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).