Sun L, Sun T T, Lavker R M
Department of Dermatology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 1999 Jun 11;274(24):17334-41. doi: 10.1074/jbc.274.24.17334.
Recently, metabolic enzymes have been observed in both the lens and corneal epithelium at levels greatly exceeding what is necessary for normal metabolic functions. These proteins have been termed taxon-specific crystallins and are thought to play a role in maintaining tissue transparency. We report here that cytosolic NADP+-dependent isocitrate dehydrogenase (ICDH) represents a new corneal crystallin. Using suppression subtractive hybridization, we identified a gene (with a deduced amino acid sequence that showed 94% identity to rat cytosolic NADP+-dependent ICDH) that is preferentially expressed in bovine corneal epithelium. Northern blots established that its mRNA level in the corneal epithelium was 31-, 39-, 133-, 230-, and 929-fold more than in the liver, bladder epithelium, stomach epithelium, brain, and heart, respectively. This mRNA was detected primarily in corneal epithelial basal cells by in situ hybridization. SDS-polyacrylamide gel electrophoresis, two-dimensional gel analysis, and Western blotting showed that this protein was overexpressed in the corneal epithelium, constituting approximately 13% of the total soluble bovine corneal epithelial proteins. Enzyme assays showed a corresponding overabundance of this protein in bovine corneal epithelium. Taken together, these data indicate that bovine cytosolic ICDH fulfills the criteria for a corneal epithelial crystallin and may be involved in maintaining corneal epithelial transparency.
最近，在晶状体和角膜上皮中均观察到代谢酶，其水平大大超过正常代谢功能所需的水平。这些蛋白质被称为分类群特异性晶状体蛋白，被认为在维持组织透明度方面发挥作用。我们在此报告，胞质NADP⁺依赖性异柠檬酸脱氢酶（ICDH）代表一种新的角膜晶状体蛋白。通过抑制性消减杂交，我们鉴定出一个基因（其推导的氨基酸序列与大鼠胞质NADP⁺依赖性ICDH显示出94%的同一性），该基因在牛角膜上皮中优先表达。Northern印迹法证实，其在角膜上皮中的mRNA水平分别比在肝脏、膀胱上皮、胃上皮、脑和心脏中高31倍、39倍、133倍、230倍和929倍。通过原位杂交主要在角膜上皮基底细胞中检测到该mRNA。SDS-聚丙烯酰胺凝胶电泳、二维凝胶分析和Western印迹显示，该蛋白在角膜上皮中过表达，约占牛角膜上皮总可溶性蛋白的13%。酶活性测定表明该蛋白在牛角膜上皮中相应过量。综上所述，这些数据表明牛胞质ICDH符合角膜上皮晶状体蛋白的标准，可能参与维持角膜上皮的透明度。
