Biochimica et Biophysica Acta, 1160 (1992) 55-62
55
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34354
Sequence comparison among muscle-specific calpain, p94, and calpain subunits Hiroyuki Sorimachi and Koichi Suzuki Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo (Japan)
Key words: Calpain; p94; Calcium; Cysteine proteinase; Evolution While conventional calpains, m- and /z-calpains named according to their calcium-dependence, are expressed in almost every tissues, mRNA of newly identified p94, which has a significant sequence similarity to the conventional calpain large subunits, is abundantly expressed only in skeletal muscle. In addition to this specific expression, p94 is distinct from conventional calpains in that it contains three unique regions showing no similarity to conventional calpain subunits. When rat and human p94 are compared, overall sequence similarity is 94.0%, which is close to those for m- and /~-calpain large subunits; 93.1% and 95.4% between human and rabbit, respectively, suggesting the evolutionary importance of p94. These calpain large subunit proteins, p94, m- and/.~-types, can be considered to constitute a super family, whose p94, m- and/~-types represent the three major types. Sequences of the calpain large-subunit family members, including the recently reported Schistosoma calpain, are compared. Their evolutionary correlation and function are discussed on the basis of the results thus far obtained.
Introduction Calpain, calcium-dependent proteinase or C A N P is a ubiquitously expressed intracellular cysteine proteinase and requires calcium ions for activity [1-7]. Two isozymes, m- and ~-calpain, have been identified and studied extensively. Both are heterodimers and consist of a distinct but similar large subunit (about 80 kDa) and an identical small subunit (about 30 kDa) [2-14].The two distinct, m- and /~-calpain large subunits can be divided into four domains as indicated in Fig. 1 [8]. Since domain II has a similarity to other cysteine proteinases such as papain and cathepsins B, H and L, especially around their active-site cysteine histidineand asparagine residues, this domain is considered as a cysteine proteinase module. Domain IV is similar to calmodulin and contains 4 EF-hand structures, indicating that this domain is a calcium-binding module possibly responsible for calcium-dependent regulation of calpain. Domains I and III have no similarity to any other sequences in protein database but the N-terminal region of domain I is autocatalytically processed during the activation, suggesting that domain I is involved in
Correspondence to: H. Sorimachi, Institute of Applied Microbiology, University of Tokyo, Yaoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan.
the regulation of activity. The function of domain III still remains to be solved. The small subunit can also be divided into two domains according to the similarity to the large subunit (Fig. 1). The C-terminal half has a significant similarity to the domain IV of the large subunit and, thus, is n a m e d domain I V ' , which contains four EF-hand structures as the large subunits. The N-terminal half, n a m e d domain V, consists of contiguous glycine residues together with hydrophobic residues which make itself hydrophobic and is suggested to interact with phospholipids [15].
p~ m-& La~ Sub
Fig. 1. Schematic protein structure of calpain subunits. Numbers above the p94 structure indicate the amino-acid residue number of rat p94 [19]. 'Cys' and 'His' in p94, m- and/z-large subunit structures represent active-site cysteine and histidine residues, respectively. 'K-rich' indicates lysine-rich region as described in this paper and NS, IS1 and IS2 indicate three unique sequences in p94. I, II, III, IV, IV' and V represent the five domains of calpain large and small subunits.
V
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m m /~ /z
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8
2 3
4 5 6 7 No. of IEmanm
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Famma
No. of Rat p94 Rat p94 1
Fig. 2. Amino-acid sequence of p94 and calpain large subunits. Lines: 1, rat p94; 2, h u m a n p94; 3, h u m a n m-calpain large subunit; 4, rabbit m-calpain large subunit; 5, chicken m-calpain large subunit; 6, h u m a n #-calpain large subunit; 7, rabbit #-calpain large subunit; 8, Schistosoma mansoni calpain. Borders of each domain are indicated by spacings and arrows. Three unique sequences of I)94, i.e., NS, IS1 and IS2, are indicated by arrows. ( ...... ), deletion at the positions; ( z~ ), positions where introns are inserted into genomic D N A of chicken m-calpain large subunit [25]. Numbers on the top and bottom of the sequences are residue n u m b e r s of rat p94 and h u m a n m-calpain large subunit, respectively. Reversed letters stand for residues that are conserved in all subunit sequences. Underlined regions (1-4) are EF-hand structures. C, H and N residues, indicated by asterisks, are active sites of calpain.
600
..
(2,PDDVIEALI{LI~)TLLDIKX~EIEQ~F L A I R D P K I ~ A I I ~ V I ~ G ~ - - ~ T L O g l P N ' F - - ~ 550 1
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50 NS
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150
No. ofHuaan m
m&osont
No. of Rat p94 Rat p94 Hu.e~'x p94 Humaa m Rabbit m Chicken m Htma~ Rabbit
8 S,
1 2 3 4 5 6 7
57 Since calpain is ubiquitously expressed in all tissues, it should play an important and fundamental role in cells. Moreover, as the activity of calpain is regulated by calcium ions essential for cellular signal transduction, calpain is believed to regulate signal transduction by modulating the activity and the structure of various proteins, e.g., protein kinase C, phospholipase C or transcription factors [16-18]. However, direct evidence of calpain as a modulator of cellular system has not yet
been obtained. Ubiquitous expression of calpain makes it difficult to identify its endogenous substrates and thus its physiological functions. In the course of cloning cDNAs for calpain large subunits, we isolated a cDNA for a putative large subunit, hopefully, the third type of the calpain large subunit and designated it as p94 [19] (Figs. 1 and 2). A putative protein encoded by the cDNA is similar to, but distinct from both m- and /z-type large subunits.
TABLE I
Similarity of amino-acid sequence of calpain large subunits I - I V in the extreme left low represent domain numbers of calpain large subunits as shown in Figs. 1 and 2. Values in the tables are identity of amino-acid sequences among chicken and human m-type, human /z-type, rat p94 and Schistosoma calpain. Three unique sequences in rat p94 were omitted for calculation. As to Domain IV, comparison with human calpain small subunit was also included. The values in parentheses indicate that partial sequence(s) was used for comparison. Domain
Rabbit m[9]
Chicken m[8]
Human /z Ill]
Human m Rabbit m Chicken m Human/z Rabbit/z Rat p94 Human p94
(85.7)
70.3 (78.6)
55.4 (59.3) 51.9
Human m Rabbit m Chicken m Human/z Rabbit/Z Rat p94 Human p94
92.9
Rabbit tz[9]
Schistosoma
Rat p94 [19]
Human p94 [19]
m. [16]
32.4 (28.6) 32.4 37.8
32.4 (30.4) 32.4 33.3
17.6 (28.6) 18.2 13.1
(98.2)
24.3 20.0
60.6 59.8 59.4 62.6
47.7 47.7 48.1 49.2
93.7
50.6 48.5
Human 30K[13]
I
-
II
III Human m Rabbit m Chicken m Human/z Rabbit/Z Rat p94 Human p94 IV Human m Rabbit m Chicken m Human/z Rabbit/Z Rat p94 Human p94
75.1 74.3
65.0 61.0 64.2 65.4
73.5 74.3 79.4
94.7
63.3 62.8
60.3 60.1 68.9
(61.4) (59.8) (65.9) (96.3)
52.6 52.6 60.9 58.9 (59.0)
48.8 48.3 55.3 53.6 (50.8) 92.1
35.1 34.8 41.2 38.8 (42.5) 37.9 35.2
92.8
55.1 53.3
50.9 51.5 66.7
50.0 50.3 65.1 94.6
45.5 48.1 56.0 52.4 50.0
47.8 48.1 56.5 53.0 50.6 95.8
31.4 29.5 27.8 29.0 32.5 32.0 32.8
(93.1)
66.3 (65.5)
62.3 (63.3) 70.3
(54.7) (54.3) (65.2) (95.4)
51.8 52.4 59.2 53.9 (51.3)
50.9 (51.3) 54.4 53.6 (51.7) 94.0
37.4 (38.1) 38.4 37.3 (35.6) 40.4 39.3
S. mansoni Total Human m Rabbit m Chicken m Human/z Rabbit/z Rat p94 Human p94
47.9 50.0 52.4 51.2 49.7 57.7 58.9 30.2
58 100
80
X D
m tin
l
60
I
40
20
i
I
iI
I
I
i
III
IV
F
T
Domain
Fig. 3. Amino-acid sequence similarities among members of the calpain large subunit family. T indicates total sequence. Sequence comparisons are: (o), rat and h u m a n p94; (z~) rabbit and h u m a n m-types; ( v ) , rabbit and h u m a n /z-types: ( n ) , chicken and h u m a n m-types; (e), rat p94 and h u m a n m;type; (A), rat p94 and h u m a n /z-type: ( • ) , rat p94 and chicken m-type; ( i ) , h u m a n m-type, and /z-type: ( x ) c h i c k e n m-type and h u m a n / z - t y p e .
