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Expression and mutagenesis of thrombospondin
J Lawler1, P Ferro, M Duquette
1Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115.
Biochemistry
|February 4, 1992
Summary
This study demonstrates that expressing human thrombospondin (TSP) in mouse cells yields a functional, secreted glycoprotein. Key structural domains influence TSP assembly and heparin-binding properties.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Thrombospondin (TSP) is a large, adhesive glycoprotein crucial in cellular processes.
- Understanding TSP's structure-function relationship is vital for its biological roles.
Purpose of the Study:
- To investigate the expression and functional characterization of human thrombospondin in a heterologous system.
- To identify key domains responsible for TSP assembly and heparin binding.
Main Methods:
- Expression of human thrombospondin cDNA in mouse NIH 3T3 cells.
- Biochemical analysis including protein synthesis, secretion, and binding assays (antibody, heparin, calcium).
- Site-directed mutagenesis to assess the role of specific TSP domains (procollagen homology, type 1/2 repeats).
Main Results:
- Successfully synthesized and secreted a 420,000-dalton TSP subunit that comigrated with native human platelet TSP.
- The expressed TSP bound to anti-TSP antibodies, heparin, and calcium.
- Truncated TSP variants with deletions in specific domains showed altered assembly and heparin-binding affinities.
- Mutations in lysine/arginine-rich motifs significantly diminished high-affinity heparin binding.
Conclusions:
- The mouse NIH 3T3 cell system is suitable for expressing functional human thrombospondin.
- Specific domains, including the procollagen homology region and type 1/2 repeats, are critical for TSP trimerization and stability.
- Heparin-binding activity is modulated by distinct sequence motifs within the TSP subunit.