Lp82 is the dominant form of calpain in young mouse lens.
The purpose of this study was to characterize Lp82 calpain in normal mouse. Lp82 is a lens-specific, calcium-activated isozyme from the calpain super family of cysteine proteases (EC 34.22.17). RT-PCR and molecular cloning were performed on total RNA from 12 day-old mice. Lp82 and m-calpain protein levels and proteolytic activities in lenses were measured by casein zymography, immunoblotting, and ELISA after partial purification by DEAE-HPLC. The 2334-bp cDNA encoding for mouse Lp82 contained a single large open reading frame encoding a protein of 709 amino acid residues with a calculated molecular weight of 82.2 kDa and a predicted pI of 5.8. The amino acid sequence of mouse lens Lp82 was 99% homologous to rat lens Lp82. As in rat, mouse lens Lp82 showed a unique N -terminus and deletion of the IS1 and IS2 regions. In contrast to rat, Lp82 was the dominant calpain in young mouse lens. Lp82 was lens-specific, and the lens nucleus contained the highest specific activity of Lp82 and very little m-calpain. Endogenous Lp82 in lens soluble proteins was activated by addition of calcium and caused limited proteolysis of crystallins even in the presence of large amounts of recombinant domain I from the natural calpain inhibitor calpastatin. Loss of Lp82 protein accompanied aging of mouse lens. Lp82 may be responsible for a major portion of crystallin proteolysis occurring during normal lens development and maturation, or during cataract formation in young mice.
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Departments of Oral Molecular Biology, Biochemistry and Molecular Biology, and Ophthalmology, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Hata
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Shih
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Fukiage
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Nakamura
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Y
Azuma
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Shearer
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TR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002154:Calpain; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003459:Crystallins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015151:Immunoblotting; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification*; D007908:Lens, Crystalline / Q000201:enzymology*; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D003459:Crystallins; D007527:Isoenzymes; D012333:RNA, Messenger; D002154:Calpain; D003546:Cysteine Endopeptidases; C118624:calpain Lp82