Identification and characterization of two novel calpain large subunit genes
Introduction
Calpains are a family of cytosolic cysteine proteases. The ‘classical’ calpain proteases consist of an isoform-specific large subunit and an invariant small subunit (CAPN4). The crystal structure of the CAPN2 large subunit (Hosfield et al., 1999, Strobl et al., 2000) reveals four distinct domains (D-I to D-IV) in the large subunit. D-II consists of two subdomains, D-IIa and D-IIb, which are probably reoriented upon Ca2+ binding to assemble a cysteine protease active site. D-III is an eight-stranded β-sandwich that has some similarity to the Ca2+-binding C2 domain (Rizo and Südhof, 1998), while D-IV binds Ca2+ through EF-hand structural elements. Atypical mammalian large subunit calpain paralogues have been identified (Sorimachi and Suzuki, 2001), some of which contain alternatives to D-IV that lack EF-hand structures (CAPN5, CAPN6, CAPN7 and CAPN10; Dear et al., 1997, Braun et al., 1999, Franz et al., 1999). In the case of CAPN6, the protease domain possesses features suggesting it may be inactive (Dear et al., 1997).
Although a great deal of biochemical information has been accumulated on calpains, their physiological function is still unclear. Some insights have come from the analysis of inactivated calpain genes. Disruption of mouse Capn4 results in embryonic lethality (Arthur et al., 2000, Zimmerman et al., 2000) while its inactivation in 3T3 cells leads to transformation and tumorigenesis (Liu et al., 2000). Disruption of Capn1 alters platelet function (Azam et al., 2001). Inactivation of mouse Capn3, the human orthologue of which is mutated in human limb-girdle muscular dystrophy type 2A (Richard et al., 1995), results in a phenotype similar to the human disease and is associated with altered apoptosis and perturbation of the IκB/NF-κB pathway (Richard et al., 2000). Genetic variation in intron 3 of CAPN10 is associated with type 2 diabetes mellitus (Horikawa et al., 2000).
In order to characterize the function of calpains, it is important to identify all members of the family and determine how similar they are in structure and expression. This is particularly relevant when knockout mice are being generated and functional redundancy may occur due to the expression of other similar members of the gene family, thus complicating the resultant phenotype. With this aim in mind, we attempted to identify further calpain genes by searching the public databases. Two potential novel calpain large subunit genes were identified and further characterized. This brings the number of calpain large subunit calpain genes to 13. As no further calpain-like genes could be identified in the recently published near-complete public (Lander et al., 2001) and private (Venter et al., 2001) human genome sequences, this suggests that all members of the calpain large subunit gene family have now been identified.
Section snippets
Sequence analyses
DNA sequences were examined for homology with the non-redundant nucleotide and EST databases of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov) and the 3.5-fold-redundant Celera mouse genome database using the BLASTN algorithm (Altschul et al., 1990). DNA sequences were assembled into contigs using SeqMan of the DNASTAR suite of programs. Gene Construction Kit 2 (Textco, West Lebanon, NH) was used for graphic representation of gene structure. Multiple
Identification and structure of CAPN13 and CAPN14
To identify new calpain genes, searches were made of the NCBI high throughput genomic sequence (htgs) database with vertebrate calpain protein sequence. Two potential new members of the human calpain large subunit gene family were identified. The first of these, named CAPN13, was present in a partially sequenced BAC (GenBank Accession number: AC068689) while the second, named CAPN14, was present in a further partially sequenced BAC (GenBank Accession number: AC015980). Exons were predicted from
Acknowledgements
We thank Tanna Franz and Melanie Hunn for technical assistance.
References (27)
- et al.
Basic local alignment sequencing tool
J. Mol. Biol.
(1990) - et al.
CAPN 8: isolation of a new mouse calpain-isoenzyme
Biochem. Biophys. Res. Commun.
(1999) - et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction
Anal. Biochem.
(1987) - et al.
A new subfamily of vertebrate calpains lacking a calmodulin-like domain: implications for calpain regulation and evolution
Genomics
(1997) - et al.
CAPN11: a calpain with high mRNA levels in testis and located on chromosome 6
Genomics
(1999) - et al.
Gene structure, chromosomal localization and expression pattern of Capn12, a new member of the calpain large subunit gene family
Genomics
(2000) - et al.
SOLH, a human homologue of the Drosophila melanogaster small optic lobes gene is a member of the calpain and zinc-finger gene families and maps to human chromosome 16p13.3 near CATM (cataract with microphthalmia)
Genomics
(1998) - et al.
Antisense RNA-mediated deficiency of the calpain protease, nCL-4, in NIH3T3 cells is associated with neoplastic transformation and tumorigenesis
J. Biol. Chem.
(2000) - et al.
Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A
Cell
(1995) - et al.
C2-domains, structure and function of a universal Ca2+-binding domain
J. Biol. Chem.
(1998)
Disruption of the murine calpain small subunit gene. Capn4: calpain is essential for embryonic development but not for cell growth and division
Mol. Cell. Biol.
Disruption of the mouse μ-calpain gene reveals an essential role in platelet function
Mol. Cell. Biol.
Structure of a Ca2+-binding domain reveals a novel EF-hand and Ca2+-induced conformational changes
Nat. Struct. Biol.
Cited by (69)
Mitochondrial localization of calpain-13 in mouse brain
2022, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Calpain-13 has domain structures similar to conventional calpains, such as calpain-1, and is classified as a classical calpain. It shares approximately 36% homology with calpain-14 [19], which is tissue-specific and is mainly expressed in the esophagus. Pathogenic genetic abnormality of CAPN14 causes eosinophilic esophagitis.
Calpains for dummies: What you need to know about the calpain family
2021, Biochimica et Biophysica Acta - Proteins and ProteomicsA study on the association of TCF7L2 rs11196205 (C/G) and CAPN10 rs3792267 (G/A) polymorphisms with type 2 diabetes mellitus in the South Western of Iran
2018, Egyptian Journal of Medical Human GeneticsEosinophilic Esophagitis: A Primary Disease of the Esophageal Mucosa
2017, Journal of Allergy and Clinical Immunology: In PracticeCalpain-14 and its association with eosinophilic esophagitis
2017, Journal of Allergy and Clinical ImmunologyAn eccentric calpain, CAPN3/p94/calpain-3
2016, Biochimie