Characterization of the sensitivity of AplCCal1 activity to N-terminal cleavage and calpain inhibitors.

<p>(A) AplCCal1 (200ng/ul) was pre-incubated for 30min with or without Ca²⁺ before addition of casein substrate (205ng/ul) with or without Ca²⁺, then incubated 30min before 33ul of the reaction was subjected to gel electrophoresis and Coomassie staining. (i) Schematic of experimental design. (ii) Representative Coomassie stained gel. (iii) Quantification of combined upper and lower casein bands, expressed as a proportion of a control that was never exposed to Ca²⁺. A one-tailed t-test for paired samples yielded p<0.05, represented by an asterisk (*). Data from 3 independent experiments. Error bars show SEM. (B) After a 20min preincubation with 100uM PD-150606, ALLM or vehicle, AplCCal1alt (325ng/ul) or porcine CAPN-1 (116ng/ul) and casein (195ng/ul) were incubated in the presence or absence of 5mM Ca2+, and in the presence or absence of 100uM PD-150606, ALLM or vehicle for one hour at room temperature. Thirty-two microliters of each reaction was subjected to SDS-PAGE and Coomassie staining. AplCCal1alt was used at higher concentrations than porcine calp-1 to compensate for its lower activity against casein.</p

Novel calpain families and novel mechanisms for calpain regulation in <i>Aplysia</i>

<div><p>Calpains are a family of intracellular proteases defined by a conserved protease domain. In the marine mollusk <i>Aplysia californica</i>, calpains are important for the induction of long-term synaptic plasticity and memory, at least in part by cleaving protein kinase Cs (PKCs) into constitutively active kinases, termed protein kinase Ms (PKMs). We identify 14 genes encoding calpains in <i>Aplysia</i> using bioinformatics, including at least one member of each of the four major calpain families into which metazoan calpains are generally classified, as well as additional truncated and atypical calpains. Six classical calpains containing a penta-EF-hand (PEF) domain are present in <i>Aplysia</i>. Phylogenetic analysis determined that these six calpains come from three separate classical calpain families. One of the classical calpains in <i>Aplysia</i>, AplCCal1, has been implicated in plasticity. We identify three splice cassettes and an alternative transcriptional start site in AplCCal1. We characterize several of the possible isoforms of AplCCal1 <i>in vitro</i>, and demonstrate that AplCCal1 can cleave PKCs into PKMs in a calcium-dependent manner <i>in vitro</i>. We also find that AplCCal1 has a novel mechanism of auto-inactivation through N-terminal cleavage that is modulated through its alternative transcriptional start site.</p></div

Characterization of endogenous PKM in homogenates.

<p>Antibodies against the PKC Apl III C-terminus (A) and B) PDK phosphorylation site (B) were used to probe membranes containing ganglion homogenate. For comparison, the neighboring lane contains purified PKC Apl III that has been incubated with mammalian CAPN-1 to induce cleavage.</p

Variants of AplCCal1.

<p>(A) Schematic of the domain structure of the nervous system variants of AplCCal1. Splice inserts a, b, and c, and the alternative N-termini are indicated by shaded blocks. The known calpain domains are shown. The positions of the catalytic triad: cysteine(C), histidine (H) and asparagine (N) are shown. (B) Amino acid sequence of the AplCCal1 variants. Splice inserts a, b, and c, and the alternative N-termini are indicated. The catalytic residues are marked with stars. Domains (catalytic (shaded), C2L (outlined), PEF (outlined and shaded)) are shown in ovals. Note that the catalytic domain starts in the alternative N-terminal exons.</p

Phylogeny of calpain families.

<p>An overview of the analysis of all Calpain families is presented. The analysis is described in the methods (the plot is from the RAxML analysis). Numbers represent the percentage of trees containing this phylogeny. Regions of the tree are expanded in the figures noted. The vertebrate CCAL 13–14 family, CCAL C family and assorted CCALs are examined more fully later in the paper. The full tree can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186646#pone.0186646.s003" target="_blank">S2 Fig</a>.</p

Organisms used in Phylogenetic analysis.

<p>Organisms used in Phylogenetic analysis.</p

Phylogeny of Tra, Atypical and Truncated families.

<p>Species abbreviations and their phylogenetic classification and common name are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186646#pone.0186646.t001" target="_blank">Table 1</a>. All reference numbers for sequences are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186646#pone.0186646.s001" target="_blank">S1 Table</a>. The analysis is described in the methods (the plot is from the RAxML analysis). <i>Aplysia</i> calpains are in larger font and bolded as are bootstrap numbers referred to in the text that define families. When multiple families are present, the families are defined by the lines and the family name on the right. (A) Tra family, (B) Atypical and Truncated families (C) Truncated family L and associated Truncated and Classical calpains.</p

Autolysis of Aplysia AplCCal1 produces a stable cleavage product.

<p>Coomassie-stained gel representing (A) <i>Aplysia</i> AplCCal1b (80ng/ul) and (B) Porcine classical CAPN-1 (80ng/ul) incubated with casein (225ng/ul) for 30 min at various Ca²⁺ concentrations. Twenty-eight microliters of the reaction was loaded. (C) <i>Aplysia</i> AplCCal1a (290ng/ul) was incubated with or without Ca²⁺ for 30 min. Thirty-one microliters of the reaction was subjected to immunoblot with an antibody against the N-terminal His-tag, stripped and reprobed with an AplCCal1 C-terminal antibody. (D) AplCCal1a or AplCCal1alt (50ng/ul) was incubated in the presence or absence of Ca²⁺. At the indicated timepoints, 25ul of each reaction was removed and inactivated by addition of 5X Laemmli sample buffer. Samples were subjected to immunoblot with an antibody against the AplCCal1 C-terminus.</p

<i>Aplysia</i> calpains.

<p><i>Aplysia</i> calpains.</p

AplCCal1 autolyses <i>in vivo</i>.

<p>(A) Desheathed paired pleural-pedal ganglia were treated for 20min with 100uM ionomycin or vehicle and then homogenized and subjected to western blot with an antibody against the C-terminal of AplCCal1. (B) Quantification of data from 4 independent experiments as shown in (A). A one-tailed t-test for paired samples yielded p<0.05, represented by an asterisk (*). Error bars show SEM.</p