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The Calpain System

Muscle Biology Group, University of Arizona, Tucson, Arizona

ABSTRACT

I. INTRODUCTION

II. BACKGROUND INFORMATION: THE WELL-CHARACTERIZED PLAYERS µ-CALPAIN, m-CALPAIN, AND CALPASTATIN

A. Properties of µ- and m-Calpain

1. cDNA sequences of µ- and m-calpain

2. Genomic sequences of µ- and m-calpain

3. Crystallographic and solution structures

4. Autolysis and the proenzyme question

B. Properties of Calpastatin

1. DNA sequencing

2. Domain structure of calpastatin

3. Binding of calpastatin to the calpains

C. Purification of the Calpains and Calpastatin and Assay of Their Activity

1. Purification of the calpains

2. Purification of calpastatin

3. Assays of calpain activity

III. OTHER MEMBERS OF THE CALPAIN FAMILY: CALPAIN-LIKE MOLECULES

A. Calpain-Like Molecules in Invertebrates

1. Schistosoma mansoni calpains

2. Drosophila calpains

3. Crustacean Ca2+-dependent proteases

4. Calpain-like molecules in C. elegans

B. Calpain-Like Molecules in Vertebrates

1. Calpain 3a (p94) and limb girdle muscular dystrophy type 2A

2. Other vertebrate calpain homologs

C. Summary

D. Nomenclature

IV. BIOCHEMICAL PROPERTIES AND REGULATION OF CALPAIN ACTIVITY

A. Expression of Proteolytically Active Molecules

B. Tissue Localization

1. Presence in different tissues

2. Immunolocalization

C. Regulation of Calpain Activity

1. The Ca2+ requirement problem

2. Ca2+-binding properties of µ- and m-calpain

3. Subsite specificity

4. Phosphorylation of the calpains

5. Calpastatin

6. Mechanisms involved in catalysis

V. PHYSIOLOGICAL FUNCTIONS OF THE CALPAIN SYSTEM

A. What Do The Transgenic Mice Show?

B. In Vitro Substrates

C. In Vivo Substrates and Some Proposed Functions

1. Cytoskeletal/membrane attachments: cell motility

2. Signal transduction pathways

3. The cell cycle

4. Regulation of gene expression

5. Apoptosis

6. Long-term potentiation

VI. PATHOLOGICAL IMPLICATIONS OF THE CALPAIN SYSTEM

VII. SUMMARY AND CONCLUSIONS

	ABSTRACT

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	I. INTRODUCTION

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A great deal of information has been obtained on properties of the calpain system since purification in 1976 of the protein that was later named m-calpain (83, 84). Books dedicated to Ca²⁺-dependent proteases and the calpains were published in 1990 (284) and 1999 (473), and a monograph on calpain methods and protocols appeared in 2000 (104). Two FASEB Summer Research Conferences devoted to the calpain system have been held: the first in Copper Mountain, CO in 1999 and the second in 2001 at Whitefish, MT. A web site that offers opportunities for exchange of information and discussion has been established: http://ag.arizona.edu/calpains/. A number of shorter review articles on different aspects of the calpain system have also appeared during the last 10 years (139, 196, 377, 418–420, 437, 438, 472 as examples of some of these reviews). The state of the calpain system up to 1991 was summarized in a comprehensive review on the calpains in this journal (76). A number of important advances have occurred since this 1991 review, including 1) cloning and successful expression of a proteolytically active m-calpain from E. coli (142) or baculovirus (268) expression systems; 2) crystallographic structures, first of domain VI (39, 247) and then of the entire m-calpain molecule in the absence of Ca²⁺ (171, 430); 3) identification of calpain-like Ca²⁺-activated proteases in invertebrate tissues and of a number of mRNAs encoding putative calpains, some of them evidently tissue specific (416, 417, 420, 438); and 4) generation of two knock-out mice demonstrating that the calpain system is essential for life (10) but that deletion of only one of the two "ubiquitous" calpains, µ-calpain, does not result in embryonic death (14).

Despite this wealth of new information, two of the primary questions concerning the calpain system remain unanswered: 1) How is calpain activity regulated in living cells? 2) What is the physiological function (or stated differently, what are the physiological substrates) of the calpain system in normal cells? The structural information obtained during the last 10 years has altered views as to how calpain activity may be regulated in living cells and has made it possible to propose specific structural changes that must occur to convert a catalytically inactive calpain molecule into an active proteolytic enzyme. On the other hand, although information is accumulating rapidly, the physiological function(s) of the calpain system remain poorly defined. This review summarizes the recent structural findings and some of the evolving concepts concerning regulation of calpain activity in cells and then describes some of the wide variety of physiological functions in which the calpain system has been implicated. The abundance of information on the calpain system makes it impossible to completely review all facets of this rapidly evolving area, and the review focuses on summarizing the structural information that has been firmly established and on those areas where the available information supports concrete concepts concerning regulation or function. Areas where controversy still exists, where results must be interpreted cautiously, and where new information is needed will be indicated.

The review begins with two sections describing properties of the well-characterized µ-calpain, m-calpain, and calpastatin molecules and the identification and some properties of the newest members of the calpain family, and follows with two sections on regulation and physiological function of the calpain system. The review ends with a brief overview of some of the potential pathological involvements of the calpain system. A chapter describing the events that led to purification of m-calpain in 1976 and summarizing some of the questions regarding the calpain system at that time was published in 1990 (135), and readers should consult this chapter for information on history of the calpain system.

	II. BACKGROUND INFORMATION: THE WELL-CHARACTERIZED PLAYERS µ-CALPAIN, m-CALPAIN, AND CALPASTATIN

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Nomenclature of the calpain system will be discussed in section III after description of the recently identified "novel" or "atypical" calpains. The terms µ-calpain and m-calpain were first used in 1989 (62) to refer to the micromolar Ca²⁺-requiring (µ-calpain) and millimolar Ca²⁺-requiring (m-calpain) proteases, respectively, and it has been recommended that these names be used to distinguish these two calpains (434); µ- and m-calpain are sometimes referred to as the "ubiquitous," "conventional," or "typical" calpains.

A. Properties of µ- and m-Calpain

The calpains that have been isolated in a protein form are Ca²⁺-dependent, cysteine proteases (Table 1). Calpastatin, the third well-characterized member of the calpain system, is a protein that specifically inhibits the proteolytic activity of µ- and m-calpain but of no other proteases tested thus far. Some general properties of the three well-characterized members of the calpain system are summarized in Table 1.

1. cDNA sequences of µ- and m-calpain

cDNAs encoding µ-calpain and m-calpain have been cloned and sequenced for human (433), monkey, mouse, rat (91), bovine, porcine (411), rabbit (109, 111; partial), and chicken (324) calpains, and a great deal is known about the properties of these two proteins (Fig. 1). The large subunit has a molecular mass near 80 kDa, with the µ-calpain 80-kDa subunit usually being slightly larger than the m-calpain 80-kDa subunit (81,889 Da compared with 79,900 Da for the human calpains for example; Refs. 8, 177). Also, molecular masses for the small subunit from a number of species have ranged from 28,100 (381) to 28,235 Da (109). The amino acid sequences of calpains from vertebrate species are highly conserved with over 90% homology among the mammalian calpains sequenced thus far (91, 411, 433), and there is no evidence of any alternative splicing during expression of the calpain genes. Hence, the large and small subunits for µ- and m-calpain have masses near 80 and 28 kDa, respectively, at least among the vertebrate species, and in their native form, the µ- and m-calpain molecules are heterodimers. Possible causes (for example, autolysis during purification) for the differences in molecular weights or subunit composition of µ- and m-calpain reported in some of the early studies have been discussed in the earlier review in this journal (76).

