Canine myocilin is associated with lipid modified by palmitic acid.

Ricard, Cynthia S; Mukherjee, Amrita; Silver, Frankie-Lynn; Wagenknecht, Philip L

PURPOSE Myocilin, the product of the GLC1A locus in humans, has been associated with primary open angle glaucoma (POAG). Mutations in myocilin exon 3 correlate with age of onset and severity of POAG. Myocilin has been under investigation in experimentally induced glaucoma in rats, mice, and monkeys, however no animal model of inherited POAG exists except in beagles. Our aim was to determine whether canine myocilin more closely resembles human myocilin than does myocilin from other species. METHODS Canine myocilin was cloned and sequenced from beagle ocular tissues and Madin-Darby Canine Kidney (MDCK) cells, a well characterized canine cell line. Immunohistochemistry was performed on beagle tissues and cultured canine ocular primary cells. RT-PCR was employed to analyze genes expressed by tissues and cells. Myocilin protein synthesized by MDCK and canine optic nerve head cells (ONH) was analyzed by metabolic radioisotope labeling. Immunoprecipitation, enzymatic digestion, and Triton X-114 phase separation were performed to determine the carbohydrate content and the hydrophobicity of canine myocilin, respectively. RESULTS Canine ONH astrocytes cultured from a beagle eye expressed myocilin and markers of type 1B astrocytes observed also in human ONH cells in culture. The sequence of canine myocilin showed close similarity with the human sequence but lacked the additional upstream coding region found only in human and rat. Myocilin was labeled with (3)H palmitic acid. This label co-eluted with (35)S label in hydroxylapatite chromatography despite purification by phase separation in Triton X-114, immunoprecipitation with myocilin-specific sera, and denaturing conditions of heat, SDS, and reducing agent. CONCLUSIONS Canine myocilin revealed no striking sequence differences from myocilin of other species. Mutations in myocilin of glaucomatous beagles may suggest a similar pattern of involvement in inherited POAG. This represents the first report of lipid associated with the myocilin protein. Many lipid-modified proteins reside in lipid rafts. This modification of myocilin with lipids supports the unusual secretion properties and may suggest a function of myocilin in sorting, cell signaling and communication between the extracellular matrix and the cell membranes.