Huang W, Jaroszewski J, Ortego J, Escribano J, Coca-Prados M
Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Ophthalmic Genet. 2000 Sep;21(3):155-69.
Mutations in the the glaucoma gene GCL1A, also known as trabecular meshwork glucocorticoid response (TIGR) or myocilin (Myoc), have been shown to be associated with juvenile-onset primary open-angle glaucoma. Very little is known about the pattern of expression of the TIGR gene in human ocular tissues. In-situ hybridization experiments demonstrated the localization of TIGR mRNA in cells throughout the iris, ciliary muscle, and the filtering portion of the trabecular meshwork of normal eye donors. The expression of TIGR protein was investigated by Western blot using an epitope-directed antibody to the carboxy terminus region of TIGR. This antibody was able to distinguish a recombinant TIGR fusion protein from a truncated TIGR form containing the naturally occurring Gln(368)-->stop mutation. In tissue extracts from the iris, ciliary body, and trabecular meshwork, the antibody recognized a major protein band of 57-kDa molecular mass. Deglycosylation treatment with PNGase F, NANase II, and O-glycosidase indicated that the 57-kDa protein in these tissues was unglycosylated. In agreement with this observation, in coupled in-vitro transcription/translation systems, the 57-kDa TIGR protein was unaffected by the presence of the processing and glycosylation activities of canine pancreatic microsomal membranes. These findings support the view that the expression of TIGR mRNA in cells of the iris, ciliary body, and trabecular meshwork correlates with that of TIGR protein, and that the 57-kDa TIGR protein was unglycosylated. These results, which are in contrast with earlier reports, raise the possibility that the TIGR protein might be processed into distinct forms in a tissue-specific manner.
青光眼基因GCL1A（也称为小梁网糖皮质激素反应蛋白（TIGR）或肌纤蛋白（Myoc））的突变已被证明与青少年型原发性开角型青光眼有关。关于TIGR基因在人眼组织中的表达模式知之甚少。原位杂交实验证明了TIGR mRNA在正常眼供体的整个虹膜、睫状肌和小梁网滤过部分的细胞中的定位。使用针对TIGR羧基末端区域的表位定向抗体，通过蛋白质印迹法研究了TIGR蛋白的表达。该抗体能够区分重组TIGR融合蛋白和含有天然存在的Gln(368)→终止突变的截短TIGR形式。在来自虹膜、睫状体和小梁网的组织提取物中，该抗体识别出一条分子量为57 kDa的主要蛋白带。用PNGase F、NANase II和O-糖苷酶进行去糖基化处理表明，这些组织中的57 kDa蛋白未被糖基化。与这一观察结果一致，在体外偶联转录/翻译系统中，57 kDa的TIGR蛋白不受犬胰腺微粒体膜加工和糖基化活性的影响。这些发现支持了这样一种观点，即TIGR mRNA在虹膜、睫状体和小梁网细胞中的表达与TIGR蛋白的表达相关，并且57 kDa的TIGR蛋白未被糖基化。这些结果与早期报道相反，增加了TIGR蛋白可能以组织特异性方式加工成不同形式的可能性。
