Human neutrophil elastase-mediated cleavage sites of MMP-9 and TIMP-1: implications to cystic fibrosis proteolytic dysfunction

Abstract

Cystic fibrosis (CF) is a lethal genetic disorder characterized by airway remodeling and inflammation, leading to premature death. Recent evidence suggests the importance of protease activity in CF pathogenesis. One prominent protease, matrix metalloprotease (MMP)-9, demonstrates increased activity in CF individuals undergoing acute pulmonary exacerbation. This is thought to be mediated by both direct MMP-9 activation and the degradation of its natural inhibitor, tissue inhibitor of metalloprotease-1 (TIMP-1). To examine if this relationship exists in nonexacerbating CF individuals, we examined protease activity in sputum from these individuals compared with nondisease controls. We demonstrated increased gelatinolytic activity in CF sputum. These samples had elevated human neutrophil elastase (HNE) levels which correlated with an increased MMP-9/TIMP-1 ratio. To determine if HNE could discretely cleave and activate MMP-9, these enzymes were coincubated and two specific cleavage sites, between Valine(38) and Alanine(39), and between Alanine (39) and glutamic acid(40) were observed. These sites corresponded with appropriate molecular weight for the activated MMP-9 isoform in CF sputum. Using N-terminal sequencing of cleavage fragments obtained with TIMP-1 incubation with HNE, we confirmed the TIMP-1 cleavage site for HNE is at Valine(69)-Cysteine(70). We also show for the first time that human neutrophils were capable of degrading TIMP-1 ex vivo and that a 16 kDa TIMP-1 fragment was identified in CF sputum, consistent with the expected cleavage of TIMP-1 by HNE. These results demonstrate increased MMP-9 activity in stable CF lung disease, and the presence of specific protease products in CF sputum highlights that HNE-mediated activity plays a role in this dysregulation.

Figure 1

(A) CF patients demonstrate elevated gelatinase activity: CF clinical samples on gelatin zymogram with increased 78 kDa band. Each lane represents a separate patient. (B) CF patients have reduced TIMP-1 levels: Sputum from CF patients (n = 9) are measured via ELISA compared with normal nondisease controls (n = 5), *P < 0.05. (C) CF patients have an elevated MMP-9/TIMP-1 ratio: Sputum from CF patients (n = 9) MMP-9/ TIMP-1 levels are compared with normal nondisease controls (n = 5), *P < 0.01.

Figure 2

(A) CF patients demonstrate increased HNE activity: Sputum from CF patients (n = 9) are measured for HNE activity and then normalized to normal nondisease controls (n = 5), *P < 0.05. (B) MMP-9/TIMP-1 ratio correlates with HNE concentration in CF patients: MMP-9/TIMP-1 ratio and HNE concentration demonstrates an r value of 0.81 (P < 0.05) in CF specimens (n = 9).

Figure 3

(A) HNE cleaves MMP-9 and produces a specific fragment: Coomassie blue stain of recombinant human MMP-9 (2 μg) (lane 1) with HNE (2 μg) (lane 2) at 15 min (lane 3), 30 min (lane 4), 45 min (lane 5) and 60 min (lane 6) demonstrated the presence of a 78 kDa isoform. (B) Residues of prodomain of MMP-9 cleaved by HNE: Arrows indicate other protease cleavage sites leading to activation. The two residues we found to be cleaved by HNE are also shown.

Figure 4

(A) HNE degrades TIMP-1 and produces a specific fragment: Coomassie blue stain of recombinant human TIMP-1 (100 ug/mL, 28 kDa black arrow) incubated with recombinant HNE (50 ug/mL, 27 kDa white arrow) for 2 h (Lane M). Lane H = HNE alone (2 h, 37°C), Lane T = TIMP-1 alone (2 h, 37°C). (B) N-terminal sequencing determines HNE-specific cleavage site of TIMP-1: 16 kDa fragment from Figure 4a was excised and underwent trypsin digestion. Trypsin-cleavage sites (noted by superscripts) were aligned; a cleavage site was found which did not correspond to trypsin specificity (underlined), corresponding to Val⁶⁹ on the TIMP-1 sequence. (C) TIMP-1 cleavage by PMN lysates is rescued by HNE inhibitor: 1 μg of TIMP-1 was incubated with a PMN lysate, PMN lysate + elastase inhibitor (500 fold molar excess), or DMSO (elastase vehicle) for 24 h. Densitometry and band imaging was performed utilizing BioRad Chemidoc XRS system. PMN lysate decreased TIMP-1 expression to 56% of control but HNE inhibitor increases this to 82% of control TIMP-1 band. (D) TIMP-1 fragment is present in CF sputum: Western blot (12% SDS nonreduced gel) demonstrates a 28 kDa TIMP-1 band in both TIMP-1 std (20 ng/mL) and in a pooled CF sputum sample (n = 9). In addition, a 16 kDa TIMP-1 band is identified in the pooled CF sputum sample (white circle).

Figure 5

(A) MMP-9 Crystal Structure (RCSB structure IRJ): The red motif represents the MMP-9 prodomain, while the yellow region represents the remainder of MMP-9 molecule. The purple region represents the catalytic site. The green residue (with white circle) shows the HNE cleavage site (Val³⁸-Ala³⁹ and Ala³⁹-Glu⁴⁰ ) as determined via mass spectrometry. (B) MMP-3 interaction with TIMP-1 (RCSB 1009): While the crystal structure for MMP-9/TIMP-1 interaction is not completely known, it is thought to be similar to MMP-3/ TIMP-1 interaction. The pink colored molecule is MMP-3 and the light blue colored molecule is TIMP-1. The red amino acid is Thr⁹⁸, an important amino acid in TIMP-1 recognition of MMPs. HNE cleavage site for TIMP-1 is Val⁶⁹ (yellow, with white circle) as determined via N-terminal sequencing of TIMP cleavage products.

Figure 6

Increased MMP-9 activity augments potential mechanisms of CF-related patho-biology: Ongoing dysregulation of MMP-9 activity in CF airways impacts various aspects of CF related pathophysiology including airway remodeling, epithelial cell damage, increased mucous production and augmentation of inflammatory response.