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T Iwata, N Sanuki, K Fujiki, HN Thanh, A Kanai, S Umeda, T Nishiyama, Y Mashima, Y Tanaka; Identification of Erythrocyte Antigen PBDX Expressed in Human Cornea Epithelial Cells and Keratocytes . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1672.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Keratocytes are spindle shape fibroblast-like cells which occupy cornea stroma as major cellular component. We have focused on cornea keratocytes and performed cDNA subtraction method to clone and characterize abundantly expressed but functionally unknown genes. One of the gene isolated by this method, pseudoautosomal-boundary divided on the X-chromosome (PBDX) gene, is discussed in detail. Methods: PCR-select cDNA subtraction method and 5'/3'-RACE methods were used to clone genes abundantly expressed in primary human keratocytes. The cDNA subtraction was performed with primary human keratocyte against pool of equally mixed mRNA from brain, kidney, liver, and skeletal muscle. Real-time quantitative PCR was used to measure and compare the expression level between various tissues and between male and female keratocytes. In situ hybridization was performed to localize PBDX transcript in the Cynomolgus monkey cornea. Results: Out of twenty-five clones isolated, one of the clone was identified as PBDX gene located in pseudoautosomal region of X-chromosome. Identical open reading frame but additional 1.3Kb of nucleotide was identified at the 3' end of mRNA compared to the previously reported full-length mRNA sequence. PBDX was abundantly expressed in epithelial and keratocytes but not in endothelial cells of human cornea. Expression level of PBDX was more than 8-fold higher in human cornea than any of eight other tissues compared. This X-chromosome gene was expressed approximately twice the amount in female keratocytes compare to male. Amino acid analysis revealed possible phosphorylation and myristoylation sites. Conclusion: X-chromosome erythrocyte antigen PBDX was found abundantly expressed in human cornea keratocyte by cDNA subtraction method. The newly identified 2.3Kb splice variant of PBDX differs in size by 1.3Kb than previously reported.
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