Disruption of the Murine Calpain Small Subunit Gene, Capn4: Calpain Is Essential for Embryonic Development but Not for Cell Growth and Division

Calpains are a family of Ca(2+)-dependent intracellular cysteine proteases, including the ubiquitously expressed μ- and m-calpains. Both μ- and m-calpains are heterodimers, consisting of a distinct large 80-kDa catalytic subunit, encoded by the genes Capn1 and Capn2, and a common small 28-kDa regulatory subunit (Capn4). The physiological roles and possible functional distinctions of μ- and m-calpains remain unclear, but suggested functions include participation in cell division and migration, integrin-mediated signal transduction, apoptosis, and regulation of cellular control proteins such as cyclin D1 and p53. Homozygous disruption of murine Capn4 eliminated both μ- and m-calpain activities, but this did not affect survival and proliferation of cultured embryonic stem cells or embryonic fibroblasts, or the early stages of organogenesis. However, mutant embryos died at midgestation and displayed defects in the cardiovascular system, hemorrhaging, and accumulation of erythroid progenitors

Transgene-host Cell Interactions Mediate Significant Influences On The Production, Stability, And Function Of Recombinant Canine Fviii.

Recombinant FVIII manufacturing is characterized by poor product stability and low yields. Codon-optimization of transgenes accelerates translation by exploiting the synonymous codon usage bias of a species. However, this can alter the performance of the final product. Additionally, the effects of transgene design across diverse cell types are not well understood and are of interest for next-generation protein and gene therapies. To investigate the effects of transgene design across different host cells, B-domain-deleted (BDD) and modified codon-optimized (CO-N6) transgenes were inserted via lentiviral delivery into cBOECs, HEK293T, and MDCK cells. The CO-N6 cFVIII transgene produced threefold more protein per transgene in HEK293T cells, and sixfold more protein in the two canine cell lines. However, pharmacokinetic analysis in hemophilia A dogs demonstrated that cFVIII produced from cBOECs transduced with the CO-N6 transgene had significantly reduced in vivo recovery. Furthermore, this product showed reduced in vitro stability and activity on thrombin activation versus the BDD product. This trend was reversed in HEK293T lines. Overall, our results demonstrate the need for an integrated approach that not only assesses protein expression levels but also considers the influence that host-cells have on preserving the molecular and biochemical properties of the naturally occurring FVIII

Crystallization and X-ray crystallographic analysis of m-calpain, a Ca²⁺-dependent protease

The absolute requirement of Ca2+ for proteolytic activity is a feature unique to the calpains, a family of heterodimeric cysteine proteases. Conditions are described which give rise to diffraction-quality crystals of m-calpain in two crystal forms, P1 and P21. Data have been collected from native crystals of m-calpain in both P1 and P21 forms, to 2.6 and 2.15 Å, respectively. Selenomethionine-containing crystals have been grown in both forms, and anomalous data from the P21 selenomethionine enzyme provided the location of 17 of the 19 Se atoms in the protein.</jats:p