View larger version (21K): [in this window] [in a new window]   FIG. 1. Schematic diagram showing the domain structure of human µ- and m-calpain as predicted by their amino acid sequence and the domain structure of human m-calpain as determined from its crystallographic structure. The six EF-hand sequences in the 80-kDa subunit and the five EF-and sequences in the 28-kDa subunit are shown as vertical bars. The crystallographic structure of m-calpain shows that the sixth EF-hand predicted from the amino acid sequence at the domain II/III boundary does not have an EF-hand structure in the calpain molecule, and it is unlikely that it binds Ca2+, at least in m-calpain. Crystallographic analysis also indicates that the EF-hand at the COOH-terminal ends of domains IV and VI of the 28- and the 80-kDa subunits, respectively, is involved in association of the two subunits and not in Ca2+ binding.

The 28-kDa subunit is identical in the µ- and mcalpain molecules and is encoded by a single gene on chromosome 19 in humans (326). The NH₂-terminal region of this subunit, domain V (Fig. 1), is Gly rich; 40 of the first 64 residues of this domain are Gly, and there are stretches of 11 and 20 Gly residues in this region (all numbers in this section are for the human calpains unless otherwise noted). The first Gly is at residue 10 in the small subunit, and residues 10–20 are Gly. The stretch of 20 Gly residues begins at residue 37 and ends at residue 56. Domain V is often referred to as an hydrophobic domain, and it has been suggested that this domain binds phospholipid (180). Because Gly is near the center of the hydrophobicity scale (103), however, the amino acid composition of domain V is not characteristic of a highly hydrophobic domain, and recent studies have indicated that domain III of the 80-kDa subunit binds phospholipid (462) and has a role in interacting with cell membranes (127). Of the 101 residues in domain V, 40 are Gly, 5 are Pro, 30 are hydrophobic, and 26 are polar or charged. In addition to the long stretches of Gly residues, a region from residues 78–83 contains all five Pro residues (PEPPPP), and a region between residues 91–97 contains four Glu residues (EANESEE). Hence, the amino acid sequence of domain V suggests an unordered structure that could serve as a tether to other molecules or structures.

The COOH-terminal part of the 28-kDa subunit (residues 101–268 in Fig. 1), domain VI, is frequently termed a calmodulin-like domain, because initial analyses of the amino acid sequence of this domain suggested the presence of four EF hand Ca²⁺-binding sequences at residues 152–163, 182–193, 217–228, and 247–258 (325). The amino acid sequence of domain VI is only marginally homologous to that of calmodulin, however (23% identity and 30% similarity for the human molecules). Moreover, the X-ray crystallographic structures of this subunit (39, 247) have revealed the presence of a fifth Ca²⁺-binding site at residues 108–119, thereby identifying the calpains as members of the penta-EF-hand family of proteins (261, 484). Members of the penta-EF-hand family of proteins form dimers that involve the fifth EF-hand and associate with membranes (484), two properties that are also characteristic of µ- and m-calpain. Subsequently in this review, the five EF-hand sequences in the 28-kDa subunit will be designated EF-1', EF-2', EF-3', EF-4' and EF-5', although EF-5' and probably EF-4' do not bind Ca²⁺ in the calpain molecule.

Recently, cloning and expression studies have identified an intronless gene that is present in both human and mice and that encodes a 248-amino acid polypeptide having a predicted molecular mass of 27,659 Da and 63% amino acid sequence identity with small, 28-kDa subunit of the calpains (393). This 248-amino acid polypeptide differs from the classical 28-kDa small subunit in that it does not have the two stretches of 11 and 20 Gly residues in domain V that the 28-kDa subunit has, and it seems to bind only weakly to the large, 80-kDa subunit in in vitro assays (393). Coexpression of the 248-amino acid polypeptide with the 80-kDa subunit of m-calpain, however, produces a proteolytically active enzyme having 70% of the activity of an expressed m-calpain. As will be described subsequently in this review, disruption of the gene encoding the 28-kDa small subunit is embryonically lethal in mice, even though the 248-amino acid polypeptide is expressed in these animals. Hence, the 248-amino acid polypetide cannot substitute for the 28-kDa subunit in cells, and the function of this polypeptide is presently unclear.

The 80-kDa subunits of µ- and m-calpain are different gene products (genes on chromosomes 11 and 1, respectively, in the human; Ref. 326), but share 55–65% sequence homology within a given species (433). The 80-kDa subunits of µ- and m-calpain were originally divided into four domains on the basis of their amino acid sequence (433; Fig. 1), but the recent crystallographic structure of mcalpain (171, 430; discussed in sect. IIA3) has shown that the 80 kDa of this calpain has six "domains" (Fig. 1). Two of these domains contain only 18 (the NH₂-terminal domain; Fig. 1) or 17 (the linker domain; Fig. 1) amino acids, and hence are not typical of domains as the term is normally used. Because the crystallographic structure of µ-calpain is not yet known, it is still unclear whether µ-calpain also contains six domains similar to those in the crystallographic structure of the Ca²⁺-free m-calpain molecule. In view of these uncertainties, the NH₂-terminal 18 amino acids will be referred to as domain I and the 17 amino acids in the linker will be referred to as the linker domain with the realization that this nomenclature may change as additional information becomes available. This nomenclature will be limited to discussion of m-calpain and will be clearly designated as the "crystallographic domain" structure to distinguish it from the domains predicted from amino acid sequence. The domain structure based on amino acid sequence will be used in the following because it is available for both µ- and m-calpain.

1. DOMAIN I. The NH₂-terminal domain has no sequence homology with any polypeptide sequenced thus far; sequence homology of domain I among different species [human, chicken, rat, porcine, rabbit (partial)] is 72–86%.
   
2. DOMAIN II. A domain with a Cys residue at position 115 (µ-calpain) or 105 (m-calpain) that is the active site Cys and with a His residue at position 272 (µ-calpain) or 262 (m-calpain) and an Asn residue at position 296 (µ-calpain) or 286 (m-calpain); these residues form a catalytic triad characteristic of cysteine proteases such as papain or cathepsins B, L, or S. Domain II, however, shares little sequence homology with these other cysteine proteases, and it is likely that it evolved from a different ancestral gene. Note that the active site Cys is in domain IIa, whereas the His and Asn that constitute the remainder of the catalytic triad are in domain IIb in the crystallographic structure of m-calpain (Fig. 1). Recent X-ray crystallographic results show that domains IIa and IIb each bind one atom of Ca²⁺ in a peptide loop consisting of 8 (domain IIa) or 9 (domain IIb) amino acids (298). Sequence homology of domain II among different species is high, ranging from 85 to 93%.
   
