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(Investigative Ophthalmology and Visual Science. 2002;43:1749-1756.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Construction of Human Corneal Endothelial cDNA Library and Identification of Novel Active Genes

Rieko Sakai1, Tadatoshi Kinouchi2, Shoko Kawamoto3, M. Reza Dana4, Toshirou Hamamoto2, Tadahiko Tsuru1, Kousaku Okubo3 and Satoru Yamagami1

1 From the Departments of Ophthalmology and 2 Biochemistry, Jichi Medical School, Tochigi, Japan; the 3 Institute for Molecular and Cellular Biology, Osaka University, Osaka, Japan; and 4 Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To describe genes expressed in human corneal endothelial cells and identify novel genes.

METHODS. Sixteen human donor corneas that had no history of corneal disease, infection, or intraocular surgery were used within 7 days of death. Total RNA was extracted from corneal endothelial cells with attached Descemet membranes. A 3'-directed cDNA library was constructed from mRNA by using a pUC19-based primer. These sequences were compared with each other to determine their frequency and were searched against GenBank for identification. To identify novel specific and abundant transcript genes in corneal endothelial cells, the novel genes were compared with an expressed sequence tag database, the expected sequence extended, and 5' rapid amplification of cDNA ends–polymerase chain reaction cloning performed.

RESULTS. The human corneal endothelial cDNA library showed that the most abundant transcript was prostaglandin D2 synthase. The remaining transcript genes that were present in abundance consisted of lactate dehydrogenase-A, gene signature (GS) 3582, which is a novel gene without a known function, and matrix Gla protein. The full-length sequence of GS3582 showed similarity to genes obtained in ovary and TESTIS.

CONCLUSIONS. A human corneal endothelial cDNA library was constructed. An expression profile of corneal endothelium provides probes to monitor physiologic and pathologic conditions of this tissue in terms of gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The corneal endothelium (CE) is a single layer of flat hexagonal cells that lies on a basement membrane, the Descemet membrane. There is no mitotic activity in the human CE after birth, which leads to a gradual decrease of the CE cell population with age as the cells migrate.1 The CE plays a critical role in controlling stromal hydration, including the barrier and pump function of the aqueous humor and ion transport. In addition, the cells have metabolic activity as indicated by the presence of mitochondria, endoplasmic reticulum, Golgi apparatus, and free ribosomes.2 Gene analysis of CE could provide molecular clues for essential functions of the CE, including the maintenance of corneal transparency.

In the human eye, gene expression profiles have been investigated for the corneal epithelium,3 conjunctival epithelium,4 trabecular meshwork,5 and retina.6 Much of the corneal stroma, the CE, and iridocorneal angle (trabecular meshwork) are derived from neural crest, whereas the corneal and conjunctival epithelium and sensory retina are derived from surface ectoderm and neural ectoderm, respectively, suggesting that the genes expressed in the CE may be distinct from those in the epithelium of the cornea or conjunctiva. A cDNA library from cultured rabbit CE7 has been characterized. However, human CE (HCE) does not regenerate in vivo, reflecting an essential difference from the regenerative capacity of rabbit CE.8 Therefore, gene expression profiles of noncultured HCE could provide important details on the in vivo function of HCE.

Unique gene expression analyses based on 3'-directed cDNA contain a short sequence of mRNA polyA+ tails through the nearest MboI site. The information is called a gene signature (GS) and consistently represents the mRNA population in a given tissue.9 10 The MboI site is used, because GATC is the most uniformly distributed recognized sequence for commercially available four-base cutters in human gene sequences. By comparing the expression profile for a particular tissue with profiles from other cells, genes can be categorized into those that are active in many other source tissues.

