Lengsin: A Novel Marker for Terminal Differentiation in the Lens

Abstract

Abstract: : Purpose: Functional Genomics of a novel gene discovered through the NEIBank project Methods: Human, mouse and rat cDNAs were obtained from EST analyses. Gene sequences were derived from the genome projects. Splice variants were examined by PCR. Expression was examined by Northern and Western blot and by ISH. Recombinant proteins were produced using the pET system. Structure was examined by electron microscopy (EM). Photo-activatable ATP was used to examine cofactor binding and mass spectroscopy was used to validate peptide sequences. The gene promoter was cloned with a GFP reporter for transgenic mouse production. Results: Lengsin is a novel and abundant transcript in a human lens cDNA library. It appears to be highly specific for the lens and is expressed in a remarkably restricted pattern in the ring of fiber cells undergoing terminal differentiation and loss of nuclei. Lengsin belongs to the glutamine synthetase (GS) superfamily. EM reveals a six-fold symmetrical oligomer similar to that of bacterial GS proteins. Recombinant protein will bind ATP, but no enzyme activity has yet been demonstrated. The mouse (and rat) gene has 5 exons. In the human gene, the third exon is detectable but is accumulating mutations and would disrupt the ORF if used. Indeed, this exon is skipped in splicing, providing another example of a "pseudo-exon" in a human lens gene (the first example being αA-crystallin). Transgenic mice are being generated that use the lengsin promoter to target genes to the terminal differentiation zone of the lens. Crystallin promoters are being used to mis-express lengsin at earlier stages in mouse lens cell differentiation and a gene KO construct is in progress.The human gene is located at chromosome 6q12, close to LCA5. Conclusions: Lens transparency depends on a highly ordered progression from undifferentiated epithelial cells to terminally differentiated fiber cells. In the final stages, the fiber cells lose nuclei and other organelles in a process with some similarities to apoptosis. Lengsin is a novel and specific marker for this stage and is a candidate for involvement in this characteristic process of lens cell maturation. The lengsin promoter should allow us to target other genes to dissect the events involved in nucleus and organelle loss.

Keywords: molecular biology • gene/expression • anterior segment