3. DOMAIN III. This domain has no sequence homology with any polypeptide sequenced thus far. In addition to linking the Ca²⁺-binding domains of the calpain molecule to the catalytic domain (domain II), domain III may be involved in binding phospholipids (462) and in regulating calpain activity by its participation in critical electrostatic interactions (171, 430). Analysis of the amino acid sequences indicates that this domain also contains two potential EF-hand Ca²⁺-binding sequences, one at the domain II/III boundary (residues 329–341, for µ-calpain; residues 318–338 for m-calpain; in domain IIb of the crystallographic structure of m-calpain), and one at the domain III/IV boundary (µ-calpain residues 554–565; mcalpain residues 541–552; in domain IV of the crystallographic structure of m-calpain; shaded areas in Fig. 1). The sequence at the domain II/III boundary does not have an EF-hand conformation in the crystallographic structure of rat or human m-calpain, and this region does not seem to bind Ca²⁺ in m-calpain. The EF-hand sequence at the domain II/III boundary in the calpain isolated from Schistosoma mansoni, however, binds Ca²⁺ (7; see sect. IIIA1). Because the crystallographic structures of µ-calpain and the other calpains that have been identified in the past 10 years (see sect. III) are not yet available, it is still unclear whether this sequence binds Ca²⁺ in these other calpains. The EF-hand sequence at the domain II/III boundary will be referred to as EF-IIb in this review with the understanding that it may not bind Ca²⁺ in some calpains.^.
   
4. DOMAIN IV. Like the sequence of domain VI, the sequence of this domain is marginally homologous to calmodulin (24–44% identity and 51–54% similarity for µ-or m-calpain) and contains four sets of sequences that predict EF-hand Ca²⁺-binding sites (Fig. 1). In contrast to the domain assignment based on amino acid sequence, the crystallographic structure of m-calpain indicates that domain IV of human m-calpain (430) begins at amino acid residue 530 and thus includes the EF-hand at residues 541–552. Hence, domain IV of the 80-kDa subunit also has five EF-hand sequences, with the fifth, COOH-terminal EF-hand involved in dimerization of the 28- and 80-kDa subunits. Subsequently in this review, the six EF-hand sequences on the 80-kDa subunit will be referred to as EF-IIb, EF-1, EF-2, EF-3, EF-4, and EF-5 from the NH₂-terminal to the COOH-terminal end of the 80-kDa subunit. Sequence homology among the species ranges from 65 to 93% for this domain.
   

2. Genomic sequences of µ- and m-calpain

Annotation of the genomic sequences for the human, mouse, and other species whose genome has been/is being sequenced will soon make this information available for all of the calpain polypeptides, including the recently identified calpain-like molecules (see sect. III) from a number of species. Genomic DNA sequences for the 80-kDa subunit of mouse µ-calpain (28 exons, 21 kb; Ref. 14), the 80-kDa subunit of rat m-calpain (21 exons, >33 kb), human calpain 3a (24 exons, 40 kb; Ref. 366), human calpain 4 (11 exons, 11 kb; Ref. 296), mouse calpain 8 (23 exons, 50 kb; Ref. 157), human calpain 10 (15 exons, 31 kb; Ref. 170), and the mouse calpain 12 (21 exons, 13 kb; Ref. 88) have been determined. Studies of the promoter region of calpain genes have been done for only the chicken and human m-calpain 80-kDa subunits (112, 155, 156) and the human 28-kDa subunit (296). The human 28-kDa subunit gene is 11 kb and contains 11 exons. Exon 1 is a noncoding sequence, and translation starts at the 16th base in exon 2. Each of the EF-2', EF-3', EF-4', and EF-5' sequences are encoded by one exon, exons 7, 8, 9, and 10, respectively, but the EF-1' sequence is split between exons 4 and 5. The positions of the intron/exon junctions are identical in domain IV of the chicken m-calpain gene and domain VI of the human 28-kDa gene. The 5'-upstream region of the human 28-kDa gene lacks a TATA or CAAT box sequence and is GC rich, containing three G-C boxes (GGGCGG). Such 5'-upstream sequences are characteristic of "housekeeping" genes.

The gene for the chicken 80-kDa m-calpain subunit is 10 kb and contains 11 exons (112). The 5'-upstream sequence of both the chicken and the human 80-kDa m-calpain subunit (only the 5'-upstream region of the human gene was sequenced, so the size and number of introns for the human 80-kDa m-calpain subunit gene are unknown) also lack a TATA or CAAT box and are GC rich, indicating that these genes also probably belong to the "housekeeping" family of genes. CAT expression analysis identified four negative elements in the 2,500 nt upstream from the transcription start site of the human 80-kDa m-calpain gene. Removal of these elements results in a 13-fold increase in CAT expression. All four negative enhancer elements respond to the same or similar cellular trans-acting factor(s). The regions from nt -202 to -160 and from nt -130 to -80 contain two promoter elements. Both the human 80-kDa m-calpain gene and the human 28-kDa gene contain AP-1 and Sp1 binding sites upstream of the translation initiation site.

Incubation of HeLa cells with the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), results in upregulation of expression of the m-calpain 80-kDa gene but has little or no effect on expression of the genes for the µ-calpain 80-kDa, the 28-kDa subunit, or calpastatin (156). Assay of human 80-kDa m-calpain-CAT constructs identified a cis-acting element in the -202/-80 upstream region of the m-calpain gene. This region of the human 80-kDa m-calpain gene contains a TGAATCA sequence at -132, which is closely related to the collagenase TPA-responsive element. It has been suggested that upregulation of both protein kinase C (PKC) and m-calpain expression by TPA is related to a role of m-calpain in regulation of PKC activity in cells.

Limited sequence data (410) have not identified obvious consensus promoter elements in the 5'-untranslated (UTR) region of the human µ-calpain gene and has shown that the 5'-UTR region of the human µ-calpain gene is highly homologous with the 5'-UTR regions of the bovine (77% identity) and porcine µ-calpain genes.

3. Crystallographic and solution structures

Crystallographic structural information on the calpains has become available in the past 5 years. First, the crystallographic structures of expressed domain VI polypeptides were solved to 2.3 Å (39) and 1.9 Å resolution (247). This was followed shortly afterward by the crystallographic structure of expressed rat (171) or human (430) m-calpain at 2.6 and 2.3 Å, respectively.

The expressed domain VI polypeptides contained amino acids Met-87/Ser-270 from the rat 28-kDa subunit (39) or amino acids His-84/Ser-266 from the porcine 28-kDa subunit (247; both expressed in Escherichia coli). The structures obtained in the two studies were essentially identical; the domain VI polypeptide crystallized as a homodimer with each monomer having five EF-hands. EF-1' (residues 108–119) and EF-2' (residues 152–163) structures were paired and were in an "open" conformation, whereas the EF-3'/EF-4' (residues 182–193 and 217–228, respectively) pair was in a "closed" conformation.