Thus, m- and /z-types together with p94 compose a super family, 'calpain large subunit family'. Recently, cDNA coding for a protein similar to the calpain large subunits has been isolated from a cDNA library of Schistosoma mansoni [20]. This putative protein can also be included in this family. It is essential to identify p94 at the protein level, m R N A of which is expressed only in skeletal muscle, to understand not only the structure and function of muscle, but also the general physiological functions of calpain. We tried to identify p94 protein by using an antibody specific to p94 but it could not be detected in muscle extract (unpublished results). In this report, the structure of p94 was compared with the other members of the large subunit family as a step to understand the physiological function and evolutionary correlation of calpain. The structure of conventional calpains
Large subunits of calpain As shown in Table I and Fig. 3, the large subunits of mammalian m- and/z-calpains are well conserved along the whole sequence when compared within the same type. All four domains are almost equally well-conserved and all the similarities are around 90%. The overall similarities of m- or /z-calpain large subunits between human and rabbit are 93.1 or 95.4%, respectively. When m-type and /z-type are compared one
another, i.e., human (or chicken) m-calpain large subunits vs. human/z-calpain large subunits, the similarity somewhat reduces. Total similarity between human /z-type and human or chicken m-type is 62.3 or 70.3%, respectively. When similarity of each domain is considered, significantly high similarity of domain II responsible for proteolytic activity can be noticed, especially when different types are compared (Fig. 3). This suggests that a module that gives proteolytic activity in the calpain large subunit is common to both types and that type-specific nature must be ascribed to the other domains, i.e., domain I, III a n d / o r IV. Total similarity between chicken and human m-types (66.3%) is slightly lower than that between chicken m-type and human/z-type (70.3%). Therefore, chicken m-type was once considered as a prototype of both mand /z-types of mammal when only a single type (mtype) was found in muscle [8]. Chicken /z-type, however, has been identified [21] and the primary structure is being elucidated (unpublished results). Now it is clear that chicken has two distinct types corresponding to mammalian m- and /z-types as discussed below.
Small subunits of calpain Unfortunately, since primary sequences of only mammalian small subunits are available at present, little information can be deduced from the sequence comparison. As shown in Table II, total similarities among human, porcine, rabbit, bovine and rat are from 90.1 to 97.4%. The similarity among the small subunits T A B L E II
Amino acid sequence similarities of calpain small subunits Domains V and I V ' indicated in Fig. 1 of human, porcine, rabbit, bovine and rat were compared. As to domain IV', mouse pMP41 was also included.
Domain V Human Porcine Rabbit Bovine Rat
Porcine [32]
Rabbit [10]
Bovine [33]
Rat
98.0
97.0 96.9
94.0 95.9 94.9
93.0 91.8 92.8 89.5
-
96.4 97.6
96.4 98.2 97.0
91.7 92.3 90.4 90.5
31.1 31.8 31.1 33.3 31.8
96.6 96.6
95.5 96.6 96.2
92.2 92.1 91.4 90.1
26.8 27.5 26.8 28.8 27.5
Domain I V ' Human 97.0 Porcine Rabbit Bovine Rat Total Human Rabbit Porcine Bovine Rat
97.4
Mouse pMP41 [34]
-
59 is as high as that of the large subunits, or even higher. No significant difference exists between the similarities of domains V and IV'. This may suggest that both domains are equally important for a function of calpain, although domain V is a small subunit-specific domain and domain IV' is not. Domain IV', together with domain IV of the large subunit was presumably derived from the same ancestral gene. To get more evolutionary information of the small subunits, determination of the primary structure of lower animals is essential. The structure of p94
Characteristics of p94 Unique characteristics of p94 when compared to those of the other members of the calpain large subunit family are; (i), its m R N A is expressed only in skeletal muscle and the amount is at least one-order of magnitude higher than that encoding m- and/z-calpain large subunits [19]; (ii), three unique sequences exist; at the N-terminal of domain I, in the middle of domain II and at the C-terminal of domain III (Figs. 1 and 2, indicated as NS, IS1 and IS2, respectively) and, therefore, (iii), the molecular mass is 94 kDa. The unique regions show no sequence similarity to any other proteins in the protein database, including m- and /z-calpain large subunits. NS is about 60 residues long and rich in proline residues. IS1 is located just before the active site histidine residue of p94, suggesting that IS1 might correlate with p94 proteinase activity. IS2 has a lysine-rich sequence similar to nuclear localization signal near the N-terminus (indicated as 'K-rich' in Fig. 1) [19,22]. Function(s) and comformational specificity of these regions are still unknown, but will be discussed in this paper. The presence of a third calcium-dependent proteolytic activity in mammalian skeletal muscle other than m- and/z-calpain has not yet been identified. Although chicken skeletal muscle contains high m-calpain together with m- and/z-calpains [23], this does not correspond to p94, since the molecular mass is 75 kDa. To elucidate the discrepancy between a high level of p94 m R N A and the absence of its protein, we analyzed the expression of the p94 cDNA in COS cells and found that IS2 plays a pivotal role in the existence of p94 protein (unpublished results). It is essential to examine the structures of both conventional calpains and novel p94 more in detail to understand the physiological function of calpain super family and the specific function of p94 in muscle. Further characterization of the p94 protein is now in progress. Does a regulatory subunit of p94 exist? Since exon-intron organizations of calmodulin domains in the large (chicken m-type [24]) and small
(human [25]) subunits (IV and IV') are identical, the gene for the C-terminal half of the small subunit was diverged from the same ancestral gene module for the large subunit. Possibly, this calmodulin gene module fused with a GC-rich D N A fragment encoding the N-terminal glycine-rich domain (domain V) to give a gene for the calpain small subunit. The N-terminal glycine-rich sequence is processed during autocatalytic activation of calpain [26,27]. The function of the small subunit is, therefore, to regulate localization and activity of calpain molecule a n d / o r to stabilize the large subunit. A small subunit for p94 as well as Schistosoma calpain has not yet been identified. Following this three possibilities can be postulated: (i), p94 associates with the same 30 kDa subunit as conventional calpains; (ii), p94 exists and functions as a monomer or homooligomer; (iii), p94 associates with some protein(s) other than the calpain small subunit. On the basis of the sequence similarity, it is highly likely that p94 interacts with the calpain small subunit. p94 cannot be detected in muscle due to its extremely rapid turnover rate. Further, its m R N A is expressed only in the fully differentiated muscle cells, not in myoblast such as rat L8 cells. Some kind of regulator, if not a subunit, might be required for stage-specific stable expression, localization and physiological function of p94.
