Identification of a 9 kDa γ- crystallin fragment in human lenses

Abstract

The degraded polypeptides ( M r < 14 kDa) were isolated by a preparative SDS-PAGE method from water soluble (WS) and water insoluble (WI) proteins of human lenses from donors of ages between 5 and 75 years. SDS-PAGE analysis showed the presence of a major 9 kDa polypeptide species that showed an agerelated increase in levels in WS-polypeptide preparations. In order to identify the parent crystallin of the 9 kDa polypeptide, the immunoreactivities of the WS- and WI-degraded polypeptides to immuno-affinitypurified anti-human α-, β- and γ-crystallin antibodies were determined by the Western blot method. The WS- and WI-9 kDa polypeptides showed immunoreactivity to only the anti-γ-crystallin antibody suggesting it to be a fragment of γ-crystallin. A 9 kDa species was purified by Sephadex G-50 chromatography from the WS-protein fraction of lenses from 20–30-year-old donors. The purified polypeptide showed a single protein band during SDS-PAGE and also an apparent single spot on two-dimensional gel-electrophoresis (IEF followed by SDS-PAGE). The purified preparation also showed a single major peak during reverse phase HPLC chromatography. The purified 9 kDa polypeptide showed immunoreactivity to only the anti-γ-crystallin antibody. A polyclonal antibody raised against the purified 9 kDa polypeptide showed immunoreactivity only to a 20 kDa γ-crystallin species. The partial N-terminal sequence analysis of the 9 kDa polypeptide showed it to be a fragment of γD-crystallin. Together these results show that a 9 kDa γD-crystallin fragment exists in increasing quantities in human lenses during aging.