Lp85 calpain is an enzymatically active rodent-specific isozyme of lens Lp82

Purposes. To clone and sequence the cDNA for Lp85 calpain from young rat le ns, and to test for Lp85 protein expression and proteolytic activity. Methods. RT-PCR and molecular cloning were performed on total RNA from 12 d ay-old rats. Lp85 protein expression was visualized by immunoblotting using a specific antibody developed to the unique peptide sequence in Lp85. Prot eolytic activity was assessed by casein zymography. Transient expression of Lp85 and previously characterized lens-specific calpain Lp82 were separate ly 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 predicte d 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 high est in the nuclear region, most concentrated in the insoluble fraction, dis appeared 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, enzymat ically active, member of the AX1 (alternative exon 1) subclass of calpains. In conjunction with Lp82 and m-calpain in lens, Lp85 may be responsible fo r proteolysis during normal lens development and maturation or during catar act formation in young rodents.