Characterization and Regulation of Lens-specific Calpain Lp82

Abstract

Eye tissues contain splice variants of muscle-preferred p94 (calpain 3), such as lens-specific Lp82 and Lp85, retina-specific Rt88, and cornea-specific Cn94. The purpose of the present experiment was to analyze the activation and regulation of the best characterized p94 splice variant, Lp82. Recombinant rat Lp82 (rLp82) was expressed using the baculovirus system, purified with Ni-NTA affinity and DEAE-ion exchange chromatographies, and characterized by SDS-PAGE, casein zymography, and immunoblotting. After incubation with calcium, rLp82 autolyzed into two major fragments at ϳ60 and 22 kDa. Sequencing of the autolytic fragments showed loss of three amino acids from the N terminus and cleavage near the IS2 region. Also, Lp82 and calpain 2 were found to hydrolyze each other. Calpastatin inhibited calpain 2 activity, but not Lp82. Homology modeling suggested that the lack of inhibition of Lp82 by calpastatin was due to molecular clashes at the unique AX1 region of Lp82. Lp82 also hydrolyzed calpastatin. These results suggested that Lp82 might regulate other calpain activities and cause hydrolysis of substrates such as crystallins during lens cataract formation.Calpains (EC 3.4.22.17) comprise a family of non-lysosomal, cysteine proteases with a neutral pH optimum and a requirement for calcium for activation (1). They are widely distributed in animal tissues, where they are involved in a variety of cellular processes involving calcium (2). Calpains consist of the ubiquitous calpains 1 (-calpain), 2 (m-calpain), and 10; and tissue-specific calpains such as 3 (muscle-specific p94), 8, and 9 (3-6). Recently, splice variants of calpain 3 such as lens-specific Lp82 and Lp85, retina-specific Rt88, and cornea-specific Cn94 were found in the eye (7-10). Tissue-specific calpains may have unique properties or structures distinct from the ubiquitous calpains. For example, mutations in muscle-preferred p94 calpain cause muscular dystrophy type 2A in man (11). p94 contains three unique regions: the novel N-terminal sequence (NS), 1 insert region 1 (IS1), and insert region 2 (IS2) (4).Lens-specific calpain Lp82 belongs to a new subclass of calpains, termed AX1, because they replace the NS region in the N-terminal domain I by using alternative exon 1, a portion of the 3Ј end of intron 1 from the p94 gene. Common promoter elements in the eye may cause the alternative transcription of AX1 from the p94 gene. Other p94 variants contain combinations of splice variations in or near the IS1 and IS2 insert regions. Lp82 activity in young rat lenses is abundant and stable, because the IS1 and IS2 regions are splice deleted (12, 13). Besides Lp82, rodent lenses contain ubiquitous calpain 2, atypical and ubiquitous calpain 10, and lens-specific Lp85. These calpains may regulate each other by proteolysis, because they are substrates for each other. Thus, the purpose of the present experiment was to study calpain interactions by analyzing the activation and regulation of Lp82 in lens. EXPERIMENTAL PROCEDURES Animals-Experiments were performed u...

Insertion Sequence 1 of Muscle-specific Calpain, p94, Acts as an Internal Propeptide

“…E-64 and leupeptin could not prevent degradation of p94 in muscle extracts or in heterologous expression systems (8), presumably because IS1 restricts access to the active site cleft. In contrast, caseinolytic activity of Lp82 (where IS1 is missing) was completely inhibited by the E-64 or EGTA (20). Furthermore, leupeptin and calpain inhibitors I and II effectively inhibit the splice variant of p94 present in peripheral blood mononuclear cells that lacks IS1 (18).…”

“…Rapid autolysis of p94 during purification from rabbit muscle fractions was not prevented by the calpain inhibitors E-64 (trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane) and leupeptin (8), whereas autolysis of purified recombinant Lp82 was totally inhibited by E-64 or EGTA (20). Moreover, a recently reported p94 isoform present in human peripheral blood mononuclear cells that lacks IS1 and the lysine-rich IS2 sequence was also inhibited by leupeptin and calpain inhibitors I and II (18).…”

“…Moreover, a recently reported p94 isoform present in human peripheral blood mononuclear cells that lacks IS1 and the lysine-rich IS2 sequence was also inhibited by leupeptin and calpain inhibitors I and II (18). In contrast to -and m-calpain, p94 and its splice variants have been shown to be insensitive to inhibition by calpastatin (8,18,20). The basis for this insensitivity is not known, but it could be due in part to the absence of the small subunit, which is one of the defined calpastatin binding sites present in m-calpain (24).…”

“…In contrast to p94, Lp82, the lens-specific calpain isoform that lacks NS, IS1, and IS2, has been detected as a full-length 82-kDa protein band in young rodent lenses, and the corresponding enzymatic activity band has been detected on casein zymograms (19). Furthermore, a recombinant construct of Lp82 was stably produced in insect cells, purified, and characterized (20). Very recently, an alternatively spliced variant of mouse skeletal muscle p94 that lacks IS1 and IS2 (p94:exon 6 Ϫ 15 Ϫ 16 Ϫ , p94⌬) has been successfully expressed in mammalian cells and shown to be proteolytically active (21).…”

Peptidase Activity of the Escherichia coli Hsp31 Chaperone

“…Gelatin-PAGE and casein-PAGE in-gel proteolysis assays were performed as described in (3,12); 2 g of Hsp31 were electrophoresed onto 12% SDS-polyacrylamide gels containing either 0.2% co-polymerized gelatin or 0.2% copolymerized casein, and proteolytic activity was visualized by clearing zones resulting from gelatin or casein hydrolysis (12). Gelatin and casein zymography was also performed using electrophoresis under native conditions as described by Fukiage et al (13). Proteolysis of citrate synthase, ␣-casein, and ␤-casein was assayed by incubation of the reaction mixtures containing 2 g of the protein substrate and 0.5 g of Hsp31 at 37°C in 20 mM Tris, pH 8, 5 mM MgCl 2 .…”