LEVELS OF CRYSTALLIN FRAGMENTS AND IDENTIFICATION OF THEIR ORIGIN IN WATER-SOLUBLE HIGH-MOLECULAR-WEIGHT (HMW) PROTEINS OF HUMAN LENSES

Purpose. The aims of this study were to determine in the human lens wa ter soluble-high molecular weight (WS-HMW)-proteins: (a) the levels of degraded polypeptides (crystallin fragments), and (b) the in vivo cle avage sites in the parent crystallins to produce the major fragments. Methods, The WS-HMW proteins (Mr > 15 x 10(6) daltons) were isolated a s a void volume peak from homogenates of lenses of donors of different ages using Agarose A 15m gel-filtration chromatography, The degraded polypeptides (Mr < 18 kDa), present in the WS-HMW proteins, were separ ated by a preparative SDS-PAGE method and quantified as a percent of t otal WS-HMW proteins. In addition, the parent crystallins of the major polypeptides were identified by the Western blot method using antibod ies raised either to the whole crystallin molecule or to desired regio ns at N- and C-terminals or middle of individual crystallins. The part ial N-terminal sequences of purified individual polypeptides were dete rmined to identity the cleavage sites in parent crystallins. Results. The levels of degraded polypeptides as percent of the total WS-HMW pro teins increased with aging, i.e. about 5% in lenses of 16 to 19 year-o ld-donors compared to 27% in the 60-80 year-old-donors. As many as thi rteen polypeptide species with Mr's between 3 to 17 kDa were separated from WS-HMW proteins by a preparative SDS-PAGE method. The Western bl ot analyses showed that the polypeptides originated from alpha-, beta- and gamma-crystallins and the cleavage sites varied in different regi ons of crystallins as identified by partial N-terminal sequence analys es. Conclusions. The data showed an age-related increase in levels of degraded polypeptides in the WS-HMW proteins and the polypeptides were derived from alpha-, beta- and gamma-crystallins.