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Classification of calpains
Structural classification

The calpain superfamily is divided into several subfamilies according to the domain structure. Calpains with a domain structure similar to that of CAPN1[µCL] and CAPN2[mCL], i.e., containing CBSW and PEF domains in addition to the CysPc domain, are termed “classical” calpains (Croall and Ersfeld, 2007) (Fig. 2). Historically, these calpains have also been called “typical” calpains, but “typical” could be misleading, because they are only distributed among vertebrates, insects, and schistosomes (see Table 1). Accordingly, “non-classical” calpains (formerly called “atypical” calpains) do not contain both CBSW and PEF domains. Of the 15 representative products of human calpain genes, nine are classical and six are non-classical (see Human calpains).

fig1

Fig. 2 Structural classification and consensus domain structures of calpain super family members

Calpain homologs have been identified in almost all eukaryotes and a few bacteria. Their structures are classified as described in the text. The consensus domain structures for their classification are shown.


Abbreviations used for domain names:
CysPc: calpain-like cysteine protease sequence motif defined in the conserved domain database at the National Center for Biotechnology Information (cd00044), which is composed of two protease core domains 1 and 2 (PC1 and PC2)
PC1: protease core domain 1
PC2: protease core domain 2
CBSW: calpain-type beta-sandwich domain (formerly called "C2-domain-like (C2L)" domain)
PEF: penta-EF-hand domain
GR: glycine-rich hydrophobic domain
MIT: microtubule interacting and transport motif
C2: C2 domain
Zn: Zn-finger motif
SOH: SOL-homology domain
TM: transmembrane domain

Most of the human classical calpains are conserved in other vertebrates (Table 1); fish have a duplicate set of most of these genes (Macqueen et al., 2010) . Only a few classical calpains have been identified in invertebrates: Schistosoma mansoni (blood fluke), Drosophila melanogaster (fruit fly), and Anopheles gambiae (mosquito) have four, three, and three, respectively (Sorimachi et al., 2011b). No classical calpain homologs have been found in Caenorhabditis elegans (nematode), trypanosomes, plants, fungi, or Saccharomyces cerevisiae (budding yeast) (Sorimachi et al., 2011b).

The non-classical calpain CysPc domains are 30-75% identical at the amino acid level and contain different domains. Some of these domains, such as the transmembrane (TM) domain, are not found in human calpains. The non-classical calpains probably function differently from classical calpains, and not all are Ca2+-dependent. These features, together with the organization of mammalian calpain genes, led to the hypothesis that calpain molecules were generated evolutionarily by combining an ancestral calpain-type cysteine protease gene with genes encoding other functions. The non-classical calpains can be further classified into several subfamilies according to their domain structures.

Human CAPN7[PalBH] is the most evolutionarily conserved human calpain, with homologs found in vertebrates, yeasts, fungi, protists, nematodes, and insects (Drosophila is an exception), but not in plants (Table 1) (Denison et al., 1995; Futai et al., 1999) . CAPN7[PalBH] homologs commonly contain two CBSW domains in tandem, each of which diverge (greatly or moderately) from those of conventional calpains, and conserved microtubule interacting and transport (MIT) motifs at the N-terminus (Fig. 2).

Human CAPN10, the longest product of the CAPN10 gene (Horikawa et al., 2000) , contains slightly divergent CBSW domains in succession at the C-terminus. CAPN10 homologs are only found in vertebrates, and do not contain an MIT domain (Croall and Ersfeld, 2007). TRA-3 is found in nematodes and vertebrates, but not in insects or lower organisms (Table 1) (Sorimachi et al., 2011b). TRA-3 homologs, including human CAPN5[hTRA-3] and CAPN6, contain CBSW and C2 domains in succession at the C-terminus (Fig. 2 and see Human calpains). Thus, CAPN10 and TRA-3 homologs can be grouped together with the CAPN7[PalBH] homologs within the PalB subfamily, all of which contain the structural consensus of two tandem CBSW and/or C2 domains at the C-terminus (Fig. 2).