The fifth EF-hand, EF-5', was involved in forming the interface between the two monomers in the homodimer. Earlier studies (181, 320) indicated that the COOH-terminal ends of domains IV and VI (Fig. 1) were involved in the noncovalent association of the 28- and 80-kDa subunits of the calpain molecule, and the crystallographic structure of the m-calpain molecule (171, 430) has now shown that the fifth EF-hands of domains IV and VI are responsible for this association (at least for m-calpain). Removal of either 22 or 25 residues from the expressed domain VI, i.e., the fifth EF-hand loop and the eighth -helix, prevents heterodimer formation (106, 294). The interactions between the two domain VI monomers in the crystal structure are principally hydrophobic and involve Ile-254, Val-256, Ile-258, and interaction of Trp-261, Leu-262, Leu-264, Met-266, and Tyr-267 on one polypeptide with Phe-243, Phe-240, Met-239, and Leu-236 on the second polypeptide. Interestingly, the same residues with only a few conservative substitutions are also present in corresponding positions of the COOH terminus of domain IV from the 80-kDa subunits of both µ- and m-calpain (Table 2). That these residues are highly conserved in the calpains that have been sequenced thus far (Table 2) indicates their importance in association of the 28- and 80-kDa subunits in both µ- and m-calpain.

The EF-1', EF-2', and EF-3' sites all contained a Ca²⁺ atom when crystallization was done at "low" Ca²⁺ (1 mM; Ref. 39), whereas the EF-4' site contained a Ca²⁺ atom only when crystallization was done at higher Ca²⁺ concentrations (20 mM; Ref. 247; or 200 mM; Ref. 39). Moreover, the Ca²⁺ atom in the EF-4' site was not located in the loop of the EF-hand but rather at the COOH-terminal end of the loop near the NH₂ terminus of the seventh -helix in this domain. Soaking crystals in ytterbium resulted in replacement of the Ca²⁺ atom in the EF-4' site, supporting the suggestion that this site does not bind Ca²⁺ with an affinity as high as the EF-1', EF-2', and EF-3' sites do (39). Ca²⁺-binding properties of the complete calpain molecules are discussed in section IVC2.

Comparison of structures obtained in the presence (Ca²⁺ in EF-1', EF-2', and EF-3') or in the absence of Ca²⁺ indicated that the Ca²⁺-induced structural changes in domain VI crystals are very small. The largest Ca²⁺-induced structural changes occur in the EF-1' region of the molecule (residues 98–115, Ref. 39). For reasons that presently are unclear, stability of the heterodimeric mcalpain molecule is reduced when the 28-kDa subunit of the expressed molecule has been truncated up to residue 115 (removing the EF-1' hand); possibly, removal of this region of the domain VI polypeptide alters folding of the remainder of the polypeptide (106).

Consequently, the crystallographic structures of domain VI suggest that binding of Ca²⁺ to the EF-1', EF-2', and EF-3' sites in this domain results in only a small structural change at the EF-1' region of this domain. If Ca²⁺ binding to the EF-1, EF-2, and EF-3 sites in domain IV results in a similar pattern of conformational changes, and if Ca²⁺ binding to domains IV and VI is the trigger for Ca²⁺-induced proteolytic activity of the calpains, then the small conformational changes in the NH₂-terminal region of domain IV would need to be transmitted through domain III to the catalytic domain II (Fig. 1). This use of a "lever" to amplify a small conformational change is reminiscent of the myosin head where small structural changes associated with binding of ATP are amplified through the "stalk" of the myosin head to produce a movement of 10 nm relative to the actin filament (497). It is still unknown whether such an amplification occurs upon Ca²⁺ binding to the calpains. Ca²⁺ binding to domains IV and VI also weakens the affinity of these two subunits for each other, causing dissociation of the 28- and 80-kDa subunits (see sect. IVC6). An inhibitor of the calpains that does not bind to the active site and that has an inhibition constant (K_i) of 0.3 µM (an -mercaptoacrylate derivative) was shown to bind to a hydrophobic pocket between helices B and D (2nd and 4th helices) in domain VI. Inhibitor binding caused minimal conformational changes in the crystal structure of this domain (247). It is possible that binding of the inhibitor "locks" domain VI (and domain IV) in a structure that prevents the small conformational changes in the NH₂-terminal region of this domain and thereby prevents transmission and amplification of this signal to the remainder of the molecule. Additional studies showing whether Ca²⁺ in the presence of this inhibitor can elicit the large conformational changes that have been observed in µ- and mcalpain in solution (later in this section) would confirm or refute this possibility.

The X-ray crystallographic structure of rat (171) or human (430) m-calpain that had been expressed in an E. coli (171) or a baculovirus (430) expression system has been solved to 2.6 or 2.3 Å, respectively (Fig. 2). The two structures, which are in a Ca²⁺-free state, are essentially identical. The 28-kDa subunit of rat m-calpain was truncated at amino acid 85 to facilitate high levels of expression in E. coli, but the first 85 amino acids of the small subunit were not represented by clear electron density in the X-ray diffraction pattern of the human m-calpain anyway. Hence, neither of the structures contains domain V. Attempts to obtain the crystallographic structure of µ-calpain have not yet been successful, although the similarity in amino acid sequence suggests that the two structures will be similar, and the structure of µ-calpain has been modeled on the basis of these similarities (365). m-Calpain is an elongated molecule with dimensions of 100 x 60 x 50 Å. Hydrodynamic measurements had earlier indicated that the calpain molecules were ellipsoidal (101), but the hydrodynamic estimates of 20 x 76 Å were smaller than those obtained from the X-ray structure. The crystal structure suggests that the 80 kDa of m-calpain has six "domains" (or, as discussed previously, four domains, aNH₂-terminal sequence and a linker) rather than the four domains predicted from amino acid sequence (Fig. 1). The crystallographic domain I (NH₂-terminal sequence) is short, 19 amino acids, and makes contact with domain VI but not with any other parts of the calpain molecule. Domain II, the catalytic domain, is divided in the crystallographic structure into two domains, domain IIa and domain IIb, that contain the Cys and the His/Asn residues, respectively, that constitute the catalytic triad of the Calpains. The crystallographic domain III is shorter than the domain III predicted from the amino acid sequence and ends with a stretch of 18 amino acids that form a "linker" with domain IV. The structure of domain IV is very similar to the structures of domain VI published previously (39, 247); indeed, the structure of domain IV in the human m-calpain resembles the Ca²⁺-bound structure of domain VI more closely than the Ca²⁺-free domain VI (430).

View larger version (54K): [in this window] [in a new window]   FIG. 2. Crystallographic structure of human m-calpain. The domains, in different colors, are labeled dI, dIIa, dIIb, dIII, dIV, dV, and dVI. I-II is the -helix linking domains I and IIa. The linker "domain" is a red line running from the gap between dIII and dIV to the bottom right of the diagram and is labeled III-IV. Locations of EF-1, EF-2, and EF-3 hands in domains IV and VI are indicated. The active site Cys, C105, is in gray as are His-262, Asn-286, and Trp-288 at the top of domain IIb. [Adapted from Reverter et al. (364).]

The crystallographic structure of m-calpain leads to several important conclusions (these conclusions strictly pertain only to the Ca²⁺-free form of m-calpain).

1. The active site, which is at the top of the structure in Figure 2, is not sterically blocked by domain I (the NH₂-terminal, 18-amino acid domain) as has been widely believed. Hence, removal of domain I by autolysis (see sect. IIA4) does not activate a proenzyme by removal of a peptide that blocks access to the active site as autolytic activation of other proenzymes does.
   