In this study, we describe the gene expression profile of the HCE by using the GS system and we identify the full-length sequence of a novel gene. These results may provide important candidate genes for the study and understanding of the physiological and metabolic functions of the HCE.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of HCE
Sixteen human corneas that had no history of corneal disease, infection, or intraocular operation, were provided from the American Eye Bank of Florida. The average age of these corneas was 59.9 ± 5.9 years (age range, 49–70). All corneas were kept in storage medium (Optisol GS; Chiron Vision, Irvine, CA) at 4°C and were used within 7 days of death. This storage medium consists of a hybrid of K-Sol (Chiron Vision) and Dexol (Chiron Vision) containing 2.5% chondroitin sulfate, 1% dextran, vitamins, precursors of adenosine triphosphate (adenosine, inosine, and adenine), gentamicin, and streptomycin.11 12 After excising the clear cornea with scissors, the CE and Descemet membrane were peeled away in a sheet from the periphery to the center with fine forceps. Based on our investigations, correlating histologic findings with our microsurgical procedures, posterior stroma adhering to the Descemet membrane leads to considerable resistance in excising the CE-Descemet complex from the remainder of the cornea. To avoid the inclusion of posterior stromal tissue, only strips of CE-Descemet tissue that were excised smoothly from the stroma, without residual stroma, were used in the experiments. The removed CE was immediately homogenized in RNA isolation agent (RNAzol B; Tel-Test. Inc., Friendswood, TX), and stored at -70°C until use.

Construction of a 3'-Directed cDNA Library
Total RNA was extracted from the homogenized solution. PolyA+ RNA was prepared by using an mRNA purification kit (QuickPrep; Amersham Pharmacia Biotech, Tokyo, Japan) according to the manufacturer’s instructions. A 3'-directed cDNA library was constructed using polyA+ RNA, as described previously.11 Briefly, pUC19-based vector primer was used for cDNA synthesis, and the 5' side of double-stranded was trimmed off with MboI (GATC) to decrease the transforming efficiency bias caused by size difference.12 Cohesive termini were created at the other end of the vector molecules by cleaving with BamH1 and were self-ligated. By transforming them into Escherichia coli DH5 (Toyobo, Osaka, Japan) and using one tenth of the mixture, we obtained approximately 2.5 x 105 independent transformants. With this shortening of the cDNAs using MboI, we could diminish the transforming efficiency bias caused by size differences.10

Sequence and Data Analysis
The transformed colonies were selected randomly and lysed. The cDNA moiety was then amplified by polymerase chain reaction (PCR), using a primer pair that flanked the insert (5'-GTTTTCCCAGTCACGACGTTG-3'; 5'-ACCATGATTACGCCAGCTTG-3'), as described elsewhere.12 The PCR products were subjected to a cycle sequencing reaction, using dye primer M13, and analyzed by autosequencer (model 373A; PE Biosystems, Foster City, CA). Inserted sequences shorter than 20 bp were eliminated from analysis because of inadequate length to discriminate among approximately 105 transcripts at their 3' termini. Inserts that had no unique sequences or had more than 5% ambiguous nucleotides were also eliminated. Sequences were compared to each other using the FastA program,13 and identical sequences were grouped together as a single gene species. Sequences with more than 95% identical bases throughout the query sequences were regarded as identical. For recurring sequences that appeared more than twice, homologies were searched in a daily updated expressed sequence tag database (dbEST) using the Basic Local Alignment Tool (BLAST) of the National Center for Biotechnology Information (NCBI, Bethesda, MD; available in the public domain at http://www.ncbi.nlm.nih.gov/BLAST).

Full-Length Sequencing
The novel and abundant transcript gene, GS3582, in our established HCE cDNA library, corresponded to the daily updated dbEST and extended the expected sequence. 5'-Rapid amplification of cDNA ends (RACE)-PCR cloning was performed using a kit (Sure-RACE; Origene Technologies, Inc., Rockville, MD) that allows isolation of the 5' sequence of the target transcript. The RACE panels in the kit consist of double-stranded cDNA from 24 individual human tissues arrayed in a multiwell plate and provides two contiguous adapter-specific primers at the 5' end. Outer primer for first-round PCR, gene-specific primer (GSP)-1: 5'-TTTCCGCAACATTCTCCTTTT-3') and inner primer for second-round (nested) PCR, GSP2: 5'-TTCTGTGTTTGGCTTGGT-3') were designed from the expected sequence.

