Influence of specific regions in Lp82 calpain on protein stability, activity, and localization within lens.
OBJECTIVE
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 mu- 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 maturational proteolysis.
Ma
H
H
Departments of Oral Molecular Biology, Oregon Health Sciences University, Portland, Oregon, USA.
Shih
M
M
Fukiage
C
C
Azuma
M
M
Duncan
M K
MK
Reed
N A
NA
Richard
I
I
Beckmann
J S
JS
Shearer
T R
TR
D000818:Animals; D019556:COS Cells; D002154:Calpain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000502:physiology; D004795:Enzyme Stability / Q000502:physiology; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D015151:Immunoblotting; D007908:Lens, Crystalline / Q000201:enzymology*; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D009154:Mutation; D010957:Plasmids; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013329:Structure-Activity Relationship
D002154:Calpain; C118624:calpain Lp82