Sequence similarity between human and rat p94 As shown in Table III, human and rat p94 are 94.0% identical, although the N-terminal 43 residues of human p94 have not yet been determined (Fig. 2). Although sequences of rat m- and/z-types have not yet been reported, as described above, m- and /z-calpain TABLE III Sequence similarityof rat and human p94 The values in parentheses indicate sequences which have not yet been determined completely. Domain I-NS, Domain II-IS1 and Domain III-IS2 represent the similarityof the domains ignoring NS, IS1 or IS2, respectively. Domains I NS I-NS
Similarity (98.2) (100.0) 97.2
II IS1 II-IS1
93.7 93.5 93.8
III IS2 III-IS2
92.1 85.5 93.7
IV
95.8
Total
(94.0)
60
Schistosoma calpain contains a short insertion sequence at the IS2 position. Moreover, Drosophila cal-
large subunits show 93.1 and 95.4% identity, respectively, between human and rabbit. Currently, available information is limited, but the sequence conservation among mammalian p94 species is as high as that among m- and /x-types, suggesting that p94 is functionally as important as m- and /x-types. On the other hand, sequences of protein kinase C, phospholipase C and c-jun, typical members of signal transduction, are 97 to 99% conserved among different mammalian species [28-30]. Calpain seems to be less conserved than other members of the signal transduction machinery, although a clear conclusion must await further structural analyses of the enzymes from various organisms. As in the case of m- and /x-types, similarity of each domain of p94 shows almost the same value when human and rat p94 are compared. More precisely, slightly lower conservation in the IS2 region (85.5%) is observed as shown in Table IV. Since the average of similarities of four domains, NS and IS1 is 95.3% (standard deviation 2.6%), the 85.5% similarity is significantly lower. The meaning of this lower conservation of IS2 remains to be clarified. As shown in Fig. 2,
pains also contain insertions of comparable lengths to p94 at the identical position. The insertions found in these two organisms, however, show no sequence homology to IS2. These observations suggest that the IS2 region might be at the surface of the molecule and the insertion at this position does not affect much the whole conformation of the calpain large subunit, e.g., presumably by forming a loop-out structure.
Proteolytic activity and calcium dependence of p94 The fact that domain II is the most conserved domain when p94 and other types are compared (Table I and Fig. 3), strongly suggests that p94 possesses proteinase activity as m- and /x-types. Although the proteinase activity of p94 has not yet been identified, the conservation, especially around the active site cysteine, histidine and asparagine residues indicates a strong evolutionary pressure. In other words, the muscle specific function of p94 must be accomplished by other p94-specific sequences, such as NS, IS1 a n d / o r IS2.
T A B L E IV
Scores of EF-hand structures of calpain subunits Numbers in the top line represent 4 E F - h a n d structures indicated in Fig. 2. 'Extra' shows EF-hand structure between domains II and III as reported in Ref. 20. Scores are an identity of sequences to EL--LL--LO-O-OG-LO--OL--LL--L sequencially, where E, L, O, G and - stand for glutamic acid, l e u c i n e / v a l i n e / i s o l e u c i n e / p h e n y l a l a n i n e / m e t h i o n i n e , aspartic a c i d / a s p a r a g i n e / g l u t a m i c a c i d / g l u t a m i n e / s e r i n e / t h r e o n i n e , glycine and any amino-acid residue, respectively. - stands for the sequence corresponding to that region does not exist or has not yet been determined. Bold n u m b e r s indicate that the positions experimentally proved to bind calcium ions previously [35]. EF-hand Structures
1
2
3
4
Extra
Total
Human Rat
13 13
11 11
10 10
11 11
12 12
45 45
m-calpain Large subunits Human Rabbit Chicken
12 12 11
11 11 11
11 11 11
13 13 11
9 10 10
47 47 45
/x-calpain Large subunits Human Rabbit
12 12
12 12
11 11
13 13
11 -
48 48
S.Mansoni
12
11
11
8
13
42
Calpain Small subunits Human Porcine Rabbit Bovine Rat
12 12 12 12 12
10 10 10 10 10
12 11 11 11 12
11 11 11 11 11
45 44 44 44 45
pMP41 Mouse
12
12
10
11
45
Average
12.1
10.9
10.9
11.4
p94
X-calpain
11.0
45.4
61 Since domain IV of p94 shows the same level of similarity among the three types, p94 should have calcium binding activity. Moreover, as shown in Table IV, EF-hand scores [31] of p94 domain IV are comparable to those of conventional calpain large and small subunits. It is, however, difficult to predict the calcium sensitivity of p94 from the scores, since m- and/z-types have a different calcium sensitivity, regardless of similar EF-hand scores. As pointed out in Ref. 20, the calpain large subunit contains an extra EF-hand structure at the boundary of domains II and III. The physiological meaning of the extra EF-hand structure is unknown. It is possible that the region folds near the domain IV and functions as an extra calcium-binding site.
Calpain large subunit family As described above, intra-mammal comparison showed clear distinction among three types of the calpain large subunit family, i.e., p94, m- and /z-type. No ambiguity exists in the classification of the three types. Chicken m-type is exceptional, since total similarity of chicken m-type to human m-type is a little lower than that to human /z-type. Newly identified chicken /z-type is more similar to human /z-type than human m-type and human /z-type is more similar to chicken /z-type than chicken m-type. Chicken /z-type is, therefore, /z-type and chicken m-type is m-type. A c D N A encoded protein isolated from Schistosoma mansoni is equally similar to the three types of calpain large subunits, p94, m- and /z-types. Evolutionary correlation among the 3 types of the large subunit will be clarified when further comparative studies on calpain from various animals are performed.
Evolutionary process of calpain molecules Since p94, m- and /z-types together with Schistosoma calpain are composed of identical domain structures, genes of these four proteins were derived from the same ancestral gene. The ancestral gene consists of genes for the cysteine proteinase module, calcium binding module and at least two unknown modules. Evolutionary distance between p94 and m- or/z-type is slightly further than that between m- and /z-types, judging from the sequence similarity, while Schistososoma calpain is equally distant from p94, m- and /ztypes (see Table I). Instead, domain IV of p94 is nearer to small subunit than that of m- and /z-types. A summary of these investigation is shown in Fig. 4. Evolutionarily closer relationship of p94 and the small subunit might correlate with the subunit structure of p94. As discussed above, it is not clear, according to present knowledge, if Schistosoma calpain is the ancestral molecule or not. The domain structure, however, is still identical to that of vertebrates, strongly suggesting that this structure is important for the function a n d / o r
Fig. 4. Evolutionaryrelationship among calpain subunits. Mm, M/~, p94, and Sm stand for mammalian m-type, mammalian ~-type, mammalian p94 and Schistosoma calpain, respectively. The length between each subunit represents qualitatively the evolutionary distance and the values near the connected bars are total sequence similarities between those two types.
stability of the calpain large subunit. To elucidate these problems it is essential to study p94 at the protein level. Further studies are now in progress to clarify the structural and functional characteristics of p94, which will also reveal evolutionary relationship among the members of the calpain large subunit family.