The RACE cDNAs were amplified with DNA polymerase (KOD-Plus; Toyobo). After incubation at 94°C for 3 minutes, the first round of PCR was performed with 5 cycles of 94°C for 3 seconds, 63°C for 30 seconds, and 72°C for 3 minutes and then 15 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 6 minutes, followed by 72°C for 6 minutes in a PCR thermal cycler (model MP; Takara, Kyoto, Japan). After incubation at 94°C for 3 minutes, the second round of PCR was performed with 35 cycles of 94°C for 30 seconds, 62°C for 30 seconds, and 72°C for 3 minutes, followed by 72°C for 6 minutes. Among human cDNAs in different tissues, PCR products 300 to 400 bp in length were circularized and subcloned into a vector (pBluescript; Stratagene, La Jolla, CA). Randomly selected plasmids were amplified and purified into DNA with a kit (Plasmid Midi; Qiagen, Hilden, Germany). The products were amplified with T7 promoter primer (T7 HT Primer; Toyobo) with 25 cycles of 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 4 minutes after incubation at 96°C for 30 seconds. Amplified products were sequenced with the autosequencer (ABI Prism 310 Genetic Analyzer; PE Biosystems). The resultant sequence was extended to the expected sequence, and the analysis of the total GS3582 sequence was concluded.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
An Expression Profile of Active Genes in Human Corneal Endothelium
The frequency distribution for the GSs is listed in Table 1 . We used 5125 transformants randomly for sequence and obtained 1460 independent GS species comprising 2663 clones by eliminating mitochondrially coded sequences, ribosomal RNAs, repetitive sequences, and low-quality sequences having more than 5% ambiguous bases. Among them, 946 (64.8%) species represented by 2019 clones, were identified in GenBank (GenBank is provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD, and is available at http://www.ncbi.nlm.nih.gov/Genbank), and the remaining 514 (35.2%) species, represented by 644 clones, were novel genes. Three hundred ninety-five GSs appeared recurrently, representing genes that are highly or moderately active in the human CE. One thousand sixty-five GSs appeared only once. All the GSs collected from the HCE were compared with sequences obtained previously from other tissues. The table listing all the GS found in the corneal tissue is referred to as a gene expression profile of corneal endothelium. It can be obtained through our BodyMap Server (Tokyo University, Tokyo, Japan; available at http://bodymap.ims.u-tokyo.ac.jp).


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  		Table 1. Distribution of Gene Signatures among Different Occurrence Groups

 
Gene Expression Profile of Human Corneal Endothelium
Part of the expression profile listing active genes in the order of abundance of transcript genes is shown in Table 2A . The most abundant transcript was prostaglandin (PG) D2 synthase (n = 68). The following transcript genes consisted of lactate dehydrogenase-A (n = 28); GS3582 (n = 21), a novel gene; and matrix Gla protein (n = 17). To reveal the gene expression specific to the HCE, the number of GSs detected in HCE were compared with that of GSs in other tissues obtained previously in our BodyMap. The gene ratio in HCE was calculated according to the following formula: gene ratio = detected gene number in the HCE divided by the total gene number of other tissues including HCE genes in BodyMap. Genes expressed with high frequency in the HCE compared with those in other tissues are listed in Table 2B . Damage-specific DNA-binding protein 2 (48 kDa; n = 5) was found only on the CE among five GSs. The following genes with high rates of expression in the CE were the novel gene, GS3582 (75% of GSs listed in the BodyMap are found in CE), highly similar to Homo sapiens mRNA for KIAA0585 protein (67%), and human carbonic anhydrase 3 (52%).


