Molecular Characterization of Novel Proteins, p132 and p128, Two Splice Variants of a Single Gene, Isolated From Human Lens Epithelial cDNA Library

Abstract

Abstract: : Purpose: Circulating autoantibodies can be detected in a wide variety of autoimmune diseases including age related cataract (ARC) which specifically recognize various cells or intracellular organelles, and thus offer unique tools for the isolation and cloning of novel molecules. To this end, we isolated two novel clones from human lens epithelium (hLEC) cDNA library with serum from cataract patient. We have characterized the functional domains and organization, and intracellular localization of two novel proteins, p132 and p128, splice variants of single gene to understand the molecular mechanism of their expression and function in eye lens. Methods: hLEC–cDNA library was screened with serum from cataract patient. Two immunologically positive clones yielded a 5.0kb and 4.9kb EcoR1 and XhoI insert. Isoloated clones were sequenced and protein coding region was determined. Full–length cDNA were cloned into prokaryotic (pGEX5X–3) and eukaryotic (pEGFP) expression vectors at predefined restriction sites using PCR. Subcellular localization using cell organelles specific markers was determined in Cos–7 or LECs following transfection with pEGFP–p132/ pEGFP–p128 under fluorescent microscope. Transcript size and tissue distribution was done with Northern blot. Western analysis and immunohistochemistry was carried out with specific anti p132 and p128 antibodies.Results:Bioinformatics based analysis showed that p132 and p128 are derived from a single gene. Sequence analysis of cDNA p132 and p128 showed long coding region and contained 3363 and 3282 bps and encoded protein was 1121 and 1094 amino acids with molecular mass of 132kD and 128kD, respectively. Northern analysis revealed two 5.5 and 5.4 kb transcripts. GFP–p132 showed perinuclear and peripheral punctuate expression, in contrast, p128 revealed linear expression pattern in the cytoplasm. p132 colocalized with mannosidase II, a Golgi marker, to Golgi body. Perinuclear GFP–p132 was scattered with nocodazol induced microtubule depolymerization. Localization pattern of GFP–p132 mutants revealed that N–terminal is essential for its Golgi–localization.Conclusions: Taken together, we propose that p132, Golgi–protein may function in the specific organization of Golgi membrane and vesicles or in sorting and transport of proteins to or from the Golgi compartment. Studies on these proteins will provide new avenue to understand the lens biology.

Keywords: cataract • gene/expression • protein structure/function