Abstract
Abstract: :
Purpose: TIGR/myocilin (MYOC) is a 504 amino acids glycoprotein implicated in primary open-angle glaucoma (POAG). Its function still remains unknown. More than 35 mutations have been reported in the gene. The majority of these mutations have been detected within its last exon which encodes an olfactomedin homology domain. A few of these COOH-terminal mutations have been tested in in vitro systems. Most of these blocked MYOC secretion. To assess if one of the few NH2-terminal mutations also altered MYOC secretion, we investigated the structures and properties of the Arg82Cys disease-causing mutation. Methods: COS-7 and human trabecular meshwork (HTM) cells in culture were transfected with expression vectors encoding wild-type (wt) or mutated Arg82Cys TIGR/myocilin cDNAs. Newly synthesized proteins were analyzed by dot blot or by Western blotting in intra- and extra-cellular fractions using a specific anti-TIGR polyclonal antibody. Results: The wild-type and Arg82Cys polypeptides were secreted into the culture media of both cell lines. The levels of mutant polypeptides detected outside the cells were similar to those of their wild-type counterparts. TIGR/MYOCwt and TIGR/MYOCArg82Cys also generated homodimeric complexes in intra- and extra-cellular fractions. On the other hand, the Arg82Cys mutation significantly altered the migration pattern of the very high molecular complexes produced inside and outside the cells. In particular, the mutation blocked the formation of a specific complex migrating at approximately 200-220 kDa. Conclusion: Our data showed that Arg82 is essential for generating TIGR/MYOC very high molecular complexes. Since Arg 82 is part of a potential coiled-coiled structure, these results suggest that the mutation may alter interactions with other polypeptides or with MYOC itself without blocking secretion of the disease-causing mutant protein.
Keywords: 527 protein structure/function • 480 mutations • 476 molecular biology