M-Calpain is required for preimplantation embryonic development in mice

Previn Dutt,Karen Williams,Teresa De Veyra,Peter A Greer,J Simon C Arthur,John S Elce,Dorothy E Croall

doi:10.1186/1471-213x-6-3

Abstract

Backgroundμ-calpain and m-calpain are ubiquitously expressed proteases implicated in cellular migration, cell cycle progression, degenerative processes and cell death. These heterodimeric enzymes are composed of distinct catalytic subunits, encoded by Capn1 (μ-calpain) or Capn2 (m-calpain), and a common regulatory subunit encoded by Capn4. Disruption of the mouse Capn4 gene abolished both μ-calpain and m-calpain activity, and resulted in embryonic lethality, thereby suggesting essential roles for one or both of these enzymes during mammalian embryogenesis. Disruption of the Capn1 gene produced viable, fertile mice implying that either m-calpain could compensate for the loss of μ-calpain, or that the loss of m-calpain was responsible for death of Capn4-/- mice.ResultsTo distinguish between the alternatives described above, we deleted an essential coding region in the mouse Capn2 gene in embryonic stems cells and transmitted this mutant allele through the mouse germline. Breeding of heterozygous animals failed to produce homozygous mutant live offspring or implanted embryos. A nested PCR genotyping protocol was established, and homozygous preimplantation mutant embryos were detected at the morula but not at the blastocyts stage.ConclusionWe conclude that homozygous disruption of the Capn2 gene results in pre-implantation embryonic lethality between the morula and blastocyst stage. This establishes that μ-calpain and m-calpain have distinct functions, and that m-calpain is vital for development of the preimplantation murine embryo.

Highlights

The two ubiquitous Ca2+-dependent, cysteine proteases known as μ-calpain and m-calpain, are the founding members of a gene family comprising 13 genes in mammals [1,2,3]
We report here that Capn2 null embryos died prior to the implantation stage, indicating that m-calpain is indispensable for early embryogenesis
Two independent laboratories observed embryonic lethality in Capn4 knockout mice, albeit at different stages of development [31,32]. These observations supported the hypothesis that the small subunit is required for both μ- and m-calpain, and suggested four possibilities regarding their requirement for embryonic development: 1) both isoforms were required; 2) μ-calpain was required; 3) m-calpain was required, or 4) μ- and m-calpain are redundant, and one or the other isoform was required

Summary

Introduction

The two ubiquitous Ca2+-dependent, cysteine proteases known as μ-calpain (calpain-1) and m-calpain (capain-2), are the founding members of a gene family comprising 13 genes in mammals [1,2,3] Both are heterodimeric enzymes consisting of distinct 80 kDa catalytic subunits, encoded (page number not for citation purposes). The μ-80 k and m-80 k subunits share 62% amino acid sequence identity, and are very similar in terms of structure, protein chemistry, and in vitro substrate specificity. Despite these similarities, the differential expression patterns of μ- and m-calpain in mammalian tissues suggest they have some isoform specific and distinct functions. Since the cytoplasmic free Ca2+ concentration is typically less than 1 μM, it is assumed that other in vivo factors must contribute to regulation of these enzymes [3]

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