Ricard Cynthia S, Mukherjee Amrita, Silver Frankie-Lynn, Wagenknecht Philip L
Department of Ophthalmology, Saint Louis University, St. Louis, MO 63104, USA.
Mol Vis. 2006 Nov 17;12:1427-36.
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.
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.
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.
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.
人GLC1A基因座的产物肌纤蛋白与原发性开角型青光眼（POAG）相关。肌纤蛋白第3外显子的突变与POAG的发病年龄和严重程度相关。肌纤蛋白已在大鼠、小鼠和猴子的实验性诱导青光眼中进行研究，然而，除了比格犬外，不存在遗传性POAG的动物模型。我们的目的是确定犬肌纤蛋白是否比其他物种的肌纤蛋白更类似于人肌纤蛋白。
从比格犬眼组织和特征明确的犬肾细胞系马-达二氏犬肾（MDCK）细胞中克隆并测序犬肌纤蛋白。对比格犬组织和培养的犬眼原代细胞进行免疫组织化学分析。采用逆转录聚合酶链反应（RT-PCR）分析组织和细胞表达的基因。通过代谢放射性同位素标记分析MDCK细胞和犬视神经乳头细胞（ONH）合成的肌纤蛋白。分别进行免疫沉淀、酶消化和Triton X-114相分离，以确定犬肌纤蛋白的碳水化合物含量和疏水性。
从比格犬眼培养的犬ONH星形胶质细胞表达肌纤蛋白，并且在培养的人ONH细胞中也观察到1B型星形胶质细胞的标志物。犬肌纤蛋白的序列与人序列显示出密切相似性，但缺少仅在人和大鼠中发现的额外上游编码区。肌纤蛋白用（3）H棕榈酸标记。尽管通过在Triton X-114中进行相分离、用肌纤蛋白特异性血清进行免疫沉淀以及加热、十二烷基硫酸钠（SDS）和还原剂的变性条件进行纯化，但该标记在羟基磷灰石色谱中与（35）S标记共洗脱。
犬肌纤蛋白与其他物种的肌纤蛋白相比没有明显的序列差异。青光眼比格犬肌纤蛋白中的突变可能提示在遗传性POAG中的类似参与模式。这是与肌纤蛋白相关的脂质的首次报道。许多脂质修饰蛋白存在于脂筏中。肌纤蛋白的这种脂质修饰支持其异常的分泌特性，并可能提示肌纤蛋白在分选、细胞信号传导以及细胞外基质与细胞膜之间的通讯中的功能。
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