Sequence chromatograms of <i>CPA6</i> in two patients showing c.107G>A (Arg36His) and c.812A>G (Asn271Ser).

<p>Patients with juvenile myoclonic epilepsy were sequenced to reveal the location of mutations in the <i>CPA6</i> gene. (A) Patient EMJ18 showed heterozygosity at base pair 107, as indicated by the presence of both an adenine and guanine peak at the same position (indicated as R). (B) Patient EMJ52 showed overlapping peaks for adenine and guanine at position 812.</p

Co-expression of WT and mutant proCPA6 in HEK293T cells.

<p>HAH6-tagged or FLAG-tagged wild-type (WT) and HAH6-tagged mutant proCPA6 were co-expressed in HEK293T cells by transient transfection and ECM was analyzed. (A) Enzyme activity of CPA6 in the ECM was assayed using 3-(2-furyl)-acryloyl-Phe-Phe. In co-expression experiments, approximately 55–65% activity was seen when WT-CPA6-FLAG was coexpressed with either of the two inactive mutants (Asn271Ser and Glu398Gln), with the Arg36His mutant, or with empty pcDNA vector; the difference in means between these 4 groups was not statistically significant. (B) HA-tag immunoreactivity in the ECM, measured by western blotting, showed a 50% reduction for Arg36His-CPA6 while levels were unchanged for Asn271Ser and Glu398Gln mutants. Error bars show standard error of the mean in panels A (n≥7) and B (n≥4). **, p<0.01.</p

Novel Carboxypeptidase A6 (<i>CPA6</i>) Mutations Identified in Patients with Juvenile Myoclonic and Generalized Epilepsy

<div><p>Carboxypeptidase A6 (CPA6) is a peptidase that removes C-terminal hydrophobic amino acids from peptides and proteins. The <i>CPA6</i> gene is expressed in the brains of humans and animals, with high levels of expression during development. It is translated with a prodomain (as proCPA6), which is removed before secretion. The active form of CPA6 binds tightly to the extracellular matrix (ECM) where it is thought to function in the processing of peptides and proteins. Mutations in the <i>CPA6</i> gene have been identified in patients with temporal lobe epilepsy and febrile seizures. In the present study, we screened for <i>CPA6</i> mutations in patients with juvenile myoclonic epilepsy and identified two novel missense mutations: Arg36His and Asn271Ser. Patients harboring these mutations also presented with generalized epilepsy. Neither of the novel mutations was found in a control population. Asn271 is highly conserved in CPA6 and other related metallocarboxypeptidases. Arg36 is present in the prodomain and is not highly conserved. To assess structural consequences of the amino acid substitutions, both mutants were modeled within the predicted structure of the enzyme. To examine the effects of these mutations on enzyme expression and activity, we expressed the mutated enzymes in human embryonic kidney 293T cells. These analyses revealed that Asn271Ser abolished enzymatic activity, while Arg36His led to a ~50% reduction in CPA6 levels in the ECM. Pulse-chase using radio-labeled amino acids was performed to follow secretion. Newly-synthesized CPA6 appeared in the ECM with peak levels between 2-8 hours. There was no major difference in time course between wild-type and mutant forms, although the amount of radiolabeled CPA6 in the ECM was lower for the mutants. Our experiments demonstrate that these mutations in <i>CPA6</i> are deleterious and provide further evidence for the involvement of <i>CPA6</i> mutations in the predisposition for several types of epilepsy.</p></div

<i>CPA6</i> mutations in JME patients.

<p><i>CPA6</i> mutations in JME patients.</p

Demographic and clinical description of JME patients and Caucasian controls.

<p>SD: Standard deviation. NA: number of patients for whom no information is available.</p><p>Demographic and clinical description of JME patients and Caucasian controls.</p

Pulse-chase analysis of HEK293T cells transfected with WT or mutant proCPA6.

<p>Cells were labeled with ³⁵S methionine and cysteine for 20 minutes (pulse), washed once, and incubated for the time indicated (chase) in media containing non-radioactive methionine and cysteine. ECM was extracted with SDS and analyzed on a denaturing polyacrylamide gel, which was dried and exposed to film for 7–10 days. CPA6, which runs ~37 kDa, was not detected in the cells transfected with pcDNA vector alone. Arg36His samples showed reduced levels of CPA6 relative to WT at all time points, which was statistically significant at all time points except 24h. Asn271Ser samples showed a trend towards reduced levels of CPA6 at all time points, with significant reductions in CPA6 levels at the 4 and 8 hour time points. Comparisons were performed across CPA6 variants for each time point; ns, not significant; *, p<0.05; **, p<0.01 relative to the same time point for the WT sample. Error bars show standard error of the mean (n = 3).</p

Alignments of Arg36 and Asn271 residues in proCPA6 and related enzymes.