2. The structure of the Ca²⁺-free enzyme shows that the enzyme is catalytically inactive because the active site Cys is 10.5 Å away from the His and Asn residues, a distance too great to allow formation of a catalytically functional complex. Hence, Ca²⁺ must induce a conformational change in m-calpain that reduces this distance from 10.5 to 3.7 Å, so that Cys-105 can interact effectively with His-262 and Asn-286 to form a catalytic triad. This conformational change likely involves a rotation of Trp-288 in domain IIb (Fig. 2). Earlier studies indicated that mutation of Trp-288 to a Tyr residue reduced proteolytic activity of the mutated calpain to 5.5% of its activity before mutation (11).
   
3. The autolytic cleavage of domain I at amino acids 19–20 occurs at a point that is 40 Å away from the catalytic site in the Ca²⁺-free enzyme, so this autolysis likely is not an intramolecular event.
   
4. The crystallographic structure of the m-calpain molecule could not be done in the presence of Ca²⁺, but the very small conformational change resulting from binding of Ca²⁺ to domain VI suggests that the "Ca²⁺ switch" for initiation of proteolytic activity may involve Ca²⁺ binding at some site in addition to or other than domains IV and VI, the calmodulin-like domains. A group of acidic residues, Glu-392, Glu-393, Glu-394, Asp-395, Glu-396, Asp-397, Glu-398, Glu-399, and Glu-401, are highly conserved in the calpains, and some of these acidic residues form salt bridges with Lys-226, Lys-230, Lys-234 and Lys-354, Lys-355, Lys-357 in the crystallographic structure of m-calpain (Fig. 2). Ca²⁺ would disrupt these salt bridges, and this disruption could permit domain IIb to move toward domain IIa, leading to formation of the catalytic triad (430). Although this electrostatic mechanism "switch" may have an important role in the Ca²⁺-induced catalytic activity of the calpains (171), recent studies (298) have shown that expressed domain IIa/IIb from µ-calpain can each bind one atom of Ca²⁺ in a peptide loop and that this binding induces a conformational change that brings the catalytic residues in the expressed domain IIa/IIb within 3.7 Å of each other. Hence, at least part of the Ca²⁺ switch for activating the calpains may reside in the catalytic domain itself (see sect. IVC6).
   

In the absence of structural information on the µ- and m-calpain molecules in the presence of Ca²⁺, limited proteolytic hydrolysis with trypsin or chymotrypsin has been used to compare the structures of the complete heterodimeric calpain molecules in the presence or the absence of Ca²⁺ in solution (297, 459). Moldoveanu et al. (297) used an expressed m-calpain that had the active site Cys mutated to Ser to prevent autolysis in the presence of Ca²⁺, whereas Thompson et al. (459) used purified preparations of both µ- and m-calpain and sodium tetrathionate oxidation (199) to reversibly inactivate calpains and prevent their autolysis in the presence of Ca²⁺. The two studies produced similar results.

1. Either trypsin or chymotrypsin digestion in the absence of Ca²⁺ for periods as long as 120 min produces a limited number of polypeptide fragments, suggesting that both the µ-calpain and m-calpain molecules have a compact structure that limits the number of peptide bonds accessible to proteolytic enzymes in the absence of Ca²⁺. Moreover, similar proteolytic fragments were produced by these two proteases, which differ in their subsite specificity. Both trypsin and chymotrypsin rapidly cleave either µ- or m-calpain at a region near the COOH-terminal end of domain II or IIb in the crystallographic structure (residues 245 or 266 in µ-calpain; residue 265 in m-calpain) and at several sites in domain III (residues 363 or 472 in µ-calpain; residues 383, 400, and 503 in mcalpain). Domain I is resistant to trypsin digestion for up to 120 min in the absence of Ca²⁺, although chymotrypsin removes small segments (6 amino acids from µ-calpain; 9 amino acids from m-calpain) from the NH₂ terminus of domain I; the remainder of domain I and domain II is resistant to tryptic or chymotryptic cleavage for 120 min, suggesting that this part of the molecule (domain IIa in the crystallographic structure) is in a compact conformation in the absence of Ca²⁺.
   
2. The COOH-terminal regions of domain III and all of domain IV in either µ- or m-calpain also are resistant to proteolytic degradation, suggesting that these regions of the calpain molecule are also in a compact conformation.
   
3. Both tryptic or chymotryptic treatment cleaved the 28-kDa subunit of either µ- or m-calpain at residues 59–61 and 85–88 within 10–20 min, leaving a 24-kDa or a 20-kDa fragment that was not degraded for 120 min.
   
4. The pattern of proteolytic digestion of µ- and m-calpain by trypsin or chymotrypsin changes significantly in the presence of 1 mM Ca²⁺, a cation that does not directly affect trypsin or chymotrypsin activity. Both µ- and m-calpain that had been proteolytically inactivated were rapidly degraded within 5 min of incubation by either trypsin or chymotrypsin to small polypeptides. Only domains IV and VI remained after 30 min of digestion in the presence of Ca²⁺. Indeed, the proteolytic fragments of the 28-kDa subunit are identical whether proteolytic digestion is done in the presence or the absence of Ca²⁺. Chymotryptic digestion of either µ- or m-calpain in the presence of Ca²⁺ results in only one major degradation product from the 80-kDa subunit; the NH₂ terminus of this fragment begins at residue 515 (µ-calpain) or 503 (mcalpain). Therefore, Ca²⁺ at the concentration of 1 mM causes a substantial change in the conformation of the calpain molecules and "opens" the molecule to make it more susceptible to proteolytic degradation. Only the conformations of domains IV and VI do not seem to be significantly altered by Ca²⁺, a conclusion that was also reached in the crystallographic studies of domain VI (39, 247).
   

In sum, the currently available evidence indicates that Ca²⁺ binds to multiple sites on both the µ- and m-calpain molecules and that not all of these sites are in domains IV and VI, the penta-EF hand ("calmodulin-like") domains. Because Ca²⁺ binding to domain VI (crystallographic evidence) and to domain IV (evidence from limited proteolysis) causes only very small conformational changes in these domains, it seems likely that Ca²⁺ binding to regions other than or in addition to these two domains is involved in the "switch" that initiates proteolytic activity in the calpains. It is clear that Ca²⁺ binding to either µ- or m-calpain causes significant conformational changes in the calpain molecules and that these changes involve a "loosening" or partial unfolding of domain II (IIa, IIb) and the NH₂-terminal part of domain III, making these domains that were previously resistant to proteolytic cleavage now highly susceptible. The exact nature of these changes is unknown, but they must include a moving together of critical residues in domains IIa and IIb to form a functional catalytic triad. Although the structural information that has become available during the last four to five years has changed the prevailing views as to how Ca²⁺ initiates proteolytic activity of the Calpains, the regions in addition to those in domains IV and VI in the calpain molecule that bind Ca²⁺ and how this binding affects conformation of the calpains in a way that results in proteolytic activity remain unclear. Finally, it should be remembered that these Ca²⁺ binding events must occur at physiological Ca²⁺ concentrations, which are in the range of 50–300 nM (151, 192, 266). The Ca²⁺ binding properties of the calpains are discussed in section IVC2.

