Adachi W, Kawamoto S, Ohno I, Nishida K, Kinoshita S, Matsubara K, Okubo K
Institute for Molecular and Cellular Biology, Osaka University, Suita, Japan.
Invest Ophthalmol Vis Sci. 1998 Sep;39(10):1789-96.
To isolate and characterize a novel cathepsin gene, as part of the systematic isolation of genes uniquely active in corneal epithelium.
For the isolation of a full-length cDNA clone, a probe was selected from a set of expressed sequence tag clones classified as unique to corneal epithelium. Inserted cDNA was introduced into insect cells using a baculovirus expression system, and the secretion of recombinant protein was identified using antisera against a synthetic peptide. Proteolytic activity was determined using bovine serum albumin (BSA) as substrate. The expressions of the novel cathepsin in human cornea and other tissues were examined by reverse transcription-polymerase chain reaction (RT-PCR).
The full-length cDNA clone encoded a peptide of 334 amino acids with 82% identity with bovine cathepsin L and 77% identity with human cathepsin L when aligned. The recombinant protein produced in the baculovirus expression system cleaves BSA, and its activity was inhibited by the cysteine proteinase inhibitors E-64 and leupeptin, but not by pepstatin A, phenylmethylsulfonyl fluoride, and EDTA. By RT-PCR, a low level of expression was observed in some other epithelial tissues of ectodermal origin, but only in cornea was it higher than cathepsin L, which is known to be a general lysosomal cathepsin. Cathepsin V protein was detected in human corneal epithelium by western blot analysis, but not in tear fluid.
The amino acid homology and proteolytic activity of the recombinant protein indicate that the novel gene is a new member of the cathepsins that have features of cysteine proteinase. Its uniquely high expression in corneal epithelium strongly implies an important role in corneal physiology.
分离并鉴定一种新型组织蛋白酶基因，作为系统分离在角膜上皮中特异性活跃基因的一部分。
为分离全长cDNA克隆，从一组分类为角膜上皮特有的表达序列标签克隆中选择探针。使用杆状病毒表达系统将插入的cDNA导入昆虫细胞，并使用针对合成肽的抗血清鉴定重组蛋白的分泌情况。以牛血清白蛋白（BSA）为底物测定蛋白水解活性。通过逆转录-聚合酶链反应（RT-PCR）检测该新型组织蛋白酶在人角膜和其他组织中的表达。
全长cDNA克隆编码一个334个氨基酸的肽段，比对时与牛组织蛋白酶L的同一性为82%，与人类组织蛋白酶L的同一性为77%。杆状病毒表达系统产生的重组蛋白可切割BSA，其活性受到半胱氨酸蛋白酶抑制剂E-64和亮抑酶肽的抑制，但不受胃蛋白酶抑制剂A、苯甲基磺酰氟和乙二胺四乙酸（EDTA）的抑制。通过RT-PCR，在一些其他外胚层来源的上皮组织中观察到低水平表达，但仅在角膜中的表达高于已知为一般溶酶体组织蛋白酶的组织蛋白酶L。通过蛋白质印迹分析在人角膜上皮中检测到组织蛋白酶V蛋白，但在泪液中未检测到。
重组蛋白的氨基酸同源性和蛋白水解活性表明该新基因是具有半胱氨酸蛋白酶特征的组织蛋白酶家族的新成员。其在角膜上皮中独特的高表达强烈暗示其在角膜生理学中具有重要作用。
