Isolation and functional analysis of genes uniquely expressed in human corneal epithelium and conjunctival epithelium

我们构建了人角膜上皮mRNA表达谱，并分析了在角膜上皮中丰度或特异性表达的基因。从这些数据中，分离出一种新的组织蛋白酶（组织蛋白酶V）、uroplakin Ib和Cl通道，并分析了它们的功能和染色体定位。构建人结膜上皮的表达谱，并与人角膜上皮的表达谱进行比较。组织蛋白酶V是一种新型组织蛋白酶，其ORF与人组织蛋白酶l同源性为77%。利用杆状病毒表达系统制备的重组组织蛋白酶V蛋白作为半胱氨酸蛋白酶具有蛋白水解活性。通过RT-PCR发现，只有在角膜中，组织蛋白酶V的表达水平高于组织蛋白酶l。组织蛋白酶V可能在角膜生理中发挥重要作用。通过FISH方法，将组织蛋白酶V基因定位到距组织蛋白酶L基因15cM的染色体9q22.2区域。这表明组织蛋白酶L和组织蛋白酶V的进化更早，更晚是通过祖先基因的基因复制进行的。Uroplakin Ib蛋白具有4个跨膜结构域。该克隆是已发表的uroplakin Ib的异构体，因为该克隆的3'-UTR与已发表的uroplakin Ib不同。通过RT-PCR，不仅在移行上皮中检测到uroplakin Ib mRNA，而且在眼表中也检测到。用抗血清免疫组化检测发现，uroplakin Ib蛋白存在于角膜、角膜缘和结膜上皮的细胞膜中，尤其是在角膜上皮的浅半层。一个新的C1通道编码943个氨基酸，并映射到染色体区域1p32。C1通道mRNA的丰度是其他C1通道的100倍，提示角膜透明的重要规律。正常结膜上皮的表达谱与角膜上皮的表达谱差异很大。在这个表达谱中，角蛋白13、β - 2微球蛋白和脂皮质素高表达。在三个或更多克隆中出现的大量转录本中，包括两个未知的结膜特异性基因。少

We constructed the mRNA expression profile from human corneal epithelium and analyzed the genes expressed abundantly or specifically in corneal epithelium. From these data, a novel cathepsin (cathepsin V), uroplakin Ib and Cl channel were isolated and analyzed as to their functions and chromosomal localizations. The expression profile of human conjunctival epithelium was also constructed and compared to that of human corneal epithelium.Cathepsin V was a novel cathepsin, which contained ORF of 77% identical homology to human cathepsin L. A recombinant cathepsin V protein produced using a baculovirus expression system has proteolytic activity as a cysteine proteinase. By RT-PCR, only in cornea, the expression level of cathepsin V was higher than that of cathepsin L. Cathepsin V may play an important role in corneal physiology. By the FISH method, cathepsin V genes was mapped to chromosomal region 9q22.2, 15cM from the cathepsin L gene. This suggests that cathepsin L and V evolved more re … More cently by gene duplication from an ancestral gene. Uroplakin Ib protein had four transmembrane domains. This clone was an isoform of uroplakin Ib, which had already published, because a 3'-UTR of this clone was differed from published uroplakin Ib. By RT-PCR, uroplakin Ib mRNA was detected not only in transitional epithelium, but also on the ocular surface. By immunohistochemistry using antiserum against uroplakin Ib peptide, uroplakin Ib protein was found in cell membranes of corneal, limbal and conjunctival epithelium, especially in the superficial half of the corneal epithelial layer. A novel C1 channel coded 943 amino acids and mapped to chromosomal region 1p32. C1 channel mRNA was 100 times more plentiful than other C1 channels, implying an important rule of corneal transparency.The expression profile of normal conjunctival epithelium was so differed greatly from those of corneal epithelium. In this expression profile, keratin 13, beta- 2 microglobulin and lipocortin were highly expressed. Among the abundant transcripts appearing in three or more clones, two unknown conjunctival specific genes were included. Less