Levels of crystallin fragments and identification of their origin in water soluble high molecular weight (HMW) proteins of human lenses.
OBJECTIVE
The aims of this study were to determine in the human lens water soluble-high molecular weight (WS-HMW)-proteins: (a) the levels of degraded polypeptides (crystallin fragments), and (b) the in vivo cleavage sites in the parent crystallins to produce the major fragments.


METHODS
The WS-HMW proteins (Mr > 15 x 10(6) daltons) were isolated as 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 separated by a preparative SDS-PAGE method and quantified as a percent of total WS-HMW proteins. In addition, the parent crystallins of the major polypeptides were identified by the Western blot method using antibodies raised either to the whole crystallin molecule or to desired regions at N- and C-terminals or middle of individual crystallins. The partial N-terminal sequences of purified individual polypeptides were determined to identity the cleavage sites in parent crystallins.


RESULTS
The levels of degraded polypeptides as percent of the total WS-HMW proteins increased with aging, i.e. about 5% in lenses of 16 to 19 year-old-donors compared to 27% in the 60-80 year-old-donors. As many as thirteen polypeptide species with Mr's between 3 to 17 kDa were separated from WS-HMW proteins by a preparative SDS-PAGE method. The Western blot analyses showed that the polypeptides originated from alpha-, beta- and gamma-crystallins and the cleavage sites varied in different regions of crystallins as identified by partial N-terminal sequence analyses.


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.
Srivastava
O P
OP
Department of Physiological Optics, School of Optometry, University of Alabama at Birmingham 35294-4390, USA.
Srivastava
K
K
Silney
C
C
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D003459:Crystallins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007908:Lens, Crystalline / Q000737:chemistry*; D008875:Middle Aged; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D012995:Solubility; D014867:Water
D000596:Amino Acids; D003459:Crystallins; D010446:Peptide Fragments; D014867:Water