Op. Srivastava et al., LEVELS OF CRYSTALLIN FRAGMENTS AND IDENTIFICATION OF THEIR ORIGIN IN WATER-SOLUBLE HIGH-MOLECULAR-WEIGHT (HMW) PROTEINS OF HUMAN LENSES, Current eye research, 15(5), 1996, pp. 511-520
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.