A Structural Modification of Ubiquitin is Present in Human Senile Total Cataracts

Abstract

Abstract: : Purpose: Human lens is composed mainly by proteins. Damaged crystallins are a particular target for the ubiquitin system which has been identified in mammalian lenses. We wanted to identify and characterize ubiquitin in the human senile cataractous lenses as compared to the transparent ones. Methods: Water soluble fractions of the nuclei of human senile totally cataractous lenses were processed by means of HPLC; low molecular weight fractions were further separated by RP-HPLC. Aliquots were analyzed by means of MALDI-TOF Mass Spectrometry (MS), peptide mapping and sequencing MS, and Western Blot analysis using a rabbit polyclonal anti-ubiquitin antibody. Controls were clear human lenses of age-matched donors, divided into cortex and nucleus by a sham surgical extracapsular approach. Results: The elution RP-HPLC pattern of a normalized very small protein fraction in the region of low molecular weight proteins showed a clear difference between the normal and the cataractous samples: in the latter a very intense peak, which was barely detectable in the normal sample, was present. This peak was characterized by an absorbance typical of a protein containing tyrosine, but not tryptophan, as another fraction which was present in both samples. MALDI-TOF MS of the purified fractions showed the presence in the cataractous lenses of an intense peak corresponding to a 8182 Da protein, which was absent in the normal lens, and peaks corresponding to 8566/8583 Da proteins in both normal and cataractous lenses. Search in sequence databases and peptide mapping and sequencing by MS identified the 8566 Da species as ubiquitin; the intense peak at 8182 m/z was identified as a shorter form due to cleavage of the C-terminal 73-76 residues. Western blot analysis confirmed the presence, in the cataractous lenses, of two forms binding anti-ubiquitin antibody. Conclusions: All the results demonstrate that in the nucleus of cataractous lenses two forms of ubiquitin, one of which is not active, is present, unlike the single form present in the nucleus of normal lenses of the same age. Calcium activated calpain II may be responsible for the alteration in the structure and function of ubiquitin, which is no longer capable of inducing degradation in damaged crystallins.

Keywords: cataract • protein modifications-post translational • protein structure/function