Humans have 15 calpain genes (CAPN1-3, 5-16) as well as two genes for regulatory subunits (CAPNS1[30K] and CAPNS2[30K-2] encoded by CAPNS1 and CAPNS2, respectively) and one for a specific inhibitor protein (calpastatin encoded by CAST) (Fig. 4 and Table 2). The longest gene products of the 15 human calpain genes are classified according to their structures or tissue distribution (see Classification of calpains).
Fig. 4 Phylogenetic tree and schematic structures of human calpains and related molecules
(A) Phylogenetic tree of human calpains. The tree was drawn using the neighbor-joining/bootstrap method after aligning all the sequences using MAFFT v6.420 (strategy: E-INS-i; see Fig. 5 for alignment). The non-classical calpains are divided into three subfamilies, one of which, the PalB subfamily, shows further divergence into the strict PalB, TRA-3, and CAPN10 groups. (B) Schematic structures. Black and highlighted letters indicate ubiquitous and tissue/organ-specific calpains, respectively (see Table 1).
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
GR: glycine-rich hydrophobic domain
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
XL: N-terminal extended domain of calpastatin
L: N-terminal domain of calpastatin
Amino acid sequences of full-length CAPN1, 2, 3, 5, 6, 8, 9, 10, 11, 12, 13, 14, and parts of CAPN7 (181 to C-term), 15 (441 to C-term), 16 (31 to 647), and CAPNS1 (33 to C-term) and S2 (13 to C-term) were aligned using MAFFT v6.420(strategy: E-INS-i). The alignments around IS1 and IS2 were slightly adjusted by eyes considering the exon-intron junctions of CAPN1 and CAPN3. The positions of intron insertion are indicated by pink triangles (i+numbers correspond to the CAPN1 introns). Red and yellow indicate residues conserved in all in more than half of the sequences, respectively. Yellow triangles: active site residues. Blue asterisks: residues involved in Ca2+-binding of the protease domain of human CAPN1[μCL]. Thin, medium thick, and thick vertical lines indicate every 10, 50, and 100 residues of human CAPN1[μCL]. Arrow heads indicate domain/region boundaries. Sequences: CAPN1[μCL], NP_005177; CAPN2[mCL], NP_001739; CAPN3[p94], NP_000061; CAPN5[hTRA-3], NP_004046; CAPN6, NP_055104; CAPN7[PalBH], NP_055111; CAPN8[nCL-2], NP_001137434; CAPN9[nCL-4], NP_006606; CAPN10, NP_075571; CAPN11, NP_008989; CAPN12, NP_653292; CAPN13, NP_653176; CAPN14, NP_001138594; CAPN15[SOLH], NP_005623; CAPN16[demi-calpain], EAW47827; CAPNS1[30K], NP_001003962; CAPNS2[30K-2], NP_115706.