4. Autolysis and the proenzyme question

It was discovered in 1981 that m-calpain autolyzes rapidly in the presence of Ca²⁺ (439, 440), and it is now known that both µ- and m-calpain autolyze when incubated with Ca²⁺ (62, 81, 101, 373, 376). Although it is not uncommon for proteolytic enzymes to autolyze, autolysis of the calpains has several unique features (autoproteolysis may be grammatically more accurate than autolysis, but the briefer autolysis will be used here with the understanding that the autolytic events are proteolytic). Brief autolysis, up to 2–3 min at 25°C and in the presence of 1 mM or greater Ca²⁺, reduces the Ca²⁺ concentration required for half-maximal proteolytic activity of µ-calpain from 3–50 to 0.5–2.0 µM and that of m-calpain from 400–800 to 50–150 µM (137; Table 3) without affecting the specific activity of either enzyme (101). This same autolysis reduces mass of the 80-kDa subunit of µ-calpain to 76 kDa, mass of the 80-kDa subunit of m-calpain to 78 kDa, and mass of the 28-kDa subunit common to µ- and m-calpain to 18 kDa (SDS-PAGE; actual mass of this fragment is 20.5 kDa; Ref. 142). Amino acid sequencing studies show that the NH₂-terminal 27 or 18 (µ- or mcalpain) amino acids are removed from the 80-kDa subunit and that the NH₂-terminal 91 amino acids (hence, the entire Gly-rich domain) are removed from the 28-kDa subunit during this autolysis (433; residue numbers are for the human calpains; the autolysis sites are similar but not identical in calpains from other species).

Although autolysis occurs rapidly, it involves several sequential steps: 1) for the 80-kDa subunit of µ-calpain, the NH₂-terminal 14 amino acids are removed first to produce a 78-kDa intermediate product followed by removal of an additional 12 amino acids to produce the 76-kDa autolytic fragment (504; molecular weights as estimated with SDS-PAGE); 2) for the 80-kDa subunit of m-calpain, the NH₂-terminal 9 amino acids are removed first followed by removal of an additional 10 amino acids to produce the 78-kDa autolytic fragment (45); and 3) for the 28-kDa subunit, the NH₂-terminal 26 amino acids are removed to produce a 26- to 27-kDa fragment, an additional 37 amino acids (to Gly-64) are then removed to produce a 22- to 23-kDa fragment, and finally 28 more amino acids (to Ala-92) are removed to produce the 18-kDa (20.5 kDa) autolytic fragment (272). The 28-kDa subunit of m-calpain is autolyzed more rapidly than the 80-kDa subunit (45, 73), whereas autolysis of the 80-kDa subunit in the µ-calpain molecule seems to proceed as rapidly as or even more rapidly than autolysis of the 28-kDa subunit in this molecule (45, 68, 504).

The small change in mass during autolysis of the 80-kDa subunit of m-calpain is difficult to detect in normal SDS-PAGE analysis of the calpains, and claims that this subunit has or has not undergone autolysis need to be substantiated either by direct sequencing or by use of an antibody that specifically recognizes the NH₂ terminus of the autolyzed molecule (78, 375). It seems likely that claims that the 80-kDa subunit of m-calpain had not undergone autolysis in some early publications were incorrect because of the difficulty in resolving the 78-kDa autolytic fragment from the 80-kDa native subunit in SDS-PAGE.

Although the biochemical changes that accompany autolysis have been well characterized, the physiological significance of autolysis remains controversial. The Ca²⁺ concentrations required for autolysis are similar to or just slightly greater than those required for proteolytic activity (62, 218, 504; Table 3) and therefore are higher than would be encountered in living cells. Because these two Ca²⁺ requirements are so similar, in vitro assays of proteolytic activity are accompanied by autolysis unless the Ca²⁺ concentrations are chosen carefully to be just below those required to initiate autolysis. Proteolytic activity is less than half-maximal (usually much less) at these concentrations, and they are rarely used in in vitro assays. Many members of the cysteine protease family are proenzymes that are activated by autolytic removal of a proenzyme polypeptide that blocks access to the active site of the enzyme. That assays of proteolytic activity were always accompanied by autolysis raised the possibility that the unautolyzed calpains also were proenzymes that required autolysis for activation (274, 435).

The concept that the calpains were proenzymes was widely accepted in the 1990s, and papers have described "activation" of the calpains as being synonymous with autolysis. Although a number of studies reported that both unautolyzed µ-calpain (62, 139, 299) and unautolyzed m-calpain (63, 73, 139) were active proteases, other kinetic studies (18, 68, 161, 218) found that autolysis preceded proteolytic activity of the calpains. Mutation of the residue that is cleaved during autolysis of m-calpain prevented autolysis, but the resulting unautolyzed m-Calpain was an active protease (107). Furthermore, SDS-PAGE has been used to show that a casein substrate is cleaved to small fragments by either unautolyzed µ-Calpain or unautolyzed m-calpain (69). Oxidation of µ-Calpain with 100 µM hydrogen peroxide or sodium hypochlorite does not prevent autolysis but completely inhibits proteolytic activity, suggesting that the two are separate events (147). Finally, the crystallographic structure of m-calpain (171, 430) shows that autolysis of m-calpain removes an -helical NH₂ terminus of the 80-kDa subunit and that this NH₂ terminus does not block the active site in the inactive enzyme. The NH₂-terminal part of the 28-kDa subunit is not visible in the X-ray pattern, but it seems likely that if it were fixed in a position blocking the active site of the enzyme, it would have diffracted sufficiently to have been detected. Consequently, autolysis of m-calpain and, to the extent that structures of the two molecules are similar, of µ-calpain, does not unblock an active site as autolysis of other cysteine proteases does.

Therefore, it presently seems likely that the unautolyzed calpains are capable of proteolytic activity and are not proenzymes that require autolysis for their activation. Whether the unautolyzed calpains are enzymatically active or not may be a moot question, however, because autolysis seems to occur consistently under conditions where the calpains are proteolytically active (139). Concomitant autolysis and proteolytic activity are especially well documented in activation of platelets (120, 217, 374, 378), where platelet activation is accompanied by cleavage of spectrin (118), talin, and filamin (117) in a calpainspecific manner and by autolysis of the µ-calpain in platelets. Hence, autolysis can be used as an indicator that the calpains have been proteolytically active in a cell, but the absence of autolysis does not guarantee that the calpains have not been active.

It seems, therefore, that autolysis has some important role in calpain function. The nature of this role, however, remains a mystery. It has been reported that the peptides released during autolysis of the large subunit of µ-calpain (225) or autolysis of the 28-kDa small subunit (226, 227) have chemotactic activities on neutrophils (225, 226) or immunocytes (227) and that peptides released by autolysis of the small subunit have spasmogenic activity on sections of guinea pig ileum (257). There are numerous instances in biology where peptides released during limited proteolysis have important physiological functions related to the initial proteolysis, such as the vasoconstrictive effects of the peptides released during cleavage of fibrinogen to produce fibrin during blood clotting. It is possible that the peptides released during autolysis of the calpains may also have important as yet undiscovered properties. Recent studies (discussed in sect. IVC6) indicate that autolysis affects stability of the calpains and may initiate dissociation of the two subunits and inactivation. The possible role(s) of autolysis in the physiological function of the calpains will remain an active area of research in the coming years.

