Calpain expression and activity during lens fiber cell differentiation.
In animal models, the dysregulated activity of calcium-activated proteases, calpains, contributes directly to cataract formation. However, the physiological role of calpains in the healthy lens is not well defined. In this study, we examined the expression pattern of calpains in the mouse lens. Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7 were expressed in lens fiber cells. Using controlled lysis, depth-dependent expression profiles for each calpain were obtained. These indicated that, unlike calpain 1, 2, and 7, which were most abundant in cells near the lens surface, calpain 3 expression was strongest in the deep cortical region of the lens. We detected calpain activities in vitro and showed that calpains were active in vivo by microinjecting fluorogenic calpain substrates into cortical fiber cells. To identify endogenous calpain substrates, membrane/cytoskeleton preparations were treated with recombinant calpain, and cleaved products were identified by two-dimensional difference electrophoresis/mass spectrometry. Among the calpain substrates identified by this approach was alphaII-spectrin. An antibody that specifically recognized calpain-cleaved spectrin was used to demonstrate that spectrin is cleaved in vivo, late in fiber cell differentiation, at or about the time that lens organelles are degraded. The generation of the calpain-specific spectrin cleavage product was not observed in lens tissue from calpain 3-null mice, indicating that calpain 3 is uniquely activated during lens fiber differentiation. Our data suggest a role for calpains in the remodeling of the membrane cytoskeleton that occurs with fiber cell maturation.
De Maria
Alicia
A
Department of Ophthalmology and Visual Sciences, Washington University, St. Louis, Missouri 63110.
Shi
Yanrong
Y
Department of Ophthalmology and Visual Sciences, Washington University, St. Louis, Missouri 63110.
Kumar
Nalin M
NM
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois 60612.
Bassnett
Steven
S
Department of Ophthalmology and Visual Sciences, Washington University, St. Louis, Missouri 63110. Electronic address: bassnett@vision.wustl.edu.
D000818:Animals; D002154:Calpain / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002386:Cataract / Q000201:enzymology / Q000235:genetics; D002454:Cell Differentiation / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D005136:Eye Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D007908:Lens, Crystalline / Q000166:cytology / Q000201:enzymology*; D051379:Mice; D008817:Mice, Mutant Strains; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013049:Spectrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000502:physiology
D005136:Eye Proteins; D011994:Recombinant Proteins; C495133:SPTBN2 protein, human; D013049:Spectrin; D002154:Calpain