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Abstract #27782 Published in IGR 13-1
Interaction of Recombinant Myocilin with the Matricellular Protein SPARC: Functional Implications
Aroca-Aguilar J-D; Sánchez-Sánchez F; Ghosh S; Fernández-Navarro A; Coca-Prados M; Escribano JInvestigative Ophthalmology and Visual Science 2011; 52: 179-189
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 immunoflorescence 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.
Classification:
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
3.6 Cellular biology (Part of: 3 Laboratory methods)