References 1 2 3 4 5 6 7
Suzuki, K. (1991) Biomed. Biochim. Acta 50, 483-484. Suzuki, K. (1987) Trends Biochem. Sci. 8, 167-169. Suzuki, K. and Ohno, S. (1990) Cell Structure Function 15, 1-6. Murachi, T. (1989) Biochem. Int. 18, 263-194. Mellgren, R.L. (1987) FASEB J. l, 110-115. Johnson, P. (1990) Int. J. Biochem. 22, 811-822. Wang, K.K.W., Villalobo, A. and Roufogalis, B.D. (1989) Biochem. J, 262, 693-706. 8 Ohno, S., Emori, Y., Imajoh, S., Kawasaki, H., Kisaragi, M. and Suzuki, K. (1984) Nature 312, 566-570. 9 Emori, Y., Kawasaki, H., Sugihara, H., Imajoh, S., Kawashima, S. and Suzuki, K. (1986) J. Biol. Chem. 261, 9465-9471. 10 Emori, Y., Kawasaki, H., Imajoh, S., Kawashima, S. and Suzuki, K. (1986) J. Biol. Chem. 261, 9472-9476. 11 Imajoh, S., Aoki, K., Ohno S., Emori, Y., Kawasaki, H., Sugihara, H. and Suzuki, K. (1988) Biochemistry27, 8122-8128. 12 Aoki, K., Imajoh, S., Ohno, S., Emori, Y. Koike, M., Kosaki, G. and Suzuki, K. (1986) FEBS Lett. 205, 313-317. 13 Ohno, S., Emori, Y. and Suzuki, K. (1986) Nucleic Acids Res. 14, 5559. 14 Sakihama, T., Kakidani, H., Zenita, K., Yumoto, N., Kikuchi, T., Sasaki, T., Kannagi, R., Nakanishi, S., Ohmori, M., Takio, K., Titani, K. and Murachi, T. (1985) Proc. Natl. Acad. Sci. USA 82, 6075-6079. 15 lmajoh, S., Kawasaki, H. and Suzuki, K. (1986) J. Biochem. 99, 1281-1284. 16 Pontremoli, S., Melloni, E., Sparatore, B., Michetti, M., Salamino, F. and Horecker, B.L. (1990) J. Biol. Chem. 265, 706-712. 17 Kishimoto, A., Mikawa, K., Hashimoto, K., Yasuda, I., Tanaka, S., Tominaga, M., Kuroda, T. and Nishizuka, Y. (1989) J. Biol. Chem. 264, 4088-4092.
62 18 Hirai, S., Kawasaki, H., Yaniv, M. and Suzuki, K. (1991) FEBS Lett. 287, 57-61. 19 Sorimachi, H., Imajoh-Ohmi, S., Emori, Y., Kawasaki, H., Ohno, S., Minami, Y. and Suzuki, K. (1989) J. Biol. Chem. 264, 2010620111. 20 Andresen, K., Tom, T.D. and Strand, M. (1991) J. Biol. Chem. 266, 15085-15090. 21 Murakami, T., Ueda, M., Hamakubo, T. and Murachi, T. (1988) J. Biochem. 103, 168-171. 22 Sorimachi, H., Ohmi, S., Emori, Y., Kawasaki, H., Saido, T.C., Ohno, S., Minami, Y. and Suzuki, K. (1990) Biol. Chem. HoppeSeyler 371, 171-176. 23 Wolfe, F.H., Sathe, S.K., Goll, D.E., Kleese, W.C., Edmunds, T. and Duperret, S.M. (1989) Biochim. Biophys. Acta 998, 236-250. 24 Miyake, S., Emori, Y. and Suzuki, K. (1986) Nucleic Acids Res. 14, 8805-8817. 25 Emori, Y., Ohno, S., Tobita, M. and Suzuki, K. (1986) FEBS Lett. 194, 249-252. 26 Imajoh, S., Kawasaki, H. and Suzuki, K. (1986) J. Biochem. 100, 633-642. 27 Kawashima, S., Inomata, M. and Imahori, K. (1986) Biomed. Res. 7, 327-331.
28 Ohno, S., Akita, Y., Hata, A., Osada, S., Kubo, K., Konno, Y., Akimoto, K., Mizuno, K., Saido, T., Kuroki, T. and Suzuki, K. (1991) Adv. Enzym. Regul. 31,287-303. 29 Emori, Y., Homma, Y., Sorimachi, H., Kawasaki, H., Nakanishi, O., Suzuki, K. and Takenawa, T. (1989) J. Biol. Chem. 264, 21885-21890. 30 Vogt, P.K. and Bos, T.J. (1990) Adv. Cancer Res. 55, 1-35. 31 Kretsinger, R.H. and Nockolds, C.E. (1973) J. Biol. Chem. 248, 3313-3326. 32 Sakihama, T., Kakidani, H., Zenita, K., Yumoto, N., Kikuchi, T., Sasaki, T., Kannagi, R., Nakanishi, S., Ohmori, M., Takio, K., Titani, K. and Murachi, T. (1985) Proc. Natl. Acad. Sci. USA 82, 6075-6079. 33 McClelland, P., Lash, J.A. and Hathaway, D.R. (1989) J. Biol. Chem. 264, 17428-17431. 34 Kageyama, H. Shimizu, M., Tokunaga, K., Hiwasa, T., and Sakiyama, S. (1989) Biochim. Biophys. Acta 1008, 225-257. 35 Minami, Y., Emori, Y., Kawasaki, H. and Suzuki, K. (1987) J. Biochem. 101, 889-895.
55
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34354
Sequence comparison among muscle-specific calpain, p94, and calpain subunits Hiroyuki Sorimachi and Koichi Suzuki Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo (Japan)
Key words: Calpain; p94; Calcium; Cysteine proteinase; Evolution While conventional calpains, m- and /z-calpains named according to their calcium-dependence, are expressed in almost every tissues, mRNA of newly identified p94, which has a significant sequence similarity to the conventional calpain large subunits, is abundantly expressed only in skeletal muscle. In addition to this specific expression, p94 is distinct from conventional calpains in that it contains three unique regions showing no similarity to conventional calpain subunits. When rat and human p94 are compared, overall sequence similarity is 94.0%, which is close to those for m- and /~-calpain large subunits; 93.1% and 95.4% between human and rabbit, respectively, suggesting the evolutionary importance of p94. These calpain large subunit proteins, p94, m- and/.~-types, can be considered to constitute a super family, whose p94, m- and/~-types represent the three major types. Sequences of the calpain large-subunit family members, including the recently reported Schistosoma calpain, are compared. Their evolutionary correlation and function are discussed on the basis of the results thus far obtained.