B. Properties of Calpastatin

It was discovered during the initial studies on purification of m-calpain (83) that muscle extracts having Calpain activity also contained an inhibitor of this activity (see Ref. 135). Initial studies established that this inhibitor was a heat-stable protein (to 100°C; Ref. 332), and it has subsequently been shown that it is resistant to a wide variety of denaturing agents such as urea, SDS, or trichloroacetic acid (126, 337). The name, calpastatin, was proposed for this inhibitor in 1979 by Takashi Murachi (309). The early attempts to purify this inhibitor produced inconsistent and variable results, and the inhibitor was described as a protein having molecular masses varying from 34 to 280–300 kDa (see Refs. 135, 259 for summaries). In retrospect, a number of factors likely contributed to these inconsistencies.

First, calpastatin is labile to proteolytic degradation (277, 283, 337), and it is likely that the harsh conditions used in some of the early attempts to purify calpastatin, many of which involved heating crude extracts that likely contained a number of endogenous proteolytic enzymes, resulted in varying degrees of proteolytic degradation even though protease inhibitors were used.

Second, it is now known that the calpastatin polypeptide is nearly completely in a random coil conformation as determined by circular dichroism (212, 403) or nuclear magnetic resonance (212, 466). Because estimates of molecular weight using size exclusion chromatography are invariably based on spherical molecules as standards, size exclusion chromatography will greatly overestimate the molecular weight of a molecule having a coil conformation. The studies that reported very large molecular weights of over 200 kDa for calpastatin all used size exclusion chromatography, and it seems likely that they substantially overestimated the molecular weight of calpastatin because of this. Some of these studies also indicated that calpastatin was a dimer or tetramer in nondenaturing solutions because SDS-PAGE of their calpastatin preparations contained polypeptides that were much smaller than those estimated with size-exclusion chromatography. The available evidence indicates that calpastatin is a monomer in nondenaturing solvents (337) and functions as a monomer in cells, although definitive sedimentation equilibrium studies have not yet been done.

Third, although only a single calpastatin gene exists in humans (chromosome 5; Ref. 183), pigs (chromosome 2; Ref. 114), and bovine (chromosome 7; Ref. 38), studies during the past 8–9 years have shown that, because of the use of different promoters (65, 178, 448, 471) or alternative splicing mechanisms (238, 239, 344, 443, 448), a large number of different calpastatin isoforms ranging in molecular mass from 17.5 kDa (471) to 46.35 kDa (178) to 84 kDa (65) are produced from this single gene. At least eight different calpastatin isoforms (Table 4) have been identified in tissues (126, 138, 344, 448). Frequently, more than one isoform exists in a single tissue (288), and different isoforms exist in erythrocyes from different species (185).

Fourth, calpastatin migrates anomalously in SDS-PAGE (259, 445), and it is difficult to relate a band migrating at a particular molecular weight in SDS-PAGE to a known calpastatin isoform. For example, the 46.35-kDa calpastatin in human erythrocytes, which was the first calpastatin to be purified (446), migrates at 70 kDa in SDS-PAGE. The expressed fragments of the calpastatin molecule each migrate more slowly in SDS-PAGE than would be predicted from their respective sizes (110, 445). Hence, the anomalously slow migration of calpastatin in SDS-PAGE is a property of the calpastatin polypeptide itself and is not due to posttranslational modifications. Consequently, although the multiple bands frequently observed in SDS-PAGE of calpastatin polypeptides are often ascribed to proteolytic degradation, it is not always clear without actually sequencing them whether the bands are proteolytic products, whether they are different calpastatin isoforms resulting from some combination of alternative splicing or promoter usage, or whether they are produced by some combination of proteolysis and different isoforms.

Calpastatin is the only known protein inhibitor specific for the calpains, although both L- and H-kininogen (only the central cystatin domain of the kininogen heavy chain) and 2-macroglobulin inhibit the calpains in addition to the other proteases they inhibit (72). The central domain of kininogen has a K_i of 1.0 nM for m-calpain, and the rate constant for inhibition of the calpains by 2-macroglobin is 3 x 10^-4 M^-1·s^-1, which is intermediate in the range of values that have been reported for the inhibition of different proteases by 2-macroglobulin (72). Calpastatin does not inhibit any other protease with which it has been tested including the cysteine proteases, papain, cathepsin B, bromelin, or ficin in addition to proteases from other classes such as trypsin, chymotrypsin, plasmin, thrombin, pepsin, cathepsin D, or thermolysin (72). Neither calpastatin nor calpastatin-like activities have been reported in invertebrate tissues, and genes having sequence homologies to calpastatin have not been detected in Drosophila, Caenorhabditis elegans, or Saccharomyces cerevisiae. Hence, calpastatin may be restricted to vertebrates.

1. DNA sequencing

cDNAs encoding the calpastatins for human (12), monkey, mouse (443), rat (188), pig (445), bovine (65, 206), and rabbit (108) have been cloned and sequenced (259). Although sequence homology among the calpastatins is not as high as it is among the calpains (188), there is >65% amino acid sequence identity among the calpastatins sequenced thus far. The calpastatin amino acid sequence is unique and is not homologous to any polypeptide that has been sequenced thus far, including the cystatins which inhibit many of the cysteine proteases but not the calpains (72). The initial studies on calpastatin cDNAs from rabbit lung or liver (108) or pig heart (445) indicated that the calpastatin polypeptide consisted of four repeating, marginally homologous (23 to 36%) domains of 140 amino acids each, plus a NH₂-terminal domain, called domain L (Fig. 3), that varies in size due to alternative splicing (238, 239). The predicted mass for these calpastatins ranged from 68 to 78 kDa, depending on the size of domain L. As indicated earlier, however, the calpastatins migrate anomalously in SDS-PAGE, and relative molecular masses ranging from 107 kDa (179), to 125 kDa (93, 403), to 145 kDa (277), and to 172 kDa (241) were reported for these different calpastatins. Protein sequencing showed that the NH₂ terminus of the 46.35-kDa human erythrocyte calpastatin was Ser-Asp-Gln-Ala-Leu-Glu-Ala-Ser-Ala-Ser-Leu (178), which corresponds to the sequence of the human hepatic calpastatin beginning at Ser-287 (Fig. 3). This Ser is the first residue of domain II in human hepatic calpastatin and is located immediately after a Met residue that is encoded in a acaATGa sequence, a good Kozak consensus sequence for translation initiation. Hence, human erythrocyte calpastatin is an example of a calpastatin isoform produced from an alternative transcription/translation start site.

View larger version (22K): [in this window] [in a new window]   FIG. 3. Schematic diagram showing the domain structure of three different calpastatin isoforms as determined from their deduced amino acid sequences. The species and tissues named under each calpastatin is the species and tissue from which the calpastatin was cloned and sequenced. The calpastatin isoforms containing a L and/or a XL domain are found in a number of tissues from a number of different species and are not unique to the species or tissue named. A, B, and C are three subdomains within each domain: the B subdomain from a number of species contains the conserved sequence indicated. *** Three sites on domain XL that are phosphorylated by protein kinase A. See text for additional details.

