Characterization of human pre-elafin mutants: full antipeptidase activity is essential to preserve lung tissue integrity in experimental emphysema

Abstract

Pre-elafin is a tight-binding inhibitor of neutrophil elastase and myeloblastin; two enzymes thought to contribute to tissue damage in lung emphysema. Previous studies have established that pre-elafin is also an effective anti-inflammatory molecule. However, it is not clear whether both functions are linked to the antipeptidase activity of pre-elafin. As a first step toward elucidating the structure/function relationship of this protein, we describe here the construction and characterization of pre-elafin variants with attenuated antipeptidase potential. In these mutants, the P1' methionine residue of the inhibitory loop is replaced by either a lysine (pre-elafinM25K) or a glycine (pre-elafinM25G) residue. Both mutated variants are stable and display biochemical properties undistinguishable from WT (wild-type) pre-elafin. However, compared with WT pre-elafin, their inhibitory constants are increased by one to four orders of magnitude toward neutrophil elastase, myeloblastin and pancreatic elastase, depending on the variants and enzymes tested. As suggested by molecular modelling, this attenuated inhibitory potential correlates with decreased van der Waals interactions between the variants and the enzymes S1' subsite. In elastase-induced experimental emphysema in mice, only WT pre-elafin protected against tissue destruction, as assessed by the relative airspace enlargement measured using lung histopathological sections. Pre-elafin and both mutants prevented transient neutrophil alveolitis. However, even the modestly affected pre-elafinM25K mutant, as assayed in vitro with small synthetic substrates, was a poor inhibitor of the neutrophil elastase and myeloblastin elastolytic activity measured with insoluble elastin. We therefore conclude that full antipeptidase activity of pre-elafin is essential to protect against lung tissue lesions in this experimental model.

Scheme 1

Figure 1. Biochemical properties of pre-elafin mutants

(A) The indicated recombinant pre-elafin peptides were incubated at 85 °C for 30 min. Resistance to heat inactivation was tested by PPE inhibition assay as described in the Experimental section. The enzyme/inhibitor ratios used were 1:0.4, 1:20 and 1:350 for pre-elafin, pre-elafin^M25K and pre-elafin^M25G respectively. (B) Recombinant pre-elafin peptides were incubated at 37 °C for 2 h in the presence of trypsin at a trypsin/protein ratio of 1:2000. Digestion products at 0 and 2 h were resolved by SDS/PAGE (15% gels) under reducing conditions and revealed by silver staining. Note that the time to process the samples (t=0 h) was sufficient to digest pre-elafin, as evidenced by the decreased intensity of intact pre-elafin and the concomitant appearance of smaller species. (C) Purified WT pre-elafin and mutated variants were resolved by SDS/PAGE (15% gels) under non-reducing conditions, transferred on to PVDF membranes, and immunodetected with a polyclonal rabbit anti-elafin antibody specifically recognizing the native and functional disulfide-bonded elafin domain. The 16.5 kDa mass marker is indicated to the left-hand side of the Figure.

Figure 2. Inhibitory profile of WT and pre-elafin mutants

The inhibitory profiles of WT pre-elafin, pre-elafin^M25K and pre-elafin^M25G were compared by measuring the relative activity of trypsin (Tryp) cathepsin G (Cat G) and α-chymotrypsin (Chym) in the presence of an excess (500:1 ratio) of the indicated inhibitor. α1-Peptidase inhibitor (α1-PI) was used as a positive control for peptidase inhibition with a peptidase/α1-peptidase inhibitor ratio of 1:1.

Figure 3. Inhibition of HNE by WT and pre-elafin mutants

The residual activity (Fractional Activity) of HNE (10 nM) was assayed at 25 °C following a 30 min incubation in the presence of the indicated concentrations of inhibitor. The inhibitors used were: ●, pre-elafin; ■, pre-elafin^M25K; dashed line, pre-elafin^M25G. The lines are the best fit of eqn (1) to the experimental data. Note that no experimental points are shown for pre-elafin^M25G as the smallest concentration showing a significant inhibition was 350 nM.

Figure 4. Progress curves for the inhibition of HNE by WT and pre-elafin mutants

Assays were conducted at 25 °C with 5 nM HNE and increasing inhibitor concentrations (from top to bottom). (A) Inhibition by WT pre-elafin (0–250 nM). (B) Inhibition by pre-elafin^M25K (0–250 nM). (C) Inhibition by pre-elafin^M25G (0–1 μM).

Figure 5. Stereo views of structural models for WT and elafin mutants complexed to HNE and PPE

(A) and (B) Molecular representations of WT elafin–enzyme complex (white) superimposed on to an elafin^M25K–enzyme complex (grey). The WT Met²⁵ residue is yellow and Lys²⁵ blue. Residues making direct contact with residue 25 from the inhibitor are in stick representation. Protein backbones are in ribbon representation. Magenta and black dashed lines represent hydrogen bonds for the complex containing pre-elafin and pre-elafin^M25K respectively. (A) Inhibitors complexed to PPE. The yellow dashed line represents the distance between Lys²⁵i side chain nitrogen atom and Arg⁶¹e ζ carbon. (B) Inhibitors complexed to HNE. (C) Elafin^M25G complexed to PPE. The yellow mesh represents the cavity left by the replacement of Met²⁵ by Gly²⁵.

Figure 6. Effect of WT and pre-elafin mutants on lung histopathology and neutrophil influx in an experimental model of emphysema

Animals treated with PPE were given the indicated inhibitor, or saline only (PPE), three times a week for 2 weeks, at which time they were killed. Control animals (Ctrl) received vehicle only. (A) Representative lung slices. (B) L_m calculated from lung slices (15 microscopic fields per animal). *, Only control animals and those receiving PPE and WT pre-elafin show statistical differences compared with animals instilled with PPE only (P≤0.003; n=4–12). (C) Neutrophil influx in BAL. Animals received the indicated inhibitor 1 h post-PPE instillation and BAL were collected 24 h post-PPE administration for neutrophil cell counts. *Statistically different from the animals who received only PPE (P≤0.012; n=4). All results are expressed as means±S.E.M. Comparisons were made using ANOVA followed by a Fisher's protected least-significant difference test with Statview for Windows version 5.0 (SAS Institute, Cary, NC, U.S.A.). Pre-elafin, WT pre-elafin; M25K, pre-elafin^M25K; M25G, pre-elafin^M25G.