Introduction Calpain, calcium-dependent proteinase or C A N P is a ubiquitously expressed intracellular cysteine proteinase and requires calcium ions for activity [1-7]. Two isozymes, m- and ~-calpain, have been identified and studied extensively. Both are heterodimers and consist of a distinct but similar large subunit (about 80 kDa) and an identical small subunit (about 30 kDa) [2-14].The two distinct, m- and /~-calpain large subunits can be divided into four domains as indicated in Fig. 1 [8]. Since domain II has a similarity to other cysteine proteinases such as papain and cathepsins B, H and L, especially around their active-site cysteine histidineand asparagine residues, this domain is considered as a cysteine proteinase module. Domain IV is similar to calmodulin and contains 4 EF-hand structures, indicating that this domain is a calcium-binding module possibly responsible for calcium-dependent regulation of calpain. Domains I and III have no similarity to any other sequences in protein database but the N-terminal region of domain I is autocatalytically processed during the activation, suggesting that domain I is involved in
Correspondence to: H. Sorimachi, Institute of Applied Microbiology, University of Tokyo, Yaoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan.
the regulation of activity. The function of domain III still remains to be solved. The small subunit can also be divided into two domains according to the similarity to the large subunit (Fig. 1). The C-terminal half has a significant similarity to the domain IV of the large subunit and, thus, is n a m e d domain I V ' , which contains four EF-hand structures as the large subunits. The N-terminal half, n a m e d domain V, consists of contiguous glycine residues together with hydrophobic residues which make itself hydrophobic and is suggested to interact with phospholipids [15].
p~ m-& La~ Sub
Fig. 1. Schematic protein structure of calpain subunits. Numbers above the p94 structure indicate the amino-acid residue number of rat p94 [19]. 'Cys' and 'His' in p94, m- and/z-large subunit structures represent active-site cysteine and histidine residues, respectively. 'K-rich' indicates lysine-rich region as described in this paper and NS, IS1 and IS2 indicate three unique sequences in p94. I, II, III, IV, IV' and V represent the five domains of calpain large and small subunits.
V
200
350
t
150 ~
Dom~tin !
V
1
KKL~GII~C~.Kt~YNBII~VSLII I ~ - - ~ S S I ~ Y E I ~ O ,
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550
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600
l $2
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.
.
.
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.
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.
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V IS1
300
I O0 ISI
i ~- ~D~Jl N P N A I ~ I F A ~ S I ~ A S
_
_
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P,LD~I~DPDDYGD
650
D
FRNIFRQIA~DI~ I
V
400
I~I'I~I'£SI[017~ ITVEDP~p....D
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'
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R
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~I~--DDPDI)SEVIiSFLV~I~I~
4.50
243
~
500
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250 V
SSV~-
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I~Er~PELV GI~D~EII V i~ L L _v
........................................
.
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.
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3
4
700
.
.
.
.
manson/
m m /~ /z
p94 m
8
2 3
4 5 6 7 No. of IEmanm
&
Rabbit Chicken ttuman Rabbit
Famma
No. of Rat p94 Rat p94 1
Fig. 2. Amino-acid sequence of p94 and calpain large subunits. Lines: 1, rat p94; 2, h u m a n p94; 3, h u m a n m-calpain large subunit; 4, rabbit m-calpain large subunit; 5, chicken m-calpain large subunit; 6, h u m a n #-calpain large subunit; 7, rabbit #-calpain large subunit; 8, Schistosoma mansoni calpain. Borders of each domain are indicated by spacings and arrows. Three unique sequences of I)94, i.e., NS, IS1 and IS2, are indicated by arrows. ( ...... ), deletion at the positions; ( z~ ), positions where introns are inserted into genomic D N A of chicken m-calpain large subunit [25]. Numbers on the top and bottom of the sequences are residue n u m b e r s of rat p94 and h u m a n m-calpain large subunit, respectively. Reversed letters stand for residues that are conserved in all subunit sequences. Underlined regions (1-4) are EF-hand structures. C, H and N residues, indicated by asterisks, are active sites of calpain.
600
..
(2,PDDVIEALI{LI~)TLLDIKX~EIEQ~F L A I R D P K I ~ A I I ~ V I ~ G ~ - - ~ T L O g l P N ' F - - ~ 550 1
I ITMRYADKI-~-I I ~ I ( ~ K I ) G D G I I~TtC/A I I TMRYADXI-~-I lII~lll ~ K I ~ I IELNVLEI4QLTHYA 3 _C,.~.~Rt~IITIIntZQ~QXI IREI I ' ~ I m ~ IV~ADDO~I- i ~ I ~ Q L D P ~ m L D L ISI~C~VL 4 Gll[~ta~IZ~IlX I O.KIQI{II I ~ I l l ~ l R I I ~ IVARFADDQLI- I ~ I F K ~ P D K I G ~ I Q L D L [SI4CFSVL S (~ILm(~IQ ~ I m I ~,'LTI ~ HI[I~A(~NVV/~¥ADAETG-~RI~T GTAV/~LAE~TMCG 8 (~m~_ _vImIlmUz e k s i F m l t i i m ~ Y MIIgIESA~'KLm~IIr~IITRYSEPDLA-V~ORIV~TLDI~L~cCrrDLFK4OLTMFA 7 (~llIh~wllWli~Itl t ~ l ~ ' I MIIMilI~A(Ib'II/(KIII~i I I'RF~DIA - V I I I ~ I I i ( ] I . I I I I E I ' I 4 1 ~ T D L I ~ ~ ' I I ~ A 8 ~(~K(;t~ql FIIg~I~i~/FSAY F l l E I I / ~ A ~ ! N A I I~YODPGTDXISIEI~[~DIK'f~-f IEAHPI~IEG'I'SLF~JR)~[P~FRVIH
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~ L K T ~ [ ~ I',~It'E~QE(~ILIVFS~ISVDI~r~KKlXPIIFVSD~OVI)Q~EEOKGKTSPDKO.KQSP(~°QPGSSIX~EEQ~ FP,N I F K Q I A ( } D I ) M E I ~ L ~ I ~ IL~B~EI[I~FClIIVFSEI~YQAVI)DEIF-.~ ......................................LE-I~"DISEDDIDDGVRRLFAQLAGEDAEISAFii(I'III.~QDIKSI~FSIETC~I~D ILV~I~][~(II)FC~iv~B~]~)YQAVDDEIEAD-.......................................... LE-rdOV~DIDD~ FRRLFAQI.~GI~AEI_S~ILRIIV~II~t'II.SIETC~IIe/~MLRSD F'KNt.IFQ(~AGED~I ~ I LI~VI ~lllm) ~I~AGTVELI)DQ I OAN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LPDEQVLSE~IDI~I FKAI.HIQ~GEI)I~I ~ I LNRI I S I { H K D L ~
300
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--KD/~SI~M~IMI~II~G~I,IO21~DGrle~GTSPSGI2~ I AI~/RNMI~LLP,DSDLDP~SDDRPSRTI~Qt~-I~ 1- -RDAIISII'~II~IEROSIIMIIIB~G'ISPSGI~G~ I AI~LLRDSDLDPROSDI~FrRTII ~ Q ~ _~:llrll2K H Q K ~ L Q ~ .................................... ~rSU,DSEAZT~
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I
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150
No. ofHuaan m
m&osont
No. of Rat p94 Rat p94 Hu.e~'x p94 Humaa m Rabbit m Chicken m Htma~ Rabbit
8 S,
1 2 3 4 5 6 7
57 Since calpain is ubiquitously expressed in all tissues, it should play an important and fundamental role in cells. Moreover, as the activity of calpain is regulated by calcium ions essential for cellular signal transduction, calpain is believed to regulate signal transduction by modulating the activity and the structure of various proteins, e.g., protein kinase C, phospholipase C or transcription factors [16-18]. However, direct evidence of calpain as a modulator of cellular system has not yet
been obtained. Ubiquitous expression of calpain makes it difficult to identify its endogenous substrates and thus its physiological functions. In the course of cloning cDNAs for calpain large subunits, we isolated a cDNA for a putative large subunit, hopefully, the third type of the calpain large subunit and designated it as p94 [19] (Figs. 1 and 2). A putative protein encoded by the cDNA is similar to, but distinct from both m- and /z-type large subunits.