Sequencing the mouse calpastatin gene has shown that this gene contains 34 exons in 60 kb of sequence (448), including 5 exons upstream from exon 2, the exon encoding the NH₂ terminus of the "prototypical" calpastatin (human hepatic calpastatin in Fig. 3). The 5 upstream exons were named 1xa, 1xb, 1y, 1z, and 1u (Fig. 4). Earlier studies (65) had identified a 5'-region of the bovine calpastatin gene containing exons 1xb, 1y, and 1z. These three exons encoded a 68-amino acid domain termed domain XL (Figs. 3 and 4) that contained three protein kinase A (PKA) phosphorylation sites. Incubation of an expressed calpastatin containing domain XL with purified PKA showed that these sites were phosphorylated in vitro. Expressed calpastatin containing the XL domain migrated as a polypeptide of 145 kDa (molecular mass of the predicted amino acid sequence is 84 kDa), and antibodies specific for the XL domain showed that a 145-kDa calpastatin containing the XL domain is expressed in bovine liver and cardiac muscle extracts (65). These extracts also contained polypeptides of 120 and 110 kDa that were labeled by a monoclonal antibody specific for domain IV of calpastatin (Fig. 3; Ref. 65). Hence, some bovine tissues contain at least three different calpastatins. The upstream AUG initiation site for the XL domain is in a better Kozak consensus context than the second, downstream AUG (Met-1 in the human hepatic calpastatin in Fig. 3) and was the preferred initiation site in transcription/translation assays (65).

View larger version (19K): [in this window] [in a new window]   FIG. 4. Genomic structure of the mouse calpastatin gene and the relation of the 34 exons (29 numbered plus 1xa, 1xb, 1y, 1z, and 1u) in this gene to the predicted domains in the mouse calpastatin protein. The mouse calpastatin gene is 60 kb, and only the exons are shown in this diagram. The 1u exon is expressed only in the type III transcript, but translation of this transcript begins in exon 2.

cDNAs encoding four different NH₂-terminal sequences have been identified in the mouse (448); these four calpastatin cDNAs were named type I, type II, type III, and type IV (Fig. 4).

1. TYPE I. Type I calpastatin started at exon 1xa; type I transcripts skip exon 1xb, continue with exons 1y and 1z, and skip exon 1u. Type I transcripts were identified in mouse brain, liver, and testis.
   
2. TYPE II. Type II calpastatin started at exon 1xb and continued with exons 1y and 1z before skipping exon 1u and proceeding to exon 2. Type II transcripts therefore encoded calpastatin containing the XL domain (Fig. 3). Type II calpastatin was detected in skeletal and cardiac muscle and in much lower levels in other mouse tissues.
   
3. TYPE III. Type III calpastatin mRNA started at exon 1u and encoded the prototypical calpastatin. Translation of type III transcripts starts in exon 2, however. Type III calpastatin was expressed ubiquitously in the mouse tissues tested.
   
4. TYPE IV. Type IV calpastatin expression was limited to testis. Initiation of transcription of type IV calpastatin mRNA began at a unique exon between exons 14 and 15 that was named exon 14_t, and that was not expressed in type I, II, or III calpastatin mRNAs. Expression of type IV calpastatin therefore was initiated in domain II just after subdomain IIA and produced a polypeptide that migrated at 68 kDa in SDS-PAGE.
   

The four mouse calpastatins are probably not unique to the mouse; the human EST database includes sequences for partial cDNAs representing types I, II, III, and IV, and calpastatin mRNA transcripts corresponding to types I, II, and III calpastatins have been identified in porcine cardiac tissue (344). Moreover, further examination of the bovine calpastatin gene (65) shows that this gene also includes a region corresponding to exon 1xa. Although type II calpastatin from the mouse and human contain the PKA phosphorylation sites described by Cong et al. (65) in the XL domain, the porcine type II calpastatin lacks these sites (344). Only type III calpastatin was detected in skeletal muscle from the pig (344).

It is unclear how many different start sites of transcription/translation may be used in expression of the calpastatin gene in different tissues or under different physiological states. In addition to the four ATGs at the 5'-ends of types I, II, III, and IV calpastatin, there is an ATG that encodes Met-189 (numbering based on Met-1 at the NH₂ terminus of type I calpastatin), which is located at the L domain/domain I junction (Figs. 3 and 4), and another that encodes Met-322, which is at the NH₂ terminus of the 46.35-kDa erythrocyte calpastatin that begins at the domain II/III boundary of calpastatin (Fig. 3). A 17.5-kDa glycoprotein with a predicted amino acid sequence identical to the COOH-terminal 186 amino acids (beginning at Gln-559 sequence number based on type I calpastatin; domain III/IV in Fig. 3) of human hepatic calpastatin has been identified in human sperm (471). This glycoprotein was not isolated, so its NH₂-terminal amino acid is unknown. Human hepatic calpastatin, however, contains a Met residue at position 542, which could be the NH₂ terminus for this "sperm calpastatin."

The 5'-promoter region of the calpastatin gene in a number of species is GC rich and lacks a TATA box (65, 344), properties that were also observed for the 5'-promoter region of the m-calpain gene and that are characteristic of "housekeeping" genes. Both the bovine (65) and the porcine (344) calpastatin promoter contain multiple Sp-1 binding sites and putative CRE motifs. Earlier partial sequencing of the human calpastatin gene (239, 260) and the recent, more extensive sequencing of the mouse calpastatin gene (448) now makes it possible to assign exons 2–8 to domain L and exons 9–13 to domain I (Fig. 4). The exon/intron boundaries of all four of the calpastatin inhibitory domains are highly conserved (448). With the exception of domain IV, that has an additional sixth exon at its COOH-terminal end, all inhibitory domains contain five exons. Because each domain is capable of inhibiting one calpain molecule (see sect. IIB2) and because removal of either subdomain A or C within a domain does not completely ablate the inhibitory ability of that domain (see sect. IIB2), the use of multiple domains and multiple exons within each domain may be a protective mechanism used to ensure that mutation within one or more exons does not completely destroy the ability of calpastatin to inhibit the calpains (448).

Sequencing of calpastatin transcripts has shown that exon 3, and less frequently, exons 4, 5, and 6, are often deleted from calpastatin mRNAs by alternative splicing. Sequences of rat calpastatin obtained in early studies of this calpastatin all lacked exon 3 (94, 238), and it was assumed initially that rodent calpastatins were unique in that they lacked this exon. Several forms of mouse type I, II, and III calpastatins, some of them containing exon 3 and some of them having exon 3 deleted (443) have been identified, however; hence, some rodent calpastatins also contain exon 3.

The use of at least four alternative promoters and the ability to alter domain L by alternative splicing mechanisms make it possible to produce a number of different calpastatin polypeptides from a single calpastatin gene. The physiological significance of these different calpastatins remains a mystery. Deletion of exon 3 would remove a stretch of basic amino acids, Lys-Lys-Arg-His-Lys-Lys (residues 106–111; residue numbers based on Met from exon 1a as number 1), from the L domain of calpastatin. This region of calpastatin has been reported to be involved in binding calpastatin to biological membranes (275, 279, 281). Less than 2% of total cellular calpastatin, however, is estimated to be bound to the sarcolemma in cardiac muscle (279). Loss of the XL dom