Lp85 calpain is an enzymatically active rodent-specific isozyme of lens Lp82.
OBJECTIVE
To clone and sequence the cDNA for Lp85 calpain from young rat lens, and to test for Lp85 protein expression and proteolytic activity.


METHODS
RT-PCR and molecular cloning were performed on total RNA from 12 day-old rats. Lp85 protein expression was visualized by immunoblotting using a specific antibody developed to the unique peptide sequence in Lp85. Proteolytic activity was assessed by casein zymography. Transient expression of Lp85 and previously characterized lens-specific calpain Lp82 were separately performed in mammalian COS-7 cells.


RESULTS
The 2410-bp cDNA for rat lens Lp85 encoded a protein of 737 amino acid residues with a calculated molecular weight of 85.0 kDa and a predicted pI of 5.67. The amino acid sequence of Lp85 was identical to Lp82 except for an insert region of 28 amino acids in domain IV of the calcium-binding region. mRNA and protein for Lp85 were present only in rat and mouse lenses and not in other tissues or species. Lp85 protein concentrations were highest in the nuclear region, most concentrated in the insoluble fraction, disappeared with lens maturation, and Lp85 exhibited migration similar to Lp82 on native PAGE gels. Lp85 was enzymatically active when expressed in COS-7 cells.


CONCLUSIONS
Lp85 is a newly classified, lens- and rodent-specific, enzymatically active, member of the AX1 (alternative exon 1) subclass of calpains. In conjunction with Lp82 and m-calpain in lens, Lp85 may be responsible for proteolysis during normal lens development and maturation or during cataract formation in young rodents.
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Departments of Oral Molecular Biology, Biochemistry and Molecular Biology, and Ophthalmology, Oregon Health Sciences University, Portland, OR 97201, USA.
Shih
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Hata
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Fukiage
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Azuma
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Shearer
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D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002154:Calpain; D002417:Cattle; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007908:Lens, Crystalline / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014018:Tissue Distribution
D018076:DNA, Complementary; D007527:Isoenzymes; D012313:RNA; D002154:Calpain; D003546:Cysteine Endopeptidases; C118624:calpain Lp82