Calpain-14 and its association with eosinophilic esophagitis

Abstract

Calpains are a family of intracellular, calcium-dependent cysteine proteases involved in a variety of regulatory processes, including cytoskeletal dynamics, cell-cycle progression, signal transduction, gene expression, and apoptosis. These enzymes have been implicated in a number of disease processes, notably for this review involving eosinophilic tissue inflammation, such as eosinophilic esophagitis (EoE), a chronic inflammatory disorder triggered by allergic hypersensitivity to food and associated with genetic variants in calpain 14 (CAPN14). Herein we review the genetic, structural, and biochemical properties of CAPN14 and its gene product CAPN14, and its emerging role in patients with EoE. The CAPN14 gene is localized at chromosome 2p23.1-p21 and is most homologous to CAPN13 (36% sequence identity), which is located 365 kb downstream of CAPN14. Structurally, CAPN14 has classical calpain motifs, including a cysteine protease core. In comparison with other human calpains, CAPN14 has a unique expression pattern, with the highest levels in the upper gastrointestinal tract, particularly in the squamous epithelium of the esophagus. The CAPN14 gene is positioned in an epigenetic hotspot regulated by IL-13, a T_H2 cytokine with increased levels in patients with EoE that has been shown to be a mediator of the disease. CAPN14 induces disruptive effects on the esophageal epithelium by impairing epithelial barrier function in association with loss of desmoglein-1 expression and has a regulatory role in repairing epithelial changes induced by IL-13. Thus CAPN14 is a unique protease with distinct tissue-specific expression and function in patients with EoE and is a potential therapeutic target for EoE and related eosinophilic and allergic diseases.

Keywords: Calpains; IL-13; calpain-14; calpainopathy; desmoglein-1; enzymes; eosinophilic esophagitis; eosinophils; epithelial barrier; food allergy; genetics; limb-girdle muscular dystrophy; myositis.

FIG 1

A, The structure of the CAPN14 gene locus. Human CAPN14 (blue) is located at chromosome 2p23, according to RefSeq human genome alignment GRCh38/hg38, in juxtaposition to the polypeptide N-acetylgalactosaminyltransferase 14 (GALNT14) and encoding homology domain containing 3 (EHD3) genes. It has 26 exons (blue vertical lines). B, Phylogenetic tree of human calpains based on multiple sequence alignment by using Clustal Omega. Classical calpains are shown in blue. CAPN14 has the closest distance to CAPN13.

FIG 2

Hypothetical connection between the 2 defense mechanisms against esophageal damage: keratinization of esophagus versus esophageal expression of protecting molecules (CAPN14).

FIG 3

Expression of mRNA of calpain family members relative to GAPDH in EPC2 cells exposed to IL-13 for the indicated times. Eotaxin-3 (CCL26) is a positive control for IL-13 stimulation.

FIG 4

A, A 3-dimensional model of human CAPN14 built by using Phyre2 based on a human CAPN2 (m-calpain; Protein Databank ID: 1KFX) template. Major domains are labeled and colored differently: gray is the N-terminal anchor, blue is the PC1 domain, yellow is the PC2 domain, purple is the C2L domain, and green is the PEF domain. The residues constituting the putative catalytic triad and the Ca²⁺-binding sites are rendered with side chains and circled in red and cyan, respectively. Cartoon representation of secondary structures includes springs (α-helices), arrowed ribbons (β-strands), and irregular coils (unstructured loops). B, Schematic primary structure of human CAPN14. The amino acid sequences are highlighted in colors corresponding to the major domains, except for the catalytic triad (red) and calcium ion-binding residues (cyan). C, Proposed mechanism of CAPN14 function. The thiol of ionized Cys101 attacks the scissile bond carbonyl carbon while protonated N_D1 of the imidazole of His254 transfers the proton to the carbonyl oxygen of the substrate to form the covalent enzyme substrate tetrahedral intermediate. Decay of the tetrahedral intermediate results in cleavage of the scissile C-N bond in the substrate, releasing fragment 1, and subsequent hydrolysis of the remaining enzyme-protein thioester releases fragment 2.

FIG 5

Manhattan plot of P values obtained from the genome-wide association analysis. Data are from 736 patients with EoE and 9246 control subjects over 1,468,075 genetic variants, with minor allele frequencies of greater than 1% in the patients with EoE. The −log₁₀ of the probability is shown as a function of genomic position on the autosomes. Indicated are genome-wide significance (pink dashed line; P ≤ 5 × 10⁻⁸) and suggestive significance (solid blue line; P ≤ 1 × 10⁻⁷). The figure is modified from Kottyan et al with permission from Nature Genetics.

FIG 6

Transcriptional and epigenetic analysis of CAPN14 in esophageal epithelial cells. A, The most significantly associated SNP rs76562819 is located in the IL-13–stimulated acetylation region of histone 3 acetylated at lysine 27 (H3K27ac) over the transcription start site of CAPN14. Shown is an electrophoretic mobility shift assay of nuclear lysates from an esophageal epithelial cell line using oligonucleotides with the risk (G) and nonrisk (A) allele of rs76562819. B, Expression of CAPN genes in esophageal biopsy specimens. The heat map shows the relative expression of CAPN and CAST genes in the biopsy specimens taken from healthy control subjects (NL; 14 patients), patients with therapy-responsive EoE (remission; 18 patients), patients with active EoE (active; 18 patients), and patients with therapy-resistant EoE (resistant; 19 patients). Yellow and blue represent increased and decreased expression, respectively. C, Quantification of CAPN14 protein in control and active EoE biopsy specimens taken from 8 patients. Error bars indicate SEMs. The figure is modified from Kottyan et al with permission from Nature Genetics.

FIG 7

Proposed role of CAPN14 activity in patients with EoE. IL-13 upregulates CAPN14, the increased expression of which leads to loss of DSG1, which results in impaired barrier function and violated homeostasis, ultimately leading to disrupted integrity of esophageal epithelium. CAPN14 exerts an effector and regulatory role in IL-13–induced responses.