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  		Table 2. Gene Expression Profiles of Corneal Endothelium

 
We categorized the genes identified in GenBank according to the function and subcellular localization. Four hundred fourteen clones (20.5%) were related to ribosomal component and translational factors, 243 clones (12%) to signal transduction, 229 (11.3%) to cytoplasmic proteins, 210 (10.4%) to nuclear proteins and transcriptional factors, 163 (7.7%) to mitochondrial protein, and 118 (7.3%) to membrane protein. Among the 945 GSs identical with known genes, those encoding signal transduction, cytoplasmic proteins, membrane proteins, secretory and extracellular proteins, and cytoskeleton proteins are listed in Table 3 .


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  		Table 3. Gene Expression Profiles Categorized by Function and Subcellular Localization

 
Isolation of a Full-Length cDNA for GS3582
We chose GS3582 to investigate the HCE-specific gene, because GS3582 is an transcript gene that is abundant in the HCE with a higher proportion rate than transcript genes in other tissues in our BodyMap. To verify the distribution of GS3582 in human organs, Northern blot analysis was performed with a kit (Human 12-Lane MTN Blot; Clontech Laboratories, Palo, Alto, CA). However, GS3582 was not detected in a panel of human mRNA (data not shown). Therefore, the products of 5' RACE-PCR cloning were examined in the human cDNA in the different human tissues. PCR products of 300 to 400 bp in length were identified in the pituitary, the prostate, the testis, and the ovary (Fig. 1) .



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  		Figure 1. The products of 5' RACE-PCR cloning are shown in the human cDNA in different tissues. PCR products are identified in pituitary, prostate, testis, and ovary. A 5x sample is five times the concentration of 1x.

 
The cDNA sequence and gene structure of GS3582 are shown in Figure 2 . This sequence contains an open reading frame of 240 amino acids, with a molecular weight estimated at 25.4 kDa. The human genome database shows that GS3582 locates to human chromosome 4q28 (the nearest gene is nicotinamide-adenine dinucleotide [NADH] dehydrogenase [ubiquinone], or NDUFC1). Each exon of the gene is scattered over 14 kbp (Fig. 1B) and is completely consistent with a sequence in the database. The sequence is rich in the acidic amino acids (glutamic and aspartic acids), which are present at a ratio of 19% (46/240), and the theoretical isoelectric point is 4.24. According to protein motif analysis (PROSITE, Swiss Institute of Bioinformatics, Geneva, Switzerland, http://expasy.nhri.org.tw/prosite/; BLOCKS, the Fred Hutchinson Cancer Research Center, Seattle, WA, http://www.blocks.fhcrc.org/; ProDom, The Protein Domain Database, Toulouse, France; http://prodes.toulouse.inra.fr/prodom/doc/prodom.html; PRINTS, Protein Fingerprint Database, University of Manchester Bioinformatics Education and Research, Manchester, UK, http://bioinf.man.ac.uk/dbbrowser/PRINTS; and Pfam, The Sanger Centre, Hinxton Hall, UK; http://www. sanger.ac.uk/Pfam/), GS3582 does not possess any characteristic sequences such as a signal peptide or other localization motifs.



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  		Figure 2. (A) The sequence of GS3582. The open reading frame was analyzed and translated into the amino acid sequence. This sequence contains 240 amino acids, and the molecular weight is estimated at 25.4 kDa. GS3582 locates on human chromosome 4 and completely matches with the sequence of the human genome database. (B) Gene structure of GS3582.

