis for the development of this protein as an anti-cancer therapeutic.
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Details
- Title
- Cloning and characterization of CSVTCG-specific receptor of thrombospondin-1
- Creators
- Jing Zhou
- Contributors
- George P. Tuszynski (Advisor) - Drexel University, Medical College of Pennsylvania and Hahnemann University (1993-1996, 1998-2002)
- Awarding Institution
- Medical College of Pennsylvania and Hahnemann University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Medical College of Pennsylvania and Hahnemann University; Philadelphia, Pennsylvania
- Number of pages
- xii, 117 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- School of Medicine (1993-1996, 1998-2002); Medical College of Pennsylvania and Hahnemann University (1993-1996, 1998-2002)
- Other Identifier
- 991021888844204721