TABLE I
Similarity of amino-acid sequence of calpain large subunits I - I V in the extreme left low represent domain numbers of calpain large subunits as shown in Figs. 1 and 2. Values in the tables are identity of amino-acid sequences among chicken and human m-type, human /z-type, rat p94 and Schistosoma calpain. Three unique sequences in rat p94 were omitted for calculation. As to Domain IV, comparison with human calpain small subunit was also included. The values in parentheses indicate that partial sequence(s) was used for comparison. Domain
Rabbit m[9]
Chicken m[8]
Human /z Ill]
Human m Rabbit m Chicken m Human/z Rabbit/z Rat p94 Human p94
(85.7)
70.3 (78.6)
55.4 (59.3) 51.9
Human m Rabbit m Chicken m Human/z Rabbit/Z Rat p94 Human p94
92.9
Rabbit tz[9]
Schistosoma
Rat p94 [19]
Human p94 [19]
m. [16]
32.4 (28.6) 32.4 37.8
32.4 (30.4) 32.4 33.3
17.6 (28.6) 18.2 13.1
(98.2)
24.3 20.0
60.6 59.8 59.4 62.6
47.7 47.7 48.1 49.2
93.7
50.6 48.5
Human 30K[13]
I
-
II
III Human m Rabbit m Chicken m Human/z Rabbit/Z Rat p94 Human p94 IV Human m Rabbit m Chicken m Human/z Rabbit/Z Rat p94 Human p94
75.1 74.3
65.0 61.0 64.2 65.4
73.5 74.3 79.4
94.7
63.3 62.8
60.3 60.1 68.9
(61.4) (59.8) (65.9) (96.3)
52.6 52.6 60.9 58.9 (59.0)
48.8 48.3 55.3 53.6 (50.8) 92.1
35.1 34.8 41.2 38.8 (42.5) 37.9 35.2
92.8
55.1 53.3
50.9 51.5 66.7
50.0 50.3 65.1 94.6
45.5 48.1 56.0 52.4 50.0
47.8 48.1 56.5 53.0 50.6 95.8
31.4 29.5 27.8 29.0 32.5 32.0 32.8
(93.1)
66.3 (65.5)
62.3 (63.3) 70.3
(54.7) (54.3) (65.2) (95.4)
51.8 52.4 59.2 53.9 (51.3)
50.9 (51.3) 54.4 53.6 (51.7) 94.0
37.4 (38.1) 38.4 37.3 (35.6) 40.4 39.3
S. mansoni Total Human m Rabbit m Chicken m Human/z Rabbit/z Rat p94 Human p94
47.9 50.0 52.4 51.2 49.7 57.7 58.9 30.2
58 100
80
X D
m tin
l
60
I
40
20
i
I
iI
I
I
i
III
IV
F
T
Domain
Fig. 3. Amino-acid sequence similarities among members of the calpain large subunit family. T indicates total sequence. Sequence comparisons are: (o), rat and h u m a n p94; (z~) rabbit and h u m a n m-types; ( v ) , rabbit and h u m a n /z-types: ( n ) , chicken and h u m a n m-types; (e), rat p94 and h u m a n m;type; (A), rat p94 and h u m a n /z-type: ( • ) , rat p94 and chicken m-type; ( i ) , h u m a n m-type, and /z-type: ( x ) c h i c k e n m-type and h u m a n / z - t y p e .
Thus, m- and /z-types together with p94 compose a super family, 'calpain large subunit family'. Recently, cDNA coding for a protein similar to the calpain large subunits has been isolated from a cDNA library of Schistosoma mansoni [20]. This putative protein can also be included in this family. It is essential to identify p94 at the protein level, m R N A of which is expressed only in skeletal muscle, to understand not only the structure and function of muscle, but also the general physiological functions of calpain. We tried to identify p94 protein by using an antibody specific to p94 but it could not be detected in muscle extract (unpublished results). In this report, the structure of p94 was compared with the other members of the large subunit family as a step to understand the physiological function and evolutionary correlation of calpain. The structure of conventional calpains
Large subunits of calpain As shown in Table I and Fig. 3, the large subunits of mammalian m- and/z-calpains are well conserved along the whole sequence when compared within the same type. All four domains are almost equally well-conserved and all the similarities are around 90%. The overall similarities of m- or /z-calpain large subunits between human and rabbit are 93.1 or 95.4%, respectively. When m-type and /z-type are compared one
another, i.e., human (or chicken) m-calpain large subunits vs. human/z-calpain large subunits, the similarity somewhat reduces. Total similarity between human /z-type and human or chicken m-type is 62.3 or 70.3%, respectively. When similarity of each domain is considered, significantly high similarity of domain II responsible for proteolytic activity can be noticed, especially when different types are compared (Fig. 3). This suggests that a module that gives proteolytic activity in the calpain large subunit is common to both types and that type-specific nature must be ascribed to the other domains, i.e., domain I, III a n d / o r IV. Total similarity between chicken and human m-types (66.3%) is slightly lower than that between chicken m-type and human/z-type (70.3%). Therefore, chicken m-type was once considered as a prototype of both mand /z-types of mammal when only a single type (mtype) was found in muscle [8]. Chicken /z-type, however, has been identified [21] and the primary structure is being elucidated (unpublished results). Now it is clear that chicken has two distinct types corresponding to mammalian m- and /z-types as discussed below.
Small subunits of calpain Unfortunately, since primary sequences of only mammalian small subunits are available at present, little information can be deduced from the sequence comparison. As shown in Table II, total similarities among human, porcine, rabbit, bovine and rat are from 90.1 to 97.4%. The similarity among the small subunits T A B L E II
Amino acid sequence similarities of calpain small subunits Domains V and I V ' indicated in Fig. 1 of human, porcine, rabbit, bovine and rat were compared. As to domain IV', mouse pMP41 was also included.