 
The N-terminal 238-amino-acid sequence of GS3582 is identical with the human ovary-specific acidic protein,14 consisting of 268 amino acids, according to the BLAST program. Two genes with high homology in human GS3582 were found in the rabbit corneal endothelial cDNA library and RIKEN (The Institute of Physical and Chemical Research, Tokyo, Japan, http://www.riken.go.jp/) full-length enriched library established from adult mouse testis cDNA. The homologue of cultured rabbit corneal endothelium codes the 3' untranslated region and exhibits a 66.5% homology for human GS3582. The mouse testis has a large number of aspartic and glutamic acid residues (61 of a total 283) and a protein, translated from the mouse testis gene, possesses significant homology with the human GS3582 at the N-terminal (amino acid positions 1–60) sequence. In the mouse testis gene, two large insertion sequences exist in the C-terminal region and there are three tandem repeats, Glu-Gly-Ala-Asp-Thr-Ser-Gln, in the first insertion sequence.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we sought to construct a human corneal endothelial cDNA library by evaluating gene expression in 16 human corneas with no history of disease. Because the donor corneas were stored in storage medium (Optisol GS; Chiron) and the HCE samples were acquired from the donors within 7 days of death, it is possible that the data may not precisely reflect the in vivo expression profile due to the possible effect of the storage medium on select genes after death. However, to the best of our knowledge, storage in Optisol GS does not affect the G1 phase of the cell cycle in the human corneal endothelium.15 Moreover, similarly (or even significantly longer) stored tissue is used routinely in clinical transplantation, and the CE functions well after grafting leading to corneal transparency. These results suggest that although the gene expression profile of eye bank tissue may not be completely identical, it is fundamentally similar to that of the in vivo cornea.

The CE is metabolically active and therefore requires nutrients for its function. Glycogen and glucose from the aqueous humor are the main energy sources for endothelial cells. Glucose is catabolized through aerobic pathways, including the tricarboxylic acid cycle and hexose monophosphate shunt, and anaerobic pathways. However, whether the CE has the capacity to store glycogen is not known.1 The active genes such as lactate dehydrogenase A (n = 28), NADH dehydrogenase (n = 10), and adenosine triphosphatase (ATPase, n = 8) express abundantly, suggest dynamic glucose metabolism in the corneal endothelium. The control of corneal stromal hydration sustained by pump and barrier function in the corneal endothelium is essential for transparency. The abundant adenosine triphosphatase (ATPase) and carbonic anhydrase gene (n = 15) may explain the pump function in relation to Na-K, ATPase.

Physiologic extracellular matrix calcification is restricted to bones, teeth, and the hypertrophic zone of growth plate cartilage. Matrix Gla (n = 17) is the protein associated with protection against calcification in soft tissues.16 Nonsense mutations in the human matrix Gla protein gene cause Keutel syndrome, a rare autosomal recessive disorder characterized by diffuse cartilage calcification.17 In mice, deletion of matrix Gla protein causes cartilage calcification and arterial calcification.16 In the CE, calcium ions in the aqueous humor are necessary for the maintenance of the barrier function of the CE. The existence of matrix Gla protein gene in the HCE suggests that the protein may metabolize calcium ions that maintain barrier function and control stromal hydration while protecting calcium deposit in the cornea. SPARC/osteonectin (n = 6) in secretory and extracellular proteins also could be associated with the barrier function of the CE.18

PGD is a neuromodulator that participates in the sleep–wake cycle, thermoregulation, and odor responses.19 There are two enzymes that catalyze the conversion of PGH2 into PGD2: hematopoietic PGD synthase and lipocalin-type PGD synthase.20 21 Not only brain, but also the epididymis and testis express a high amount of lipocalin-type PGD synthase protein.22 23 24 In the eye, lipocalin-type PGD synthase is synthesized within the epithelial cells of the iris-ciliary body and pigment epithelium and is secreted into the aqueous and vitreous humors, respectively.25 26 27 The gene transcript of PGD2 synthase (n = 68) is the most abundant transcript gene in the present study, suggesting that CE covering the anterior chamber is also the major source of PGD2 synthase secretion and that PGD2 may be involved in the regulation of intraocular pressure.28

It is of interest that ß-amyloid protein precursor gene (APP, n = 10) is detected in the CE. Disruption of the normal function of the APP is known as a leading cause of Alzheimer disease.29 After a wound to the cornea, APP is upregulated in the basal cells of the corneal epithelium that is actively migrating,30 31 but not in the normal human corneal epithelium,3 implying a role for mediating epithelial migration during re-epithelialization. Because in vivo the HCE does not show any regenerative ability, it heals only by migration, suggesting a critical role for APP in CE migration. Further study is needed to reveal the exact mechanism that mediates migration in the HCE.

