Identification and characterization of two novel calpain large subunit genes

Abstract

Calpains are a family of related proteins, some of which have been shown to function as calcium-dependent cysteine proteases. CAPN1 and CAPN2, the most well characterized calpains, consist of a large (80 kDa) and a small (30 kDa) subunit. In mammals, 11 different paralogous genes encoding calpain large subunits have been identified. We report the identification of two further genes, CAPN13 and CAPN14, potentially encoding calpain large subunits. Radiation hybrid mapping localized both genes within a region mapped to 2p21-2p22. The CAPN13 mRNA exhibits a restricted tissue distribution with low levels of expression detected only in human testis and lung while CAPN14 mRNA could not be detected in any of the 76 tissues examined. Examination of the human genome sequence in the public and private consortia databases did not detect any further members of this gene family. Thus, there would seem to be 13 large subunit calpain genes in the human genome. Phylogenetic analysis reveals that the putative calpain large subunit proteins can be divided into three major groups. The 13 human large subunit genes and the single small subunit gene are located in eight syntenic groups on chromosomes 1, 2, 3, 6, 11, 15, 19 and X.

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.