Structure-function analysis of CCL28 in the development of post-viral asthma

Abstract

CCL28 is a human chemokine constitutively expressed by epithelial cells in diverse mucosal tissues and is known to attract a variety of immune cell types including T-cell subsets and eosinophils. Elevated levels of CCL28 have been found in the airways of individuals with asthma, and previous studies have indicated that CCL28 plays a vital role in the acute development of post-viral asthma. Our study builds on this, demonstrating that CCL28 is also important in the chronic post-viral asthma phenotype. In the absence of a viral infection, we also demonstrate that CCL28 is both necessary and sufficient for induction of asthma pathology. Additionally, we present the first effort aimed at elucidating the structural features of CCL28. Chemokines are defined by a conserved tertiary structure composed of a three-stranded β-sheet and a C-terminal α-helix constrained by two disulfide bonds. In addition to the four disulfide bond-forming cysteine residues that define the traditional chemokine fold, CCL28 possesses two additional cysteine residues that form a third disulfide bond. If all disulfide bonds are disrupted, recombinant human CCL28 is no longer able to drive mouse CD4+ T-cell chemotaxis or in vivo airway hyper-reactivity, indicating that the conserved chemokine fold is necessary for its biologic activity. Due to the intimate relationship between CCL28 and asthma pathology, it is clear that CCL28 presents a novel target for the development of alternative asthma therapeutics.

Keywords: Asthma; Chemokine; Disulfide; Mass Spectrometry (MS); Nuclear Magnetic Resonance (NMR); Protein Structure.

FIGURE 1.

CCL28 is a key mediator in post-viral asthma. A, CCL28 is significantly elevated on day 21 post-Sendai virus infection, with a continued significant increase to day 49 post-Sendai virus infection. Blocking CCL28 with anti-CCL28 monoclonal antibody (mAb) inhibits the development of post-viral mucous cell metaplasia (MCM) (B) and specific airway reactivity (sRaw, measure of airway hyper-responsiveness) (C) at day 49 post-virus. Values represent the mean ± S.E. *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001; n = 6 mice/group. PAS, periodic acid-Schiff stain; BM, basement membrane; Mch, methacholine.

FIGURE 2.

Recombinant human CCL28 is suitable for NMR structural analysis. A two-dimensional ¹H,¹⁵N HSQC spectrum with sequence-specific assignments for the backbone of CCL28 is shown.

FIGURE 3.

CCL28 adopts the conserved chemokine fold and may possess a unique structural domain. A, a graph generated by PECAN indicating the probability of secondary structural elements in CCL28 based on sequence and chemical shift information. Red bars are indicative of α-helix formation and, blue bars are indicative of β sheets. B, CCL28 sequence alignment and predicted secondary structure. Amino acid sequences of CCL28, CCL27 (36% identity), and CCL5 (18% identity) were aligned using Clustal Omega. CCL27 secondary structure elements are indicated below the alignments. Locations of CCL28 β-sheets and α-helices predicted by PECAN are indicated above the alignments. C, ¹H,¹⁵N heteronuclear NOE plotted as a function of the CCL28 sequence. Red bars emphasize the elevated NOE values seen for amino acids around Cys-80 that are indicative of a more ordered structure.

FIGURE 4.

A novel disulfide bond links the extended C terminus to the chemokine domain. A, graphic depiction of recombinant CCL28 with cysteines highlighted. The image was generated in Protter (34). B, MS1 and MS2 spectra of the dipeptide resulting from a disulfide bridge between Cys-30 and Cys-80. C, MS3 spectra of peptides liberated from ETD fragmentation of the dipeptide using HCD fragmentation to confirm sequence. D, MS1 spectrum of tri-peptide resulting from bridges between Cys-11 and Cys-39 and between Cys-12 and Cys-54. MS2 spectrum using ETD fragmentation of the tri-peptide results in single-charged ions consistent with masses of the liberated peptides ICVSPHNHTVK and ADGDCDLAAVILHVK.

FIGURE 5.

Effect of CCL28 tertiary structure on CD4⁺ T-cell migration in vitro. A, MALDI comparing molecular weights of native (red; hCCL28) and alkylated (blue; uCCL28) CCL28. B, one-dimensional ¹H NMR spectra overlay of both native (red; hCCL28) and alkylated (blue; uCCL28). The solid lines highlight the spectral changes between native and alkylated CCL28, which indicate that alkylated CCL28 is unfolded. Migration of purified mouse CD4⁺ T-cells in response to native (C) or unfolded (D) CCL28 is shown. Cells migrated with chemokine in the lower chamber are represented by black bars. Cells migrated with chemokine in upper chamber are represented by white bars. Values represent the mean ± S.E. *, p ≤ 0.05; **, p ≤ 0.01; samples were run in duplicate with data from n ≥ 2 separate experiments for hCCL28 and uCCL28.

FIGURE 6.

Effect of CCL28 tertiary structure on mouse asthma pathology in vivo. Mice received intranasal hCCL28 or uCCL28 daily for 3 days. 3 μg of hCCL28 but not uCCL28 drives (A) airway hyper-responsiveness (as measured by increased sRaw and decreased specific airway conductance, sGaw). Mch, methacholine. In addition, hCCL28 also drove development of mucous cell (B) metaplasia. BM, basement membrane. Values represent mean ± S.E.; 2 Way analysis of variance for (A) with * p ≤ 0.001, and NS = not significant. For (B) Student's t test with p value as indicated; n ≥ 3 mice/group.

FIGURE 7.

CCL28 is a key player in the pathogenesis of post-viral asthma. Schematic of how CCL28 activity alone can drive airway hyper-responsiveness and mucous cell metaplasia primarily through airway recruitment of a variety of IL-13 producing CCR3 and/or CCR10 expressing cells.

FIGURE 8.

Sequence alignment of all six-cysteine chemokines. The extra disulfide of CCL28 is shown in blue, CCL1 in green, CCL21 in pink, and CCL15 and CCL23 in red due to their similarity. All alignments were generated by Clustal Omega.