GSs found only in corneal endothelium included damage-specific DNA-binding protein 2 (DDB2; n = 5). Ultraviolet (UV) radiation-damaged DNA-binding activity requires the expression of two subunits, p127, DDB1 (n = 8), and p48, DDB2,32 which are detected in the HCE library, but not in corneal epithelium.3 Clinically, xeroderma pigmentosum group E cells, induced by a nonsense mutation of DDB2,33 are deficient in global genomic repair.34 Moreover, decreased cell density and increased coefficient of variation in cell area have been reported in patients with xeroderma pigmentosum.35 Nonregenerative HCE, different from regenerative corneal epithelium, may be necessary to prepare an efficient genomic system for repair of DNA damaged by UV radiation, with the aid of DDB2 protein.

As for the previously established cDNA library of anterior segment tissues of the eye, our library of noncultured HCE is not entirely compatible with that of cultured rabbit CE.7 This may be due to not only species differences, but also to the samples’ originating from cultured versus noncultured cells. There are few common gene expressions among the cDNA libraries derived from HCE and human corneal epithelium.3 Perhaps this is not surprising, given the differences in embryologic origins between these two cell types, the former derived from neural crest and the latter from surface ectoderm. In contrast, abundant genes, including lactate dehydrogenase and matrix Gla protein, in the CE coincide well with those in the human trabecular meshwork5 cDNA library, which also has a neural crest origin. Moreover, cDNA libraries of human iris-ciliary body and CE have similar gene expression profiles in our BodyMap (data not shown). These results suggest that ocular tissues originating from the neural crest surrounding the anterior chamber can provide common proteins for the maintenance of aqueous humor physiology.

We chose GS3582 (n = 21), a novel gene with a high rate of expression in the HCE, and isolated a full-length cDNA. As shown in Figure 2 , GS3582 does not possess a signal peptide at N termini, indicating that GS3582 is not a secretory protein and could be classified in the intracellular organelle or cytoplasm. By using the Web server at NCBI, significant homology (E value of 3e-98) was observed between GS3582 and the ovary-specific acidic protein (GenBank accession number, AF329088).14 The ovary-specific acidic protein was so named because of the restricted expression to the ovary by Northern blot analysis of the other systemic tissues not including human corneas. Moreover, a similar full-length sequence was also obtained in the mouse testis (accession number: AK006339 in NCBI). These results suggest apparent polymorphisms for GS3582. GS3582 is found in the human corneal epithelium, but not retina or iris in our BodyMap, and in the human cDNA of pituitary, prostate, testis, and ovary. Further studies should be conducted to reveal possible biological functions shared among these tissues and cornea. It is of interest that the ovary, testis, and anterior chamber all represent immune-privileged tissues. As such, the sharing of specific gene products among these tissues may provide some insight into similarities they share in terms of immunity.36

In summary, we have constructed a cDNA library of the HCE based on analysis of 2663 clones and have reported a full-length sequence of a novel gene showing the similarity to genes obtained in the ovary and the testis. Our cDNA library of HCE could provide new insights into the physiology of HCE.


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  		Table 2A. (continued).

 

   Footnotes
 
Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2001.

Supported by grants from Scientific Research from the Ministry of Education, Japan, (RS, SY), Comprehensive Research on Aging and Health, Ministry of Health, Labour and Welfare, Japan (TK), and National Eye Institute Grant EY-12963 (MRD).

Submitted for publication August 23, 2001; revised January 10, 2002; accepted January 25, 2002.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Satoru Yamagami, Department of Ophthalmology, Jichi Medical School, Yakushiji 3311-1, Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan; yamagami{at}jichi.ac.jp.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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