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SJ Lee, S Ghosh, B DeBroff, MB Shields, M Coca-Prados; Genetic and Molecular Characterization of TIGR/Myocilin in human aqueous humor . Invest. Ophthalmol. Vis. Sci. 2002;43(13):272.
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Purpose:To determine whether the various low molecular weight protein forms detected with TIGR epitope-specific antibodies in human aqueous humor are posttranslationally modified forms of TIGR, TIGR-like proteins and/or cleaved forms of full-length TIGR. Methods:Aqueous humor samples from glaucomatous and normal human eyes were obtained intraoperatively from patients undergoing trabeculectomy and cataract surgery, respectively, by the Department of Ophthalmology and Visual Science at Yale-New Haven Hospital under informed consent. SDS-PAGE and Western Blot analysis was performed to identify TIGR species in human aqueous humor using a combination of anti-peptide antibodies (Abs) E14L specific for the amino terminus of TIGR (residues 153-166), R14T specific for exon 2 of TIGR (residues 272-285), and C21A specific for the carboxy terminus of TIGR (residues 468-488). Species detected were analyzed through protein isolation and MALDI-MS amino acid (aa) sequencing. Additionally, a human ciliary body cDNA library was differentially screened at low stringency using radioactively labeled DNA probes to identify, select and clone TIGR species of interest and resolve them through DNA sequencing. Results:Western blot analysis of both normal and glaucomatous aqueous humor specimens revealed the predicted 55 kDa doublet of normal TIGR plus an additional discrete 30 kDa band recognized by R14T Ab and a 15 kDa band by C21A and E14L Abs. Amino acid sequencing analysis, however, could not clearly resolve the identity of these novel bands. On the genetic level, only 1 out of nearly 200 TIGR species isolated and sequenced from the human ciliary body cDNA library demonstrated a novel sequence which was found to lack the olfactomedin region of normal TIGR from nt 581 to 1723 (LZ+OLF-), resulting in a semi-canonical alternative splicing variant whose gene product would be 175 aa in length and contain the N-terminal LZ region of normal TIGR plus five novel hydrophobic aa's at the C-terminal region. Conclusion:No biologically significant variant forms of TIGR could be identified on the genetic level, suggesting that, unlike in other species, neither genetically related nor alternative splicing pathway forms of TIGR appear to be present in the human ciliary body. In addition, no posttranslationally modified or cleaved forms could be definitively detected on the protein level, perhaps due to quantitative limitations and co-migration with other proteins that may mask TIGR. Future studies to isolate TIGR in vivo may help further elucidate the complex and elusive nature of TIGR in human aqueous humor.
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