<p>(A) Human <i>CPA6</i> is translated as a 437 amino acid protein that includes a signal peptide (SP) and a prodomain (Pro). The prodomain, containing Arg36His, acts as an intramolecular chaperone and also maintains the enzyme in an inactive state. The mature enzyme is secreted, and contains only the carboxypeptidase (CP) domain. Carboxypeptidases in the M14 family have several conserved residues, often referred to by their position in the classical numbering system (italics, top numbers), which is based on the active form of CPA1. The residue number in the preproCPA6 sequence are also indicated (bottom numbers), which begins with the translation-initiating methionine. Several critical active site residues are shown. These residues are important for coordination of the zinc (<i>His69</i> = His196, <i>Glu71</i> = Glu199, <i>His196</i> = His324); substrate binding (<i>Arg127</i> = Arg254, <i>Asn144</i> = Asn271, <i>Arg145</i> = Arg272); and catalysis (<i>Glu270</i> = Glu398). (B) Clustal Omega was used for multiple sequence alignments to analyze mutated residues. Arg36 is conserved in most proCPA6 orthologs, with the exception of several primates, which have a cysteine at this residue. (C) Asn271 and its neighboring residues are 100% conserved from humans to zebrafish. (D) Across CPA-like family members, Arg36 is not conserved, and is in a region with few conserved residues. (E) Asn271 is conserved in every CPA-like enzyme in humans, as are several neighboring residues.</p

Modeling of Arg36His and Asn271Ser mutations within proCPA6 protein structure.

<p>The structure of proCPA6 was modeled based on the crystal structure of related family members as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123180#pone.0123180.ref030" target="_blank">30</a>]. PreProCPA6 contains a signal peptide, which is removed during synthesis of the protein. Once the signal peptide is removed, Arg36 (green sticks) is one of the most N-terminal residues of the proenzyme, and is located within the prodomain (blue beta sheets, yellow loops, red alpha helices). Arg36 is part of a loop with no known function. Its charge is outward-facing, and not predicted to interact with nearby residues. Asn271 (green sticks) is located within the active site of the enzyme. It is known as <i>Asn144</i> in the classical numbering system and is a critical active site residue involved in substrate binding. It faces inwards towards the other residues that participate in substrate binding (blue sticks), zinc-coordination (white sticks) and catalysis (white sticks).</p

Biochemical analyses of Arg36His and Asn271Ser mutant proCPA6.

<p>Wild-type (WT) and mutant proCPA6 tagged on the C-terminus with the HA epitope and His6 sequence (HAH6) were expressed in HEK293T cells by transient transfection. (A) Catalytic activity of ECM-bound CPA6 was assayed with the substrate 3-(2-furyl)-acryloyl-Phe-Phe. The Arg36His mutant proCPA6 produced CPA6 with approximately 50% activity relative to WT, while the Asn271Ser mutant proCPA6 had no detectable activity. Empty vector (pcDNA 3.1) and Glu398Gln proCPA6, an active site mutation, were included as negative controls. (B,C) Western blot analysis of CPA6 in cells and ECM, detected using an anti-HA antibody. All proCPA6 constructs express at equal levels in the cells. Alpha-tubulin immunoreactivity was used as a loading control. ECM levels of CPA6 produced from mutant Arg36His-proCPA6 were ~50% of WT while Asn271Ser CPA6 was expressed at ~75% of WT. The addition of heparin greatly reduced the level of CPA6 in the ECM. (D,E) Analysis of media. Under basal conditions, the level of CPA6 in medium was low, and this was increased by the presence of 400 μg/ml heparin in the culture medium. Levels of the Arg36His and Asn271Ser forms were reduced in the heparin-treated media relative to WT. Abbreviations: ND, not detectable; ns, not significant; *, p<0.05; **, p<0.01 relative to the WT control. Error bars indicate standard error of the mean in panels A (n = 10), C (n≥7), and E (n = 7).</p

Kaplan-Meier plots illustrating time to withdrawal for any cause for the groups of convulsive and non-convulsive seizures.

<p>Kaplan-Meier plots illustrating time to withdrawal for any cause for the groups of convulsive and non-convulsive seizures.</p