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Hong Ma, Marjorie Shih, Chiho Fukiage, Mitsuyoshi Azuma, Melinda K. Duncan, Nathan A. Reed, Isabelle Richard, Jacques S. Beckmann, Thomas R. Shearer; Influence of Specific Regions in Lp82 Calpain on Protein Stability, Activity, and Localization within Lens. Invest. Ophthalmol. Vis. Sci. 2000;41(13):4232-4239.
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purpose. To determine the influence of specific regions within Lp82 calpain on
protein stability, enzymatic activity, and localization within lens and
to test the influence of an Lp82 knockout mouse on normal maturational
proteolysis in lens.
methods. DNA constructs for Lp82 and Lp82-related proteins were subcloned into
the pcDNA 3.1 vector. The constructs contained a substitution of the
novel sequence (NS) region from p94 for the AX1 N-terminal region of
Lp82 and insertions of the p94 IS1 and IS2 regions into Lp82. Transient
expression of these Lp82-related proteins was performed in COS-7
mammalian cells. Immunoblotting and casein zymography were used to
measure protein stability and enzymatic activity of the expressed
proteins. Homologous recombination was used to knock out p94 gene
expression and p94 splice variants such as Lp82 and Lp85 in the lenses
of 10-day-old mice. Confocal microscopy revealed the
immunohistochemical localization Lp82 and Lp85 within lens.
results. Insertion of IS1 into Lp82 resulted in a lack of stable protein and
loss of enzymatic activity. In contrast, substitution of the NS region
for AX1 and insertion of IS2 into Lp82 had no effect on the stability
of the Lp82-related proteins. p94 knockout mice at 10 days of age
exhibited a total absence of Lp82 activity in the lens but normal
activity for the separate μ- and m-calpain gene products.
Calcium-induced in vitro proteolysis was retarded in these Lp82/p94
knockout lenses. Lp82 and Lp85 immunostaining was intense throughout
the cytoplasm of the cortical and nuclear fibers of newborn mouse
lenses with little staining in the epithelium. In contrast,
immunostaining for the ubiquitous m-calpain was highest in the
epithelium and bow region, with much lower levels in the nucleus. The
naturally occurring IS3 insert in Lp85 also promoted the association of
Lp85 with the perinuclear region of the nucleated lens fibers.
conclusions. The lack of the IS1 region in Lp82 accounts for the stability and
abundance of enzymatically active Lp82 protein in rodent lenses.
Conversely, the presence of the IS1 region is responsible for the
lability of p94 and Rt88 calpains in muscle and retina, respectively.
The insert in Lp85 may promote membrane association. A consequence of
the specific loss of Lp82 in the lens may be to retard normal
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