期刊
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
卷 52, 期 1, 页码 179-189出版社
ASSOC RESEARCH VISION OPHTHALMOLOGY INC
DOI: 10.1167/iovs.09-4866
关键词
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资金
- Spanish Ministry of Science and Innovation
- Regional Ministry of Health
- Instituto de Salud Carlos III [SAF2008-02228, GCS-2006_C/12, PAI-05-002, PCI08-0036, RD07/0062/0014]
- National Institutes of Health [EY00785]
- Research to Prevent Blindness
- Fundacion Maria Cristina Masaveu Paterson
- Regional Ministry of Science and Technology of the Board of the Communities of Castilla-La Mancha
- NATIONAL EYE INSTITUTE [P30EY000785] Funding Source: NIH RePORTER
PURPOSE. Myocilin is an extracellular glycoprotein with unknown function that is associated with glaucoma. Calpain II cleaves recombinant myocilin within the linker region of the protein, releasing the C-terminal olfactomedin domain from the N-terminal domain. The authors previously reported that myocilin interacts with the C-terminal region of hevin, a secretory glycoprotein belonging to the SPARC family of matricellular proteins. This study aims to investigate the interaction of myocilin with SPARC. METHODS. Protein-protein interactions were evaluated by the yeast two-hybrid system. The positive interactions were confirmed by solid-phase binding assays using Ni-chelating HPLC purified recombinant proteins and coexpression of recombinant proteins in HEK-293T cells. Coexpression of myocilin, SPARC, and hevin in ocular tissues was identified by immuno-florescence microscopy, Western blot, and array-based gene profiling. RESULTS. Yeast two-hybrid analyses showed that myocilin interacted with the highly conserved C-terminal extracellular calcium binding (EC) domain within SPARC and hevin. Solid-phase binding assays confirmed these interactions and showed that both myocilin and its C-terminal olfactomedin fragment interacted noncovalently with SPARC and a peptide containing the EC domain of SPARC. Full-length myocilin interacted with higher affinity with SPARC and its EC domain than the myocilin C-terminal fragment. Coexpression of the two recombinant proteins in HEK-293T cells also indicated their intracellular interaction. CONCLUSIONS. Recombinant myocilin and SPARC interact through their C-terminal domains. The data suggest that the proteolytic processing of myocilin modulates this interaction as well as the interactions of myocilin with other extracellular matrix and matricellular proteins, further supporting a functional role for this proteolytic cleavage. (Invest Ophthalmol Vis Sci. 2011; 52: 179-189) DOI: 10.1167/iovs.09-4866
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