Naturally Occurring Carboxypeptidase A6 Mutations: Effect on Enzyme Function and Association with Epilepsy

Carboxypeptidase A6 (CPA6) is a member of the A/B subfamily of M14 metallocarboxypeptidases that is expressed in brain and many other tissues during development. Recently, two mutations in human CPA6 were associated with febrile seizures and/or temporal lobe epilepsy. In this study we screened for additional CPA6 mutations in patients with febrile seizures and focal epilepsy, which encompasses the temporal lobe epilepsy subtype. Mutations found from this analysis as well as CPA6 mutations reported in databases of single nucleotide polymorphisms were further screened by analysis of the modeled proCPA6 protein structure and the functional role of the mutated amino acid. The point mutations predicted to affect activity and/or protein folding were tested by expression of the mutant in HEK293 cells and analysis of the resulting CPA6 protein. Common polymorphisms in CPA6 were also included in this analysis. Several mutations resulted in reduced enzyme activity or CPA6 protein levels in the extracellular matrix. The mutants with reduced extracellular CPA6 protein levels showed normal levels of 50-kDa proCPA6 in the cell, and this could be converted into 37-kDa CPA6 by trypsin, suggesting that protein folding was not greatly affected by the mutations. Interestingly, three of the mutations that reduced extracellular CPA6 protein levels were found in patients with epilepsy. Taken together, these results provide further evidence for the involvement of CPA6 mutations in human epilepsy and reveal additional rare mutations that inactivate CPA6 and could, therefore, also be associated with epileptic phenotypes. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc

Increased <i>CPA6 </i>promoter methylation in focal epilepsy and in febrile seizures

Focal epilepsy (FE) is one of the most common forms of adult epilepsy and is usually regarded as a multifactorial disorder. Febrile seizures (FS) often appear during childhood in a subtype of FE patients, i.e. with temporal lobe epilepsy (TLE) and hippocampal sclerosis (HS). FS are the most common human convulsive event associated with fever. Genetic evidences for FS have suggested a complex mode of inheritance. Until now, to investigate genes at the genomic level, linkage analysis of familial forms and association studies have been performed, but nothing conclusive has been clearly related to FE and FS. As complex disorders, environmental factors might play a crucial role through epigenetic modification of key candidate genes such as CPA6, which encodes Carboxypeptidase A6, an extracellular protein. Therefore, we assessed DNA methylation in promoter of CPA6. In 186 FE patients and 92 FS patients compared to 93 healthy controls and 42 treated controls with antiepileptic drugs (AEDs), we found significant higher levels of methylation for epileptic patients. Methylation status were 3.4% (±3.2%) for FE cases and 4.3% (±3.5%) for FS cases, whereas healthy individuals and treated controls with AEDs showed a level of 0.8% (±2.9%) and 1.5% (±3.9%), respectively (p≤0.001 for all comparisons). These results let growing evidence for DNA methylation involvment in FE and FS

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

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

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

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