Another evolutionarily conserved subfamily is SOL, which is found in all vertebrates, insects, nematodes, and green algae, but not in fungi or yeasts. The structure of SOL homologs is characterized by varying numbers of Zn-finger motifs within the N-terminal domain. They also share an SOL-homology (SOH) domain (Fig. 2 and see Human calpains).

Plant calpain, called phytocalpain, was first identified in Saccharum officinarum (sugarcane) in 2001 (Correa et al., 2001). The maize calpain homolog, DEK1 (defective kernel 1), was shown to be involved in aleurone cell development in 2002 (Lid et al., 2002) . DEK1 homologs are found in various plants, including rice plants and Arabidopsis. They contain TM and CBSW domains at their N- and C-termini, respectively. A DEK1 homolog is also found in Tetrahymena thermophila (Sorimachi et al., 2011b). Human CAPN10:ex1-8|9B|10[calpain-10b] and Drosophila CALPA’, which are alternative splicing gene products of CAPN10 and CalpA, respectively, and some nematode calpains have a domain structure similar to that of DEK1 (CysPc-CBSW-COOH), but lack the TM domain (Sorimachi et al., 2011b). These calpains are grouped together to form the DEK1 subfamily (Fig. 2).

Bacterial calpain was first identified in Porphyromonas gingivalis in 1992 and is known as "tpr" (thiol protease) (Bourgeau et al., 1992). Subsequent bacterial genome projects identified several other calpain homologs. These are the most divergent calpain species, sharing similarity only within the CysPc domain (see Other calpains).

Classification according to tissue specificity

In addition to their structural features, calpains are also independently categorized according to their tissue and organ distribution. Some human calpains are ubiquitously expressed, whereas others are expressed only in specific tissues or organs (Fig. 2 and see Human calpains).

Fig.3

Fig. 3 Classification of human calpains

Black and highlighted letters indicate ubiquitous and tissue/organ-specific calpains, respectively. See also Fig. 1.

Abbreviations used for domain names:
CysPc: calpain-like cysteine protease sequence motif defined in the conserved domain database at the National Center for Biotechnology Information (cd00044), which is composed of two protease core domains 1 and 2 (PC1 and PC2)
PC1: protease core domain 1
PC2: protease core domain 2
CBSW: calpain-type beta-sandwich domain (formerly called "C2-domain-like (C2L)" domain)
PEF(L): penta-EF-hand domain in the catalytic large subunit
PEF(S): penta-EF-hand domain in the regulatory small subunit
NS, IS1, IS2: CAPN3[p94]-characteristic sequences
MIT: microtubule interacting and transport motif, C2: C2 domain
Zn: Zn-finger motif
SOH: SOL-homology domain
IQ: a motif interactive with calmodulin

It is widely assumed that ubiquitous calpains play a fundamental role in all cells, whereas tissue-specific calpains, as the name suggests, have tissue-specific roles. Defects in ubiquitous calpains may be lethal (for example, Capn2-/- and Capns1-/- mice) (Arthur et al., 2000; Dutt et al., 2006; Kashiwagi et al., 2010; Tan et al., 2006; Zimmerman et al., 2000) , whereas defects in tissue-specific calpains may cause tissue-specific symptoms; for example, muscular dystrophy can be caused by a defective CAPN3 or Capn3 gene (Kramerova et al., 2004; Ojima et al., 2010; Richard et al., 2000; Richard et al., 1995) .

In addition, conventional calpains tend to be over-activated in muscular dystrophies, cardiomyopathies, traumatic ischemia, and lissencephaly, probably due to the compromised intracellular Ca2+ homeostasis caused by these diseases. Since over-activity often exacerbates the disease state, conventional calpain inhibitors are currently used to prevent the progression of such diseases (Yamada et al., 2009).


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