Abstract:
The major purpose of this research was to characterize partially degraded proteins
appearing in the ocular lens during aging and cataract, and to identify the responsible
proteolytic activities. This research is significant, because increased protein
degradation is associated with lens opacification and cataract. Determining the sites
where lens proteins become truncated and identification of the responsible proteases is
important because this information could be used to develop anticataract agents, such
as new protease inhibitors.
The methods used in this research include the application of several techniques
employed in the field of proteomics. Two-dimensional electrophoresis (2-DE) was
used to separate crystallins, the major proteins of the lens, and the modifications to
these proteins were then determined by electrospray ionization mass spectrometry
(ESI-MS). Lenses from mice of increasing age were used for these studies. Lp82 and
m-calpain, the proteases hypothesized to cause the observed truncation of mouse
crystallins, were purified from fetal calf lenses or expressed recombinantly, and their
enzymatic properties compared. The cleavage sites produced in α-crystallin by Lp82
and m-calpain in vitro were then compared to truncated α-crystallins from normal rat
lens, and rat lens with selenite-induced cataract.
The results included data showing that crystallins in mouse lenses underwent
extensive truncation after 6-weeks of age that was associated with their
insolubilization. Characteristics of purified Lp82 included: a lower calcium
requirement for activation, a decreased sensitivity to the endogenous inhibitor
calpastatin, and a greater resistance to autolysis than m-calpain. Analysis of truncated
α-crystallins isolated from both normal and cataractous rat lens indicated that Lp82
was more active in young rat lens than was m-calpain. Lp82 specifically removed 5
residues from the C-terminus of αA-crystallin. This αA degradation product was far
more abundant in the lenses of young rats than was the m-calpain specific aA product
missing 11 residues from its C-terminus.
In conclusion, this study showed that insolubilization of crystallins and cataract
may result in animal lenses from uncontrolled crystallin degradation by the protease
Lp82. This has led to a fundamental change in our view of experimental cataract
formation, since activation of the protease m-calpain was previously believed to cause
cataract.
