Autolysis, Ca2+ Requirement, and Heterodimer Stability in m-Calpain

Abstract

The roles of N-terminal autolysis of the large (80 kDa) and small (28 kDa) subunits in activation of rat m-calpain, in lowering its Ca 2؉ The calpains (EC 3.4.22.17) are cytoplasmic cysteine proteinases, which are thought to be regulated by means of their Ca 2ϩ dependence. While much work has been done on their biochemical properties, many aspects of autolysis, activation, and Ca 2ϩ requirement remained unresolved (1-3). Two mammalian forms, -and m-calpain (calpain I and II), have been most studied, since they can be isolated from animal tissues. Some other calpain forms are known so far only from their mRNA (3, 4), and the chicken calpains are not further considered here since they appear to be slightly different (5, 6). Complete purification of calpain in adequate amounts from tissue extracts is difficult, and a bacterial expression system has been described, which with the aid of a His-tag provides larger amounts of pure enzyme in about 3 days and provides a means for mutational and structural work (7). A baculovirus-based expression system has also been reported (8).The calpains consist of an 80-kDa catalytic subunit (the large subunit), containing four domains, I-IV, and a 28-kDa regulatory subunit (the small subunit), containing two domains, V and VI (9, 10). Domains I and V are involved in autolysis; domain II contains the most obvious active site residues (11); the function of domain III is not yet known, although it must also take part in the conformational changes induced by Ca 2ϩ ; and the C-terminal domains of both subunits, IV and VI, contain putative E-F hand motifs, some of which bind Ca 2ϩ (12)(13)(14)(15). No structural information is available for the complete calpain heterodimer, but the crystal structures of domain VI, with and without bound Ca On exposure to sufficient Ca 2ϩ , the calpains are assumed to undergo a conformational change which permits the following four events: limited autolysis of the small subunit from 28 to 20 kDa; limited N-terminal autolysis of the large subunit; proteolysis of a substrate such as casein if it is present; and further inactivating proteolysis of the large subunit. However, the order of these events and the Ca 2ϩ concentrations required have been difficult to establish. The Ca 2ϩ requirements ([Ca 2ϩ ] 0.5 ) for casein hydrolysis by (initially) non-autolyzed calpains are usually reported to lie in the ranges of 5-50 M Ca 2ϩ for -calpain and 250 -1000 M Ca 2ϩ for m-calpain. These ranges are lowered by prior autolysis to 1-5 M Ca 2ϩ for -calpain and 100 -200 M Ca 2ϩ for m-calpain (16 -25). The reported values vary widely for at least two reasons, first because the casein assay reflects the net effects of activation, autolysis, and inactivation, and is not well suited for kinetic studies (26); and second because, as shown here, the observed values are highly dependent on experimental conditions. While it is fairly clear that autolysis lowers [Ca 2ϩ ] 0.5 of both -and m-calpain, it has not been clear whether large and/or small subunit cleavage is req...

Regulating cell migration: calpains make the cut

“…The truncated large subunit is catalytically active and has a lower requirement for calcium (Baki et al, 1996;Imajoh et al, 1986;Suzuki and Sorimachi, 1998;Suzuki et al, 1981b). However, this event is clearly not required for catalytic activity (Cong et al, 1989;Elce et al, 1997;Guttmann et al, 1997;Molinari et al, 1994), which suggests that it functions more in the progression of activation than in its initiation.…”

“…Several different modes of regulation have been identified, although their contributions in vivo have not yet been determined. The large subunits of some calpains are autolyzed on activation, which removes domain I and abolishes the N-terminal link between the large and small subunits, thereby allowing movement of domain II (Baki et al, 1996;Cong et al, 1989;Elce et al, 1997;Guttmann et al, 1997;Imajoh et al, 1986;Molinari et al, 1994;Suzuki and Sorimachi, 1998;Suzuki et al, 1981a). The truncated large subunit is catalytically active and has a lower requirement for calcium (Baki et al, 1996;Imajoh et al, 1986;Suzuki and Sorimachi, 1998;Suzuki et al, 1981b).…”

Autolytic activation and localization in Schneider cells (S2) of calpain B from Drosophila

“…During activation of mammalian µ-and m-calpains by Ca 2+ , autolysis occurs at the N-termini of both large and small subunits [23][24][25]. There is no consensus about the mechanistic role of these autolyses: some authors regard them as consequences of activation [6,26], while others hold that they are essential for activation; at least, this is strongly suggested by the close parallelism between the amount of autolysed protein and enzyme activity in the case of µ-calpain [27]. Whatever holds true for mammalian calpains, it should be remembered that their domain I is very short compared with that of calpain B.…”

Activity profile of calpains I and II in chronically infarcted rat myocardium – influence of the calpain inhibitor CAL 9961

“…The enzyme activity for calpains I and II was measured in all samples in a total volume of 200 ml, containing 170 ml of the enzyme fraction, 10 ml Suc-Leu-Tyr-AMC (®nal concentration 500 mM) and 20 ml CaCl 2 (®nal concentration 5 mM to optimally activate both calpain isoforms, Ca 2+ -dependent activity). Details for the Ca 2+ -titrations and the relationships between hydrolysis of Suc-Leu-Tyr-AMC and Ca 2+ -requirements have been described previously by Elce et al (1997b). To measure Ca 2+ -independent activity, CaCl 2 was replaced by 20 ml EDTA (neutralized to pH 7.5 with NaOH) to give a ®nal concentration of 10 mM.…”