Interaction of myocilin with the C-terminal region of hevin.
Myocilin, a matricellular protein, is mutated in glaucoma. Here we report the identification and characterization, by the yeast two-hybrid system, of a putative interacting protein with myocilin. One of the positive clones exhibited 100% identity with the carboxyl-terminal (C-t) region of hevin, a member of the BM-40/SPARC/osteonectin family of extracellular matrix proteins. Protein interaction was assayed, in doubly transfected 293-T cells, by Western blot and fluorescent microscopy. Western blot analysis of the culture medium and lysates from cotransfected cells indicated that myocilin causes intracellular accumulation of hevin-C-t and impairs its secretion. This effect on hevin-C-t was augmented when coexpressed with the myocilin P370L mutant, known to cause a severe form of glaucoma. By fluorescent microscopy, myocilin localizes with hevin-C-t in the Golgi in cotransfected 293-T cells and with hevin-wt in the ocular ciliary epithelium. Overall, these results suggested that the C-t of hevin contains important determinants for interaction with myocilin.
Li
Yanxia
Y
Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, CT, USA.
Aroca-Aguilar
J Daniel
JD
Ghosh
Sikha
S
Sánchez-Sánchez
Francisco
F
Escribano
Julio
J
Coca-Prados
Miguel
M
D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000378:metabolism*; D005136:Eye Proteins / Q000737:chemistry* / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014018:Tissue Distribution; D000099179:Myocilin
D002135:Calcium-Binding Proteins; D003598:Cytoskeletal Proteins; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D000099179:Myocilin; C503126:SPARCL1 protein, human