Domain V Human Porcine Rabbit Bovine Rat
Porcine [32]
Rabbit [10]
Bovine [33]
Rat
98.0
97.0 96.9
94.0 95.9 94.9
93.0 91.8 92.8 89.5
-
96.4 97.6
96.4 98.2 97.0
91.7 92.3 90.4 90.5
31.1 31.8 31.1 33.3 31.8
96.6 96.6
95.5 96.6 96.2
92.2 92.1 91.4 90.1
26.8 27.5 26.8 28.8 27.5
Domain I V ' Human 97.0 Porcine Rabbit Bovine Rat Total Human Rabbit Porcine Bovine Rat
97.4
Mouse pMP41 [34]
-
59 is as high as that of the large subunits, or even higher. No significant difference exists between the similarities of domains V and IV'. This may suggest that both domains are equally important for a function of calpain, although domain V is a small subunit-specific domain and domain IV' is not. Domain IV', together with domain IV of the large subunit was presumably derived from the same ancestral gene. To get more evolutionary information of the small subunits, determination of the primary structure of lower animals is essential. The structure of p94
Characteristics of p94 Unique characteristics of p94 when compared to those of the other members of the calpain large subunit family are; (i), its m R N A is expressed only in skeletal muscle and the amount is at least one-order of magnitude higher than that encoding m- and/z-calpain large subunits [19]; (ii), three unique sequences exist; at the N-terminal of domain I, in the middle of domain II and at the C-terminal of domain III (Figs. 1 and 2, indicated as NS, IS1 and IS2, respectively) and, therefore, (iii), the molecular mass is 94 kDa. The unique regions show no sequence similarity to any other proteins in the protein database, including m- and /z-calpain large subunits. NS is about 60 residues long and rich in proline residues. IS1 is located just before the active site histidine residue of p94, suggesting that IS1 might correlate with p94 proteinase activity. IS2 has a lysine-rich sequence similar to nuclear localization signal near the N-terminus (indicated as 'K-rich' in Fig. 1) [19,22]. Function(s) and comformational specificity of these regions are still unknown, but will be discussed in this paper. The presence of a third calcium-dependent proteolytic activity in mammalian skeletal muscle other than m- and/z-calpain has not yet been identified. Although chicken skeletal muscle contains high m-calpain together with m- and/z-calpains [23], this does not correspond to p94, since the molecular mass is 75 kDa. To elucidate the discrepancy between a high level of p94 m R N A and the absence of its protein, we analyzed the expression of the p94 cDNA in COS cells and found that IS2 plays a pivotal role in the existence of p94 protein (unpublished results). It is essential to examine the structures of both conventional calpains and novel p94 more in detail to understand the physiological function of calpain super family and the specific function of p94 in muscle. Further characterization of the p94 protein is now in progress. Does a regulatory subunit of p94 exist? Since exon-intron organizations of calmodulin domains in the large (chicken m-type [24]) and small
(human [25]) subunits (IV and IV') are identical, the gene for the C-terminal half of the small subunit was diverged from the same ancestral gene module for the large subunit. Possibly, this calmodulin gene module fused with a GC-rich D N A fragment encoding the N-terminal glycine-rich domain (domain V) to give a gene for the calpain small subunit. The N-terminal glycine-rich sequence is processed during autocatalytic activation of calpain [26,27]. The function of the small subunit is, therefore, to regulate localization and activity of calpain molecule a n d / o r to stabilize the large subunit. A small subunit for p94 as well as Schistosoma calpain has not yet been identified. Following this three possibilities can be postulated: (i), p94 associates with the same 30 kDa subunit as conventional calpains; (ii), p94 exists and functions as a monomer or homooligomer; (iii), p94 associates with some protein(s) other than the calpain small subunit. On the basis of the sequence similarity, it is highly likely that p94 interacts with the calpain small subunit. p94 cannot be detected in muscle due to its extremely rapid turnover rate. Further, its m R N A is expressed only in the fully differentiated muscle cells, not in myoblast such as rat L8 cells. Some kind of regulator, if not a subunit, might be required for stage-specific stable expression, localization and physiological function of p94.
Sequence similarity between human and rat p94 As shown in Table III, human and rat p94 are 94.0% identical, although the N-terminal 43 residues of human p94 have not yet been determined (Fig. 2). Although sequences of rat m- and/z-types have not yet been reported, as described above, m- and /z-calpain TABLE III Sequence similarityof rat and human p94 The values in parentheses indicate sequences which have not yet been determined completely. Domain I-NS, Domain II-IS1 and Domain III-IS2 represent the similarityof the domains ignoring NS, IS1 or IS2, respectively. Domains I NS I-NS
Similarity (98.2) (100.0) 97.2
II IS1 II-IS1
93.7 93.5 93.8
III IS2 III-IS2
92.1 85.5 93.7
IV
95.8
Total
(94.0)
60
Schistosoma calpain contains a short insertion sequence at the IS2 position. Moreover, Drosophila cal-
large subunits show 93.1 and 95.4% identity, respectively, between human and rabbit. Currently, available information is limited, but the sequence conservation among mammalian p94 species is as high as that among m- and /x-types, suggesting that p94 is functionally as important as m- and /x-types. On the other hand, sequences of protein kinase C, phospholipase C and c-jun, typical members of signal transduction, are 97 to 99% conserved among different mammalian species [28-30]. Calpain seems to be less conserved than other members of the signal transduction machinery, although a clear conclusion must await further structural analyses of the enzymes from various organisms. As in the case of m- and /x-types, similarity of each domain of p94 shows almost the same value when human and rat p94 are compared. More precisely, slightly lower conservation in the IS2 region (85.5%) is observed as shown in Table IV. Since the average of similarities of four domains, NS and IS1 is 95.3% (standard deviation 2.6%), the 85.5% similarity is significantly lower. The meaning of this lower conservation of IS2 remains to be clarified. As shown in Fig. 2,
pains also contain insertions of comparable lengths to p94 at the identical position. The insertions found in these two organisms, however, show no sequence homology to IS2. These observations suggest that the IS2 region might be at the surface of the molecule and the insertion at this position does not affect much the whole conformation of the calpain large subunit, e.g., presumably by forming a loop-out structure.
Proteolytic activity and calcium dependence of p94 The fact that domain II is the most conserved domain when p94 and other types are compared (Table I and Fig. 3), strongly suggests that p94 possesses proteinase activity as m- and /x-types. Although the proteinase activity of p94 has not yet been identified, the conservation, especially around the active site cysteine, histidine and asparagine residues indicates a strong evolutionary pressure. In other words, the muscle specific function of p94 must be accomplished by other p94-specific sequences, such as NS, IS1 a n d / o r IS2.
T A B L E IV
Scores of EF-hand structures of calpain subunits Numbers in the top line represent 4 E F - h a n d structures indicated in Fig. 2. 'Extra' shows EF-hand structure between domains II and III as reported in Ref. 20. Scores are an identity of sequences to EL--LL--LO-O-OG-LO--OL--LL--L sequencially, where E, L, O, G and - stand for glutamic acid, l e u c i n e / v a l i n e / i s o l e u c i n e / p h e n y l a l a n i n e / m e t h i o n i n e , aspartic a c i d / a s p a r a g i n e / g l u t a m i c a c i d / g l u t a m i n e / s e r i n e / t h r e o n i n e , glycine and any amino-acid residue, respectively. - stands for the sequence corresponding to that region does not exist or has not yet been determined. Bold n u m b e r s indicate that the positions experimentally proved to bind calcium ions previously [35]. EF-hand Structures
1
2
3
4
Extra
Total
Human Rat
13 13
11 11
10 10
11 11
12 12
45 45
m-calpain Large subunits Human Rabbit Chicken
12 12 11
11 11 11
11 11 11
13 13 11
9 10 10
47 47 45
/x-calpain Large subunits Human Rabbit
12 12
12 12
11 11
13 13
11 -
48 48
S.Mansoni
12
11
11
8
13
42
Calpain Small subunits Human Porcine Rabbit Bovine Rat
12 12 12 12 12
10 10 10 10 10
12 11 11 11 12
11 11 11 11 11
45 44 44 44 45
pMP41 Mouse
12
12
10
11
45
Average
12.1
10.9
10.9
11.4
p94
X-calpain
11.0
45.4
61 Since domain IV of p94 shows the same level of similarity among the three types, p94 should have calcium binding activity. Moreover, as shown in Table IV, EF-hand scores [31] of p94 domain IV are comparable to those of conventional calpain large and small subunits. It is, however, difficult to predict the calcium sensitivity of p94 from the scores, since m- and/z-types have a different calcium sensitivity, regardless of similar EF-hand scores. As pointed out in Ref. 20, the calpain large subunit contains an extra EF-hand structure at the boundary of domains II and III. The physiological meaning of the extra EF-hand structure is unknown. It is possible that the region folds near the domain IV and functions as an extra calcium-binding site.
Calpain large subunit family As described above, intra-mammal comparison showed clear distinction among three types of the calpain large subunit family, i.e., p94, m- and /z-type. No ambiguity exists in the classification of the three types. Chicken m-type is exceptional, since total similarity of chicken m-type to human m-type is a little lower than that to human /z-type. Newly identified chicken /z-type is more similar to human /z-type than human m-type and human /z-type is more similar to chicken /z-type than chicken m-type. Chicken /z-type is, therefore, /z-type and chicken m-type is m-type. A c D N A encoded protein isolated from Schistosoma mansoni is equally similar to the three types of calpain large subunits, p94, m- and /z-types. Evolutionary correlation among the 3 types of the large subunit will be clarified when further comparative studies on calpain from various animals are performed.
Evolutionary process of calpain molecules Since p94, m- and /z-types together with Schistosoma calpain are composed of identical domain structures, genes of these four proteins were derived from the same ancestral gene. The ancestral gene consists of genes for the cysteine proteinase module, calcium binding module and at least two unknown modules. Evolutionary distance between p94 and m- or/z-type is slightly further than that between m- and /z-types, judging from the sequence similarity, while Schistososoma calpain is equally distant from p94, m- and /ztypes (see Table I). Instead, domain IV of p94 is nearer to small subunit than that of m- and /z-types. A summary of these investigation is shown in Fig. 4. Evolutionarily closer relationship of p94 and the small subunit might correlate with the subunit structure of p94. As discussed above, it is not clear, according to present knowledge, if Schistosoma calpain is the ancestral molecule or not. The domain structure, however, is still identical to that of vertebrates, strongly suggesting that this structure is important for the function a n d / o r
Fig. 4. Evolutionaryrelationship among calpain subunits. Mm, M/~, p94, and Sm stand for mammalian m-type, mammalian ~-type, mammalian p94 and Schistosoma calpain, respectively. The length between each subunit represents qualitatively the evolutionary distance and the values near the connected bars are total sequence similarities between those two types.
stability of the calpain large subunit. To elucidate these problems it is essential to study p94 at the protein level. Further studies are now in progress to clarify the structural and functional characteristics of p94, which will also reveal evolutionary relationship among the members of the calpain large subunit family.
References 1 2 3 4 5 6 7
Suzuki, K. (1991) Biomed. Biochim. Acta 50, 483-484. Suzuki, K. (1987) Trends Biochem. Sci. 8, 167-169. Suzuki, K. and Ohno, S. (1990) Cell Structure Function 15, 1-6. Murachi, T. (1989) Biochem. Int. 18, 263-194. Mellgren, R.L. (1987) FASEB J. l, 110-115. Johnson, P. (1990) Int. J. Biochem. 22, 811-822. Wang, K.K.W., Villalobo, A. and Roufogalis, B.D. (1989) Biochem. J, 262, 693-706. 8 Ohno, S., Emori, Y., Imajoh, S., Kawasaki, H., Kisaragi, M. and Suzuki, K. (1984) Nature 312, 566-570. 9 Emori, Y., Kawasaki, H., Sugihara, H., Imajoh, S., Kawashima, S. and Suzuki, K. (1986) J. Biol. Chem. 261, 9465-9471. 10 Emori, Y., Kawasaki, H., Imajoh, S., Kawashima, S. and Suzuki, K. (1986) J. Biol. Chem. 261, 9472-9476. 11 Imajoh, S., Aoki, K., Ohno S., Emori, Y., Kawasaki, H., Sugihara, H. and Suzuki, K. (1988) Biochemistry27, 8122-8128. 12 Aoki, K., Imajoh, S., Ohno, S., Emori, Y. Koike, M., Kosaki, G. and Suzuki, K. (1986) FEBS Lett. 205, 313-317. 13 Ohno, S., Emori, Y. and Suzuki, K. (1986) Nucleic Acids Res. 14, 5559. 14 Sakihama, T., Kakidani, H., Zenita, K., Yumoto, N., Kikuchi, T., Sasaki, T., Kannagi, R., Nakanishi, S., Ohmori, M., Takio, K., Titani, K. and Murachi, T. (1985) Proc. Natl. Acad. Sci. USA 82, 6075-6079. 15 lmajoh, S., Kawasaki, H. and Suzuki, K. (1986) J. Biochem. 99, 1281-1284. 16 Pontremoli, S., Melloni, E., Sparatore, B., Michetti, M., Salamino, F. and Horecker, B.L. (1990) J. Biol. Chem. 265, 706-712. 17 Kishimoto, A., Mikawa, K., Hashimoto, K., Yasuda, I., Tanaka, S., Tominaga, M., Kuroda, T. and Nishizuka, Y. (1989) J. Biol. Chem. 264, 4088-4092.
62 18 Hirai, S., Kawasaki, H., Yaniv, M. and Suzuki, K. (1991) FEBS Lett. 287, 57-61. 19 Sorimachi, H., Imajoh-Ohmi, S., Emori, Y., Kawasaki, H., Ohno, S., Minami, Y. and Suzuki, K. (1989) J. Biol. Chem. 264, 2010620111. 20 Andresen, K., Tom, T.D. and Strand, M. (1991) J. Biol. Chem. 266, 15085-15090. 21 Murakami, T., Ueda, M., Hamakubo, T. and Murachi, T. (1988) J. Biochem. 103, 168-171. 22 Sorimachi, H., Ohmi, S., Emori, Y., Kawasaki, H., Saido, T.C., Ohno, S., Minami, Y. and Suzuki, K. (1990) Biol. Chem. HoppeSeyler 371, 171-176. 23 Wolfe, F.H., Sathe, S.K., Goll, D.E., Kleese, W.C., Edmunds, T. and Duperret, S.M. (1989) Biochim. Biophys. Acta 998, 236-250. 24 Miyake, S., Emori, Y. and Suzuki, K. (1986) Nucleic Acids Res. 14, 8805-8817. 25 Emori, Y., Ohno, S., Tobita, M. and Suzuki, K. (1986) FEBS Lett. 194, 249-252. 26 Imajoh, S., Kawasaki, H. and Suzuki, K. (1986) J. Biochem. 100, 633-642. 27 Kawashima, S., Inomata, M. and Imahori, K. (1986) Biomed. Res. 7, 327-331.
28 Ohno, S., Akita, Y., Hata, A., Osada, S., Kubo, K., Konno, Y., Akimoto, K., Mizuno, K., Saido, T., Kuroki, T. and Suzuki, K. (1991) Adv. Enzym. Regul. 31,287-303. 29 Emori, Y., Homma, Y., Sorimachi, H., Kawasaki, H., Nakanishi, O., Suzuki, K. and Takenawa, T. (1989) J. Biol. Chem. 264, 21885-21890. 30 Vogt, P.K. and Bos, T.J. (1990) Adv. Cancer Res. 55, 1-35. 31 Kretsinger, R.H. and Nockolds, C.E. (1973) J. Biol. Chem. 248, 3313-3326. 32 Sakihama, T., Kakidani, H., Zenita, K., Yumoto, N., Kikuchi, T., Sasaki, T., Kannagi, R., Nakanishi, S., Ohmori, M., Takio, K., Titani, K. and Murachi, T. (1985) Proc. Natl. Acad. Sci. USA 82, 6075-6079. 33 McClelland, P., Lash, J.A. and Hathaway, D.R. (1989) J. Biol. Chem. 264, 17428-17431. 34 Kageyama, H. Shimizu, M., Tokunaga, K., Hiwasa, T., and Sakiyama, S. (1989) Biochim. Biophys. Acta 1008, 225-257. 35 Minami, Y., Emori, Y., Kawasaki, H. and Suzuki, K. (1987) J. Biochem. 101, 889-895.
