Abstract
Abstract: :
Purpose:To learn more about the gene anatomy, expression pattern and possible function of Lengsin (LGS), an abundant transcript in the human lens that codes for a protein resembling Glutamine Synthetase (GS). Methods: Gene searches were carried out with the Ensembl Genome and NCBI browsers. Phylogenetic trees were constructed with the neighbour–joining algorithm. cDNA was prepared by RT–PCR of total RNA extracted from transparent human epithelial–capsule samples. LGS–specific amplification primers were designed on the cDNA sequence available at NCBI (AF242388). The resulting amplicons were cloned and one was chosen for expression in the yeast P. pastoris. The recombinant protein was purified by metal–affinity chromatography. GS activity was assayed with γ–glutamylhydroxamate (‘transferase reaction’). Results:The LGS gene is only present in vertebrates and belongs to a unique cluster that is closer to prokaryotic (type I) than to eukaryotic (type II) GSs. Standard DNA sequence analysis programs predict three introns in the LGS gene from Homo sapiens. However, the identification of three distinct LGS cDNAs indicates that the human gene undergoes extensive alternative splicing. The LGS polypeptide is similar GSI and sequence alignment shows that the sites involved in oligomerization, glutamate and Mg2+ cation (but not ATP) binding are highly conserved. Strong support to these bioinformatic findings was provided by the observation that polyclonal anti–GS antibodies do recognize recombinant LGS. This indicates that at least some epitopes are shared by LGS and authentic GS. Despite this similarity, however, LGS was found to be completely devoid of glutamine synthetase activity; a finding that is likely explained by the lack in its sequence of many of the amino acid residues that are thought to be involved in the binding of the ATP co–substrate. Conclusions: Despite new information on lengsin organism distribution, biogenesis and structural similarity with GS, the reason for its accumulation (in a catalytically inactive form) in the lens and the functional role it may play in such an organ are as yet unknown. LGS is reminiscent of ‘crystallin–enzymes’ recruited to the lens for various purposes (e.g., chaperones, UV filters, etc.) that we are utilizing as ‘working models’ for ongoing experiments aimed at the identification of the non–GS role of this fairly abundant lens protein.
Keywords: protein structure/function