Overexpression of eCLCA1 in small airways of horses with recurrent airway obstruction

Abstract

The human hCLCA1 and murine mCLCA3 (chloride channels, calcium-activated) have recently been identified as promising therapeutic targets in asthma. Recurrent airway obstruction in horses is an important animal model of human asthma. Here, we have cloned and characterized the first equine CLCA family member, eCLCA1. The 913 amino acids eCLCA1 polypeptide forms a 120-kDa transmembrane glycoprotein that is processed to an 80-kDa protein in vivo. Three single nucleotide polymorphisms were detected in the eCLCA1 coding region in 14 horses, resulting in two amino acid changes (485H/R and 490V/L). However, no functional differences were recorded between the channel properties of the two variants in transfected HEK293 cells. The eCLCA1 protein was detected immunohistochemically in mucin-producing cells in the respiratory and intestinal tracts, cutaneous sweat glands, and renal mucous glands. Strong overexpression of eCLCA1 was observed in the airways of horses with recurrent airway obstruction using Northern blot hybridization, Western blotting, immunohistochemistry, and real-time quantitative RT-PCR. The results suggest that spontaneous or experimental recurrent airway obstruction in horses may serve as a model to study the role of CLCA homologs in chronic airway disease with overproduction of mucins.

Figure 1

Amino acid sequence of the primary eCLCA1 translation product (GenBank accession no. AY524856). SS, hydrophobic signal sequence. Conserved cysteine residues are marked with arrows. Asterisks indicate potential sites for N-linked glycosylation; solid circles indicate consensus phosphorylation sites for calcium/calmodulin protein kinase II. Amino acid changes at positions 485 (His/Arg) and 490 (Val/Leu) from single nucleotide polymorphisms are indicated by two letters at the respective positions.

Figure 2

Phylogenetic tree of CLCA family members, including the first equine homolog, eCLCA1. The eCLCA1 polypeptide sequence is part of a cluster (gray box) also containing its direct orthologs, the human hCLCA1, the murine mCLCA3 (alias gob-5), and the porcine pCLCA1. The scale bar represents 5% polypeptide sequence diversity. The tree was constructed using a Clustal W alignment in the DNAStar sequence analysis software.

Figure 3

Single nucleotide polymorphisms (SNPs) within the eCLCA1 open reading frame. Three SNPs were detected in the eCLCA1 mRNA at nucleotides (nt) 1518, 1533, and 1837 in fourteen different horses. Three horses were homozygous for genotype A (eCLCA1/v1), seven horses were heterozygous (genotype B), and four horses were homozygous for genotype C (eCLCA1/v2). The SNPs were verified by sequencing of at least three different cDNA clones.

Figure 4

Characterization of the eCLCA1 protein in vitro and in vivo. The eCLCA1 protein was detected by (A) autoradiography after in vitro translation in the presence of ³⁵S-methionine and (B) Western blotting using specific anti-eCLCA1 antibody α-eCa1. (A) A primary translation product of ~100 kDa was translated in vitro in the absence of microsomal membranes (−MM), which was glycosylated in the presence of microsomal membranes (+MM) to form an ~120 kDa glycoprotein. (B) The in vitro translated (IVT) primary translation product of 100 kDa (−MM) was identified by antibody α-eCa1, but not by the preimmune serum (PI) in a Western blot after SDS-PAGE. In contrast, two smaller proteins of α70 and 80 kDa were identified in protein lysates from equine colon mucosa, suggesting posttranslational cleavage of the primary translation product in vivo, similar to other CLCA proteins (Gruber et al. 2002).

Figure 5

Cl⁻ currents were evoked in eCLCA1-expressing cells by ionomycin-stimulated calcium influx. (A) Slow time-base record shows that ionomycin evoked an inward current in this eCLCA1/v1-expressing cell that was blocked by niflumic acid. The holding potential was −50 mV. Arrows indicate currents evoked in this cell by a series of voltage steps (20 mV steps, 150 msec) applied in control medium (left) and after application of ionomycin (right). There was a break in the slow time base record while the current/voltage relationship was assessed. The record terminates when the recording was abruptly lost. (B) Mean current/voltage relationship for the steady state difference current in cells expressing eCLCA1/v1 (n=13). Difference currents were obtained by subtracting step-evoked currents obtained before application of ionomycin from the currents obtained after ionomycin application. X-axis, current (pA); Y-axis, voltage (mV). (C) Mean current/voltage relationship for the steady state difference current in cells expressing eCLCA1/v2 (n=8).

Figure 6

Upregulation of the eCLCA1 mRNA (A) ³⁵S-exposure of a Northern blot) and protein (B) Western blot probed with antibody α-eCa1 or preimmune serum (PI) in equine lung tissue with recurrent airway obstruction (RAO). (A) The eCLCA1 mRNA was undetectable in the normal equine lung whereas a strong signal was obtained from a lung with RAO. (B) Likewise, the processed eCLCA1 protein of ~80 kDa was only weakly detectable in the lung of a healthy horse, whereas a strong 80-kDa signal and a weaker 120-kDa signal were detected in the lung of a horse with RAO. EF-1a, housekeeping mRNA elongation factor-1a.

Figure 7

Tissue and cellular distribution pattern of the eCLCA1 protein. The protein was detected by immunohistochemistry (brown staining) on formalin-fixed, deparaffinized tissue sections from normal equine nasal mucosa (A,B). (A) Control stain with preimmune serum, nasal turbinate mucosa (C), tracheal mucosa (D), subtracheal glands (E), and mucosa of a large bronchus (F,G). (G) Stained with the periodic acid–Schiff (PAS) reaction to demonstrate colocalization with mucin producing goblet cells (here stained purple), submucosal bronchial glands (H), cutaneous tubular sweat glands surrounding a hair follicle (I), renal papilla (J), small intestine (K), and large intestine (L). When compared with a normal small bronchiolus that does not express eCLCA1 (M), bronchioli from all three horses with recurrent airway obstruction had severe goblet cell metaplasia with strong staining for eCLCA1 (N), including staining of the mucus in the lumen of the airways (arrow). The insets show colocalization of eCLCA1-positive cells (top inset) with mucin-producing goblet cells (bottom inset) that were stained with the PAS reaction. Tissue sections were incubated with antibody α-eCa1, (A–F,I–N) diluted at 1:500), or with preimmune serum (A) (diluted at 1:500), and counterstained with hematoxylin blue. Bars: A–C,E,I–N = 100 μm; D,F–H: 50μm. Inset bar: =20 μm.

Figure 8

Quantitative mRNA expression analysis by real time RT-PCR of eCLCA1 in normal equine tissues and tissues from the equine respiratory tract with recurrent airway obstruction (RAO). Only a subset of all tissues investigated is shown (see text). Expression levels are given as copy numbers of eCLCA1 per copy number of the housekeeping gene elongation factor-1a (EF-1a). Columns represent mean values plus standard deviations. Asterisk indicates statistically significant difference between normal and RAO